R a d io lo g y a n d O n c o lo g y I V o lu m e 5 7 I N u m b e r 2 I P a g e s 1 4 1 -2 7 8 I J u n e 2 0 2 3 june 2023 vol.57 no.2 Radiol Oncol 2023; 57(2): A. June 2023 Vol. 57 No. 2 Pages 141-278 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Publisher Association of Radiology and Oncology Aims and Scope Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high-quality scientific papers and review articles, pertinent to 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 Kovač, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Clinical Oncology, Radiotherapy) Deputy Editors Andrej Cör, University of Primorska, Faculty of Health Science, Izola, Slovenia (Subject Areas: Clinical Oncology, Experimental Oncology) Božidar Casar, Institute of Oncology Ljubljana, Department for Dosimetry and Quality of Radiological Procedures, Ljubljana (Subject Area: Medical Physics) Maja Čemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Igor Kocijančič, University Medical Center Ljubljana, Institute of Radiology, Ljubljana, Slovenia (Subject Areas: Radiology, Nuclear Medicine) Karmen Stanič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy; Clinical Oncology) Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy, Clinical Oncology) Editorial Board Subject Areas: Radiology and Nuclear Medicine Sotirios Bisdas, University College London, Department of Neuroradiology, London, UK Boris Brkljačić, University Hospital “Dubrava”, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Maria Gődény, National Institute of Oncology, Budapest, Hungary Gordana Ivanac, University Hospital Dubrava, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luka Ležaić, University Medical Centre Ljubljana, Department for Nuclear Medicine, Ljubljana, Slovenia Katarina Šurlan Popovič, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Jernej Vidmar, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Subject Areas: Clinical Oncology and Radiotherapy Serena Bonin, University of Trieste, Department of Medical Sciences, Cattinara Hospital, Surgical Pathology Blg, Molecular Biology Lab, Trieste, Italy Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Blaž Grošelj, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Luka Milas, UT M. D. Anderson Cancer Center, Houston, USA Miha Oražem, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Gaber Plavc, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Center Ljubljana, Division of Internal Medicine, Department of Gastroenterology, Ljubljana, Slovenia Andrea Veronesi, Centro di Riferimento Oncologico- Aviano, Division of Medical Oncology, Aviano, Italy Branko Zakotnik, Institute of Oncology Ljubljana, Department of Medical Oncology, Ljubljana, Slovenia Subject Area: Experimental Oncology Metka Filipič, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Janko Kos, University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Tamara Lah Turnšek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Ida Ira Skvortsova, EXTRO-lab, Dept. of Therapeutic Radiology and Oncology, Medical University of Innsbruck, Tyrolean Cancer Research Institute, Innsbruck, Austria Gillian M. Tozer, University of Sheffield, Academic Unit of Surgical Oncology, Royal Hallamshire Hospital, Sheffield, UK Subject Area: Medical Physics Robert Jeraj, University of Wisconsin, Carbone Cancer Center, Madison, Wisconsin, USA Mirjana Josipovic, Rigshospitalet, Department of Oncology, Section of Radiotherapy, Copenhagen, Denmark Häkan Nyström, Skandionkliniken, Uppsala, Sweden Ervin B. Podgoršak, McGill University, Medical Physics Unit, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psyhology, Department of Life Sciences, Trieste, Italy Vassil Hadjidekov, Medical University, Department of Diagnostic Imaging, Sofia, Bulgaria Marko Hočevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Miklós Kásler, National Institute of Oncology, Budapest, Hungary Maja Osmak, Ruder Bošković Institute, Department of Molecular Biology, Zagreb, Croatia Radiol Oncol 2023; 57(2): B. Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217 SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369 Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. Reader for English Vida Kološa Secretary Mira Klemenčič, Zvezdana Vukmirović, Vijoleta Kaluža, Uroš Kuhar Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanović Layout Matjaž Lužar Printed by Tiskarna Ozimek, Slovenia Published quarterly in 400 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJE Zaloška cesta 2 1000 Ljubljana Slovenia Beneficiary bank account number: SI56 02010-0090006751 IBAN: SI56 0201 0009 0006 751 Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2, 1520 Ljubljana; Slovenia SWIFT: LJBASI2X Subscription fee for institutions EUR 100, individuals EUR 50 The publication of this journal is subsidized by the Slovenian Research Agency. Indexed and abstracted by: • Baidu Scholar • Case • Chemical Abstracts Service (CAS) - CAplus • Chemical Abstracts Service (CAS) - SciFinder • CNKI Scholar (China National Knowledge Infrastructure) • CNPIEC - cnpLINKer • Dimensions • DOAJ (Directory of Open Access Journals) • EBSCO (relevant databases) • EBSCO Discovery Service • Embase • Genamics JournalSeek • Google Scholar • Japan Science and Technology Agency (JST) • J-Gate • Journal Citation Reports/Science Edition • JournalGuide • JournalTOCs • KESLI-NDSL (Korean National Discovery for Science Leaders) • Medline • Meta • Microsoft Academic • Naviga (Softweco) • Primo Central (ExLibris) • ProQuest (relevant databases) • Publons • PubMed • PubMed Central • PubsHub • QOAM (Quality Open Access Market) • ReadCube • Reaxys • SCImago (SJR) • SCOPUS • Sherpa/RoMEO • Summon (Serials Solutions/ProQuest) • TDNet • Ulrich's Periodicals Directory/ulrichsweb • WanFang Data • Web of Science - Current Contents/Clinical Medicine • Web of Science - Science Citation Index Expanded • WorldCat (OCLC) This journal is printed on acid- free paper On the web: ISSN 1581-3207 https://content.sciendo.com/raon http://www.radioloncol.com Radiol Oncol 2023; 57(2): C. review 141 Bleomycin electrosclerotherapy (BEST) for the treatment of vascular malformations. An International Network for Sharing Practices on Electrochemotherapy (InspECT) study group report Tobian Muir, Giulia Bertino, Ales Groselj, Lakshmi Ratnam, Erika Kis, Joy Odili, Ian McCafferty, Walter A Wohlgemuth, Maja Cemazar, Aljosa Krt, Masa Bosnjak, Alessandro Zanasi, Michela Battista, Francesca de Terlizzi, Luca G Campana, Gregor Sersa nuclear medicine 150 Correlation of mean apparent diffusion coefficient (ADC) and maximal standard uptake value (SUVmax) evaluated by diffusion-weighted MRI and 18F-FDG-PET/CT in children with Hodgkin lymphoma: a feasibility study Nicolas Rosbach, Sebastian Fischer, Vitali Koch, Thomas J. Vogl, Konrad Bochennek, Thomas Lehrnbecher, Scherwin Mahmoudi, Leon Grünewald, Frank Grünwald, Simon Bernatz radiology 158 CT-guided biopsies of unspecified suspect intrahepatic lesions: pre- procedure Lipiodol-marking improves the biopsy success rate Marcel Christian Langenbach, Thomas Joseph Vogl, Amelie Buchinger, Katrin Eichler, Jan-Erik Scholtz, Renate Hammerstingl, Tatjana Gruber-Rouh 168 Radiological assessment of skeletal muscle index and myosteatosis and their impact postoperative outcomes after liver transplantation Miha Petric, Taja Jordan, Popuri Karteek, Sabina Licen, Blaz Trotovsek, Ales Tomazic 178 ADC values as a biomarker of fetal brain maturation Lucija Kobal, Katarina Surlan Popovic, Jernej Avsenik, Tina Vipotnik Vesnaver 184 Longitudinal monitoring of Apparent Diffusion Coefficient (ADC) in patients with prostate cancer undergoing MR-guided radiotherapy on an MR-Linac at 1.5 T: a prospective feasibility study Haidara Almansour, Fritz Schick, Marcel Nachbar, Saif Afat, Victor Fritz, Daniela Thorwarth, Daniel Zips, Felix Bertram, Arndt-Christian Müller, Konstantin Nikolaou, Ahmed E Othman, Daniel Wegener clinical oncology 191 Awake craniotomy for operative treatment of brain gliomas – experience from University Medical Centre Ljubljana Tilen Zele, Tomaz Velnar, Blaz Koritnik, Roman Bosnjak, Jasmina Markovic-Bozic 201 Cognitive functioning in a cohort of high-grade glioma patients Andreja Cirila Skufca Smrdel, Anja Podlesek, Marija Skoblar Vidmar, Jana Markovic, Jana Jereb, Manja Kuzma Okorn, Uros Smrdel contents contents Radiol Oncol 2023; 57(2): D. 211 Changes in the quality of life of early breast cancer patients and comparison with the normative Slovenian population Cvetka Grasic Kuhar, Tjasa Gortnar Cepeda, Christian Kurzeder, Marcus Vetter 220 Association between PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma treated with adjuvant systemic therapy Domen Ribnikar, Valentina Jeric Horvat, Ivica Ratosa, Zachary W Veitch, Biljana Grcar Kuzmanov, Srdjan Novakovic, Erik Langerholc, Eitan Amir, Bostjan Seruga 229 Subpleural fibrotic interstitial lung abnormalities are implicated in non-small cell lung cancer radiotherapy outcomes Makoto Ito, Takuma Katano, Hiroaki Okada, Ami Sakuragi, Yoshitaka Minami, Souichiro Abe, Sou Adachi, Yukihiko Oshima, Wataru Ohashi, Akihito Kubo, Takayuki Fukui, Satoru Ito, Kojiro Suzuki 239 The influence of BCL2, BAX, and ABCB1 gene expression on prognosis of adult de novo acute myeloid leukemia with normal karyotype patients Zlatko Pravdic, Nada Suvajdzic Vukovic, Vladimir Gasic, Irena Marjanovic, Teodora Karan-Djurasevic, Sonja Pavlovic, Natasa Tosic 249 CD56-positive diffuse large B-cell lymphoma: comprehensive analysis of clinical, pathological, and molecular characteristics with literature review Gorana Gasljevic, Lucka Boltezar, Srdjan Novakovic, Vita Setrajcic-Dragos, Barbara Jezersek-Novakovic, Veronika Kloboves-Prevodnik 257 Quantitative dynamic contrast-enhanced parameters and intravoxel incoherent motion facilitate the prediction of TP53 status and risk stratification of early-stage endometrial carcinoma Hongxia Wang, Ruifang Yan, Zhong Li, Beiran Wang, Xingxing Jin, Zhenfang Guo, Wangyi Liu, Meng Zhang, Kaiyu Wang, Jinxia Guo, Dongming Han 270 Two-stage hepatectomy in resection of colorectal liver metastases − a single-institution experience with case-control matching and review of the literature Spela Turk,Irena Plahuta, Tomislav Magdalenic, Tajda Spanring, Kevin Laufer, Zan Mavc, Stojan Potrc, Arpad Ivanecz I slovenian abstracts contents Radiol Oncol 2023; 57(2): 141-149. doi: 10.2478/raon-2023-0029 141 review Bleomycin electrosclerotherapy (BEST) for the treatment of vascular malformations. An International Network for Sharing Practices on Electrochemotherapy (InspECT) study group report Tobian Muir1, Giulia Bertino2, Ales Groselj3,4, Lakshmi Ratnam5, Erika Kis6, Joy Odili7, Ian McCafferty8, Walter A Wohlgemuth9, Maja Cemazar10,11, Aljosa Krt11, Masa Bosnjak10, Alessandro Zanasi13, Michela Battista13, Francesca de Terlizzi13, Luca G Campana14, Gregor Sersa10,15 1 Department of Reconstructive Plastic Surgery, James Cook University Hospital, Middlesbrough, United Kingdom 2 Department of Otolaryngology Head Neck Surgery, University of Pavia, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo Foundation, Pavia, Italy, 3 Department of Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 5 Department of Interventional Radiology, St George’s University Hospitals NHS Foundation Trust, London, United Kingdom 6 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary 7 Department of Plastic Surgery, St. Georges University Hospitals NHS Trust, London, United Kingdom 8 Birmingham Women’s and Children’s Hospital NHS Foundation Trust, Birmingham, United Kingdom 9 Universitätsklinik und Poliklinik für Radiologie, Universitätsmedizin Halle, Halle, Germany 10 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 11 Faculty of Health Sciences, University of Primorska, Slovenia 12 Department of Otorhinolaryngology, Izola General Hospital, Izola, Slovenia 13 IGEA S.p.A., Clinical Biophysics Lab. Carpi, Modena, Italy 14 Department of Surgery, Manchester University NHS Foundation Trust, Manchester, United Kingdom 15 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(2): 141-149. Received 26 May 2023 Accepted 3 June 2023 Correspondence to: Mr. Tobian Muir, Department of Reconstructive Plastic Surgery, James Cook University Hospital, Middlesbrough, United Kingdom. E-mail: tobian.muir@nhs.net and Prof. Gregor Sersa, Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 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 distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Biomedical applications of electroporation are expanding out of the field of oncology into vaccina- tion, treatment of arrhythmias and now in the treatment of vascular malformations. Bleomycin is a widely used scle- rosing agent in the treatment of various vascular malformations. The application of electric pulses in addition to bleo- mycin enhances the effectiveness of the drug, as demonstrated by electrochemotherapy, which utilizes bleomycin in the treatment of tumors. The same principle is used in bleomycin electrosclerotherapy (BEST). The approach seems to be effective in the treatment of low-flow (venous and lymphatic) and, potentially, even high-flow (arteriovenous) malformations. Although there are only a few published reports to date, the surgical community is interested, and an increasing number of centers are applying BEST in the treatment of vascular malformations. Within the International Network for Sharing Practices on Electrochemotherapy (InspECT) consortium, a dedicated working group has been constituted to develop standard operating procedures for BEST and foster clinical trials. Conclusions. By treatment standardization and successful completion of clinical trials demonstrating the effective- ness and safety of the approach, higher quality data and better clinical outcomes may be achieved. Key words: vascular malformations; bleomycin electrosclerotherapy; bleomycin; electrochemotherapy Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations142 Classification of vascular malformations Vascular malformations are a rare condition caused by abnormally developed blood vessels. They can occur anywhere in the body and range from simple and benign to complex conditions, with an incidence of around 1.5% in the general population. The latest and most used categorization is the International Society for the Study of Vascular Anomalies (ISSVA) classification (Table 1).1 This classification divides vascular anomalies into two main categories: tumors (true proliferative neo- plasms) and malformations (morphogenetic de- fects). These two categories are further subcatego- rized: tumors are divided into benign, locally ag- gressive/borderline and malignant, whereas mal- formations are subdivided into simple, combined or associated with other anomalies. Clinically, vas- cular anomalies can also be divided into low-flow and high-flow malformations.2 Treatment of vascular malformations Current approaches vary depending on the type and anatomical location of the vascular malforma- tion. Treatment options include observation, scle- rotherapy, laser therapy, embolization, and sur- gery.3,4 Observation is recommended for asymp- tomatic superficial or low-flow malformations that pose no immediate risk to the patient and are stable in size. Sclerotherapy involves the injection of sclerosing agents, such as bleomycin, or other agents (pingyangmycin, absolute ethanol, ethanol- amine oleate, polidocanol, doxycycline, cyanoacr- ylate, sodium morrhuate and sodium tetradecyl sulfate (STS)).5,6 Laser therapy is used to treat su- perficial vascular malformations and involves the use of a laser to heat the affected area and reduce vessel size. Embolization is a minimally invasive procedure in which small particles, metal coils, or solidifying liquid agents are injected into the mal- formation to block the flow of blood and reduce its size. This treatment is typically used for high- flow malformations. Surgery may be necessary for high-flow malformations that are difficult to treat with the other methods mentioned. Depending on the size and anatomical location, the surgeon may only remove part of the lesion.7 The use of bleomycin combined with electroporation (electrochemotherapy) in oncology There are several biomedical applications of electroporation. Reversible and irreversible elec- troporation are distinguished by the amplitude, timing and number of electric pulse applications. Irreversible electroporation is based on irreversi- ble disruption of the cell membrane causing desta- bilization of cell physiology to the extent that cells die either by apoptosis, necrosis or even immuno- genic cell death.8 In contrast, reversible electropo- ration does not cause cell death but temporarily disrupts the cell membrane in such a way that it becomes permeable for molecules that have ham- pered transport through the membrane.9–12 This TABLE 1. International Society for the Study of Vascular Anomalies (ISSVA) classification for vascular anomalies 2018 VASCULAR TUMORS VASCULAR MALFORMATIONS Benign Locally aggressive Malignant Simple Combined Infantile hemangioma Kaposiform hemangioendothelioma Epithelioid hemangioendothelioma Angiosarcoma Capillary malformation (CM) CVM, CLM Congenital hemangioma Retiform hemangioendothelioma Lymphatic malformation (LM) LVM. CLVM Tufted hemangioma PILA. Dabska tumor Venous malformation (VM) CAVM Spindle-cell hemangioma Composite hemangioendothelioma Arteriovenous malformation (AVM) CLAVMEpithelioid hemangioma Kaposi sarcoma Arteriovenous fistula Pyogenic granuloma AVM = arteriovenous malformation; CAVM = capillary arteriovenous malformation; CLAVM = capillary lymphatic arteriovenous malformation; CLM = capillary lymphatic malformations; CLVM = capillary lymphatic venous malformation; CM = capillary malformation; CVM = capillary venous malformations; LM = lymphatic malformation; LVM = lymphatic venous malformation; PILA – papillary intralymphatic angioendothelioma; VM - venous malformation Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 143 phenomenon can be exploited for enhanced drug, DNA or RNA delivery into cells. If we deliver nu- cleic acids, it is called gene electrotransfer, which can be used for cancer immune-gene therapy; for example, if introduced, DNA or mRNA encodes immunomodulatory molecules. It can also be used for vaccination purposes.13,14 The biomedical ap- plications of gene electrotransfer can be used in the treatment of cancer, as well as other diseases, including vaccination for infectious diseases such as SARS CoV-2 virus.15 In the case of electrochemo- therapy, electroporation is used to enhance the de- livery of cytotoxic molecules for cancer treatment. The principle is to inject cytotoxic drugs such as bleomycin or cisplatin into the cancer patient and apply electric pulses at the site of the tumor, where enhanced drug uptake is desired.16 Therefore, elec- trochemotherapy is a local treatment since drug cytotoxicity is enhanced only at the site of electric pulse applications. This approach is being used widely in the treatment of either cutaneous tumors or deep-seated tumors in internal organs, such as liver and pancreas.16 Several electrodes were de- signed that are best suited for the delivery of elec- tric pulses to specific anatomical locations. Due to its simple principle, i.e., the use of highly cyto- toxic drugs and the application of electric pulses for its enhanced cytotoxicity, electrochemotherapy is effective on tumors of different histological ori- gins. Its objective response rate ranges between 70-80%.17 Electrochemotherapy is listed in many national and international guidelines as a local ablative therapy and is applied in more than 200 centers across Europe.16 Electrochemotherapy mechanisms of action There are three underlying mechanisms of elec- trochemotherapy. The first is enhanced drug de- livery to tumor cells, which die due to the cytotox- icity of the drugs, either by apoptosis or necrosis. This is predominantly related to the drug used and its mode of action. Bleomycin, for example, induces mitotic cell death, which induces slow resolution of the tumor mass. Experimental data on tumors in mice demonstrate that the drug concentration in tumor cells after electroporation is increased 10 times or more, depending on the tumor type, in the case of bleomycin electrochemotherapy.18 The second mechanism is the induction of the immune response due to immunogenic tumor cell death induced by the drug. It is well known that certain ablative therapies induce immunogenic cell death that can attract and boost the immune response of the organism. This mechanism has been described in radiation therapy, thermal ab- lative techniques, and electrochemotherapy.10,12,19 Several groups have investigated the role of im- munogenic cell death in the effectiveness of elec- trochemotherapy. Now, the experimental data indicate that the response of the tumors varies depending on the immunogenicity of the tumors; more immunogenic tumors respond better to elec- trochemotherapy than less immunogenic tumors, which was linked to more pronounced immuno- genic cell death after electrochemotherapy.10,12,20 Additionally, clinical data on the treatment of melanoma demonstrate that local treatment of cutaneous metastases can boost or interact with treatment using immune checkpoint inhibitors, such as pembrolizumab.21 Patients treated with both electrochemotherapy and immune check- point inhibitors had lower disease progression rates and longer survival than those who received pembrolizumab only. The third mechanism is the vascular disrupting effect of electrochemotherapy. In early preclinical research, it was established that the application of electric pulses only temporarily abrogates blood flow within tumors. This phenomenon was termed vascular lock and lasts less than an hour.22,23 Furthermore, the effect is enhanced when the drug is present during application of the electric pulses. Investigations have shown that it results in vascular disruption that occurs within hours in tumors. Endothelial cells start to die, blood flow is obstructed, and secondary tumor cell death is induced within days due to induced tumor hy- poxia.24–27 The phenomenon is predominantly con- fined to the tumor vasculature, sparing the normal vasculature around the tumors. The reason for this is because of the high proliferation rate of endothe- lial cells in tumors compared to the vasculature in normal tissues, where the endothelial proliferation rate is very slow. The vascular disrupting effect of electrochemotherapy is not fully understood. To date, we do not know in what proportion this vas- cular disrupting effect contributes to the overall effectiveness of electrochemotherapy in specific tumor types. We know that it is dependent on the distribution and extent of tumor vascularization, and better vascularized tumors respond better to electrochemotherapy.28,29 Preclinical data also indi- cate that tumor perfusion influences the effective- ness of the treatment, with well perfused tumors showing an improved response.30 Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations144 Combination of bleomycin with reversible electroporation for treatment of vascular malformations Electrochemotherapy is safely applied to palliate bleeding cutaneous metastases and treat vascular tumors e.g., Kaposi sarcoma, superficial angio- sarcoma31-34 and highly vascularized liver metas- tases.35,36 Bleeding after the insertion of needle electrodes quickly stops due to vascular lock.37 Additionally, both superficial and liver tumors themselves also do not bleed after electrochemo- therapy due to the vascular disrupting effect. These observations indicate that electrochemo- therapy indeed exerts vascular effects, the above- mentioned vascular lock and the vascular disrupt- ing effect. Furthermore, several reports indicate that electrochemotherapy can be safely applied to control or treat bleeding tumors.37 Of note, bleed- ing stops almost immediately after the application of electric pulses; therefore, treatment of bleeding tumors is also one of the indications for electro- chemotherapy (Figure 1). These data support the potential advantage of bleomycin electrosclerotherapy in the treatment of vascular malformations. Bleomycin is already one of the most frequently used sclerotherapy agents in the treatment of these lesions.5,6 Therefore, the combination with electric pulses may only add to the effectiveness of bleomycin since it would in- crease the uptake of the drug into the endothelial lining of the affected blood vessels. In many cases, blood vessels are abnormal and endothelial cell proliferation is higher than that in normal blood vessels.38 Therefore, the vasculature in vascular malformations is impaired as it is in tumors, and electrochemotherapy is supposed to be effective in both. Histological evidence from liver biopsies in- dicates that venules are more sensitive to elec- trochemotherapy than arterioles in normal liver parenchyma.39 This would indicate that venous malformations would have been more susceptible to BEST. We recently performed a study on pigs inves- tigating vascular changes in large blood vessels such as the portal vein, inferior vena cava, and lineal vein. In this study, the vessels were directly exposed to electroporation and electrochemo- therapy by the application of electric pulses us- ing plate electrodes that embraced the vessels. Electrochemotherapy may temporarily disrupt the endothelial lining and disrupt the vasa vasorum of the vessels (unpublished data). This effect may, however, be a desired one in the treatment of all vascular malformations, specifically because we have not observed thrombi formation in the treat- ed vessels in the pig model. All this evidence supports the use of bleomycin electroporation in the treatment of vascular mal- formations, also called bleomycin electrosclero- therapy (BEST). In principle, BEST could be a safe and effective approach for the treatment of vascu- lar malformations; however, more clinical data are needed to confirm this approach. To date, there are only a handful of clinical reports on the treatment of vascular malformations with BEST. Finally, and importantly, the technique needs to be standard- ized through the development of dedicated stand- ard operating procedures. FIGURE 1. Treatment of vascularized melanoma metastasis by electrochemotherapy. (A) Highly vascularized tumor before treatment. (B) Bleeding due to electrode insertion after application of electric pulses to the tumor. (C) Bleeding stopped immediately after electric pulse application. A B C Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 145 Overview of current clinical BEST reports Publications on BEST are still limited in number. Table 2 summarizes the clinical studies and case reports published thus far.40–45 In summary, dif- ferent types of vascular malformations have been treated, with favorable clinical outcomes. Most of the studies used intralesional bleomycin, either di- luted or mixed with lidocaine. Bleomycin dosage varied between studies, but in most reports, it was lower than in traditional sclerotherapy. In addi- tion, the number of treatments required was much lower when BEST was used than when bleomycin was used alone. Drug dosage, the number of treat- ments needed, and route of drug administration are all aspects that need to be explored to develop recommendations for the future use of BEST. Another relevant aspect of BEST is the applica- tion of electric pulses. Due to the blood accumu- lated in the malformation, the electrical conduc- tivity of the treated tissue is high. Therefore, it is assumed that the coverage of the target lesion with electric pulses does not need to be so strict as in electrochemotherapy. In this regard, some clini- cians who perform BEST on patients report that fewer applications of electric pulses to the lesions are needed. This aspect that requires clarification in future studies. Studies on BEST should also report the num- ber and amplitudes of pulse applications and the electrical parameters (current). The predominantly used generator is from one producer, and vary- ing electrodes specific to this generator are used. Usually, in each electric pulse application 8 pulses of 1000−1300 V/cm in frequency 5 kHz are applied. Since different electrodes are used for different clinical situations, the reports should describe which electrodes were used. Furthermore, when standard operating procedures for BEST will be prepared, recommendations for the use of specific types of electrodes should also be prepared. BEST standardization To date, there are no standardized guidelines for BEST. As a result, each center applies the treatment according to local protocols and clinical experi- ence. Therefore, standardization of some proce- dural aspects is needed. The spread of a new technology depends on its safety. Safety aspects have already been cor- roborated, since there are already some reports of significant morbidity with bleomycin only, but not with BEST treatment.46 We must be aware that cy- totoxic drugs are used for the treatment of benign disease, that can be spilled and are sometimes also used in very young patients. This safety aspect is also related to the experience in image-guided ble- omycin and electric pulse application (Figure 2). Currently, BEST must be practiced in the frame- work of clinical studies, where patient referral and suitability for BEST treatment need to be defined. The patients need to be informed about other treatment options and their suitability for the treatment evaluated by a multidisciplinary team (MDT). It is expected that in the early stage of develop- ment, BEST will be considered in recurrences after previous treatments, whereas subsequently more precise indications for specific types of vascular malformations will be individuated. FIGURE 2. Patient treated by BEST. Axial T2-weighted, fat-saturated MRI with hyperintense (arrow) gluteal venous malformation before treatment (A). Axial T2-weighted, fat-saturated MRI of the same region 4 months after treatment. The main part of the venous malformation is occluded (B). Photography before (C) and after the treatment (D). A B C D Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations146 Other relevant procedural aspects that need to be clarified include drug injection, questions of dosage, the need for general anesthesia, electrode placement and delivery of electric pulses. Exclusion criteria need to be defined as well, such as pregnancy, lactation and allergy or hyper- sensitivity to bleomycin, or abnormal respiratory parameters. The application of electric pulses, especially with needle electrodes, is painful. Therefore, local or general anesthesia is needed. In this regard, the experience accumulated with electrochemo- therapy can be informative. Generally, the choice of the type of anesthesia is at the discretion of each center. However, there is a recent report that continuous intravenous sedation is also an option that requires less anesthetic and is much shorter in time.47 Another important issue is the route of bleo- mycin injection. In electrochemotherapy, the pre- ferred route is intravenous injection; however, in BEST treatment, this is a limited option since in- travenous sclerotherapy with bleomycin is not the standard of care. Instead, direct injection is pre- ferred, possibly under image guidance and poten- tially with a lower dose to avoid systemic toxicity. In electrochemotherapy, the bleomycin dose in intravenous or intratumoral administration is defined.48 In elderly patients older than 65 years or with renal impairment, the intravenously ad- ministered dose can be decreased by 1/3.48 In BEST treatment, the dose can be substantially decreased. The lowest effective dose of bleomycin for BEST treatment still needs to be established. The con- centration of bleomycin in the solution needs to be standardized. Another peculiar aspect is that the volume of the drug solution is dependent on the type and volume of the malformation. Furthermore, it de- pends on whether bleomycin is diluted either with foam, lidocaine or contrast agent and whether there is drainage from the malformation that needs to be stopped, either by compression or in- travascular techniques. By all means, the concen- tration and volume of the drug injected needs to be recorded and reported. The dose and route of administration may also differ in the case of fast or slow flow malformations. The treatment can be repeated up to a cumulative dose for bleomycin of 400 000 IU.48 The interval between the intralesional bleo- mycin injection and the application of electric TABLE 2. Clinical studies and case reports using bleomycin electrosclerotherapy40–45 Reference Type of malformation N of pts Bleomycin dose and concentration Electrodes used N of pulse applications Response Comment McMorrow et al., Br J Oral and Maxillofacial Surg 201744 Venous malformation 1 Reduced dose: 1/3 of the standard dose Not reported Not reported Considerable improvement after 6 unsuccessful sessions with bleomycin Case report with poor respiratory function Horbach et al., Dermatologic Surgery 202045 Hypertrophic capillary malformations 5 pts. (out of 20 planned) 0.25 mg or units/cm3 Plate & needle Not reported 7-8 weeks DEROI (changes in colorimetry) Flux in ROI (in Perfusion Units) Randomized controlled pilot trial Dalmady et al., Pediatrics 202043 Lymphatic malformation 1 0.5 mg/kg (5.4 mg) Needle 1st session: 68 applications 2nd session: 74 applications 63% growth-corrected volume decrease. No recurrence at 18 months Follow up Case report Wohlgemuth et al., Journal of Vascular Surgery 202140 Venous malformations 17 pts. (20 lesions) Calculated based on the size of the lesion. C = 0.25 mg/mL Intralesional injection (25% concentration of standard bleomycin sclerotherapy) Needle & finger Not reported 3-month post-therapy Changes in volume MRI: Volume reduction,%: > 90% 9 lesions > 70% in < 90% 6 lesions > 50% in < 70% 2 lesions < 35% 2 lesions No response 1 lesion Retrospective observational case study Kostusiak et al., Dermatologic Surgery 202242 Various vascular malformations 30 pts. VM: 17 AVM: 3 CVM LM: 2 Mixed: 2 Calculated based on the size of the lesion. Bleomycin mixed with 1 mL plain 1% lidocaine Dose not reported Needle & finger Not reported 17 Complete Response 7: significant improvement 1: moderate improvement 1: minor response 1: no response 3: active follow up Prospective observational case study Electrosclerotherapy offered to non-responding patients to standard bleomycin Krt et al., Front Oncol 202241 Arteriovenous malformation 1 750 IU BLM intralesional Plate 15 CR 18 months after BEST Case report AVM = arteriovenous malformation; BLM = bleomycin; CVM = capillary venous malformations; VM - venous malformation; LM = lymphatic malformation; Radiol Oncol 2023; 57(2): 141-149. Muir T et al. / Bleomycin electrosclerotherapy of vascular malformations 147 pulses needs to be short, while the drug is pre- sent in the treated tissue. The one-minute interval would be enough. A similar situation is also ob- served in electrochemotherapy, where the time in- terval is different when bleomycin is injected either intratumorally or intravenously. In the case of in- travenous injection, the time interval is 8 minutes, and after intratumoral injection, the time interval is just 1-3 minutes.48 The choice of the electrodes used is dependent on many factors. The type, lo- cation and size of the malformation are the most important factors. Needle electrodes with shorter or longer needles in fixed geometry are available. Some centers have concerns about so-called hex- agonal electrodes since they deliver many electric pulses between the needles with a high risk of skin hyperpigmentation at the puncture sites. Some malformations require so-called variable geom- etry electrodes. These are single long needle elec- trodes that can be placed separately and can cover deeper mostly subfascial malformations. Vascular malformations do not need to be cov- ered entirely with electric pulses, in contrast to electrochemotherapy. In the case of BEST, damage to the endothelium and vessels needs to be done throughout the malformation but not necessarily with dense and complete coverage of the whole lesion, since the goal is to improve the symptoms more than eradicating the lesion. In this way, the risk of tissue swelling and mucosal ulceration could be reduced. BEST treatment can be performed as a day case procedure unless pain, bleeding or swelling is an- ticipated. Generally, follow-up is recommended at approximately three-month intervals. These are recommendations and considerations for BEST treatment according to the experiences gained by the authors of this manuscript. The members of this working group will continue to share experiences and discuss results to identify the procedural aspects associated with the best re- sults. When a more formal consensus is reached, we will propose our best practice in the form of standard operating procedures (SOPs). Role of InspECT in BEST applications InspECT is an international network of 42 clinical centers using electrochemotherapy for the treat- ment of cancer. This is the largest group of experts on electroporation-based treatments. Some of them are acquainted with BEST and report a posi- tive experience as other external centers. Together, these centers form a network that can promote BEST worldwide. A dedicated working group for vascular malformations has been formed within InspECT. This group will promote clinical studies with BEST and seek collaboration with other cent- ers. This paper aims to raise interest and aware- ness in the treatment of vascular malformations with BEST and provide an overview of the current status of development of this approach. Future directions and conclusions The number of clinicians using BEST to treat vascu- lar malformations is growing. Due to the first and positive experiences in various vascular malfor- mations, BEST application is being practiced in an increasing number of centers throughout Europe and the UK. This article summarizes the rationale and underlying mechanisms of BEST, along with the initial clinical experiences. Additionally, it highlights the main controversial procedural as- pects and the need for dedicated SOPs. Acknowledgments The authors acknowledge the financial support from the state budget by the Slovenian Research Agency, program no. P3-0003. References 1. Classification International Society for the Study of Vascular Anomalies. [Internet]. [cited 2023 May 16]. Available at: https://www.issva.org/clas- sification 2. Wu IC, Orbach DB. Neurointerventional management of high-flow vascular malformations of the head and neck. 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Updated standard operating procedures for electrochemotherapy of cu- taneous tumours and skin metastases. Acta Oncol 2018; 57: 874-82. doi: 10.1080/0284186X.2018.1454602 Radiol Oncol 2023; 57(2): 150-157. doi: 10.2478/raon-2023-0021 150 research article Correlation of mean apparent diffusion coefficient (ADC) and maximal standard uptake value (SUVmax) evaluated by diffusion- weighted MRI and 18F-FDG-PET/CT in children with Hodgkin lymphoma: a feasibility study Nicolas Rosbach1, Sebastian Fischer1, Vitali Koch1, Thomas J. Vogl1, Konrad Bochennek2, Thomas Lehrnbecher2, Scherwin Mahmoudi1, Leon Grünewald1, Frank Grünwald3, Simon Bernatz1 1 Department of Diagnostic and Interventional Radiology, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany 2 Division of Paediatric Haematology and Oncology, Hospital for Children and Adolescents, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany 3 Department of Nuclear Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany Radiol Oncol 2023; 57(2): 150-157. Received 02 February 2023 Accepted 27 March 2023 Correspondence to: Dr. Nicolas Rosbach, Institute for Diagnostic and Interventional Radiology, University Hospital Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. E-mail: nicolas.rosbach@kgu.de Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. The objective was to analyse if magnetic resonance imaging (MRI) can act as a non-radiation ex- posure surrogate for (18)F-Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/ CT) in children with histologically confirmed Hodgkin lymphoma (HL) before treatment. This was done by analysing a potential correlation between apparent diffusion coefficient (ADC) in MRI and the maximum standardized uptake value (SUVmax) in FDG-PET/CT. Patients and methods. Seventeen patients (six female, eleven male, median age: 16 years, range: 12–20 years) with histologically confirmed HL were retrospectively analysed. The patients underwent both MRI and (18)F-FDG PET/ CT before the start of treatment. (18)F-FDG PET/CT data and correlating ADC maps in MRI were collected. For each HL-lesion two readers independently evaluated the SUVmax and correlating meanADC. Results. The seventeen patients had a total of 72 evaluable lesions of HL and there was no significant difference in the number of lesions between male and female patients (median male: 15, range: 12–19 years, median female: 17 range: 12–18 years, p = 0.021). The mean duration between MRI and PET/CT was 5.9 ± 5.3 days. The inter-reader agreement as assessed by the intraclass correlation coefficient (ICC) was excellent (ICC = 0.98, 95% CI: 0.97–0.99). The correlated SUVmax and meanADC of all 17 patients (ROIs n = 72) showed a strong negative correlation of -0.75 (95% CI: -0.84, – -0.63, p = 0.001). Analysis revealed a difference in the correlations of the examination fields. The correlated SUVmax and meanADC showed a strong correlation at neck and thoracal examinations (neck: -0.83, 95% CI: -0.93, – -0.63, p < 0.0001, thoracal: -0.82, 95% CI: -0.91, – -0.64, p < 0.0001) and a fair correlation at abdominal examinations of -0.62 (95% CI: -0.83, – -0.28, p = 0.001). Conclusions. SUVmax and meanADC showed a strong negative correlation in paediatric HL lesions. The assessment seemed robust according to inter-reader agreements. Our results suggest that ADC maps and meanADC have the potential to replace PET/CT in the analysis of disease activity in paediatric Hodgkin lymphoma patients. This may help reduce the number of PET/CT examinations and decrease radiation exposure to children. Key words: Hodgkin lymphoma; diffusion weighted imaging; apparent diffusion coefficient; MRI; PET/CT Radiol Oncol 2023; 57(2): 150-157. Rosbach N et al. / MRI as surrogate for PET/CT in children with Hodgkin lymphoma 151 Introduction Hodgkin lymphoma (HL) accounts for approxi- mately 6% of all paediatric cancers. It has an in- cidence rate of 12 cases per million per year in the age group 0–14 with a male predominance.1,2 Clinical trials and advances in therapy lead to an improvement of the 5-year survival rate for children newly diagnosed with HL.3,4 The cur- rent National Comprehensive Cancer Network (NCCN) guidelines do not address HL in paediat- ric patients.5 Therefore, initial radiological staging examinations depend on study protocols. Most pa- tients with HL receive (18)F-Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) scans as initial staging and during follow-up to assess early response and to identify responders or non-responders to chemotherapy.6-8 Over 95% of children with HL will become long-time survivors.4 Currently the Deauville five-point scale is recommended for FDG-PET/CT-based response assessment in pa- tients with lymphoma. It is a visual scale using mediastinal and liver blood pool FDG-uptake as reference points.9 The therapeutic improvements lead to increasing live expectancy and increasing number of dose-intense follow-up examinations with PET/CT. Several studies examined methods to reduce the radiation exposure for paediatric patients in whole body PET/CTs, but FDG-PET/CT is still the preferred examination to evaluate the treatment response of HL patients.10 Magnetic res- onance imaging (MRI) plays an important role in a wide field of paediatric specialities, ranging from acute trauma to oncology.11-14 In HL patients MRI is used to evaluate soft tissue lesions. In contrast to PET/CT imaging there is no radiation exposure in MRI examinations, which is especially benefi- cial in paediatric patients. In MRI with diffusion weighted imaging (DWI) apparent diffusion coef- ficient maps can be calculated. Apparent diffusion coefficient (ADC) maps have been utilized in dif- ferent setting such as ischemic stroke, heart imag- ing and differentiation between several types of cancer and cancer detection.15-18 The potential of MRI-derived apparent diffusion coefficient meas- urements as radiation free surrogate for SUVmax has not yet been evaluated. In the present study, we retrospectively evaluated the correlation be- tween ADC and SUVmax in paediatric patients with HL. Patients and methods This retrospective study was approved by the in- stitutional review board (IRB) of the University Hospital Frankfurt (IRB; 2022-603). Inclusion criteria were (I) histologically con- firmed Hodgkin lymphoma with (II) pretherapeu- tic MRI and (III) (18)F-FDG PET/CT on the same MRI or PET/CT in (IV) patients < 18 years with a (V) maximum duration between MRI and PET/CT of 30 days. FIGURE 1. Flowchart for recruitment of study subjects according to the Standards for Reporting of Diagnostic Accuracy (STARD) studies. Radiol Oncol 2023; 57(2): 150-157. Rosbach N et al. / MRI as surrogate for PET/CT in children with Hodgkin lymphoma152 Exclusion criteria were (I) missing ADC assess- ment, (II) duration between MRI and PET/CT > 30 days, (III) imaging artifacts (Figure 1). MR imaging acquisition and examination Examinations of this retrospective single centre study took place at University Hospital Frankfurt am Main/Germany at a single 1.5-T MRI Scanner in clinical routine using a standard 18-channel body-coil (Magnetom Aera; Siemens Healthineers, Forchheim/Germany) and at a single PET/CT Scanner (Biograph 6; Siemens Healthineers, Forchheim/Germany). Neck MRI examinations were performed using the following sequences: (a) T2-weighted (T2w) Turbo inversion recovery magnitude (TIRM) in transversal orientation, (b) T1-weighted (T1w) turbo spin echo (TSE) in transversal (with fat sup- pression) and coronal orientation (without fat sup- pression, substraction images were calculated) with and without contrast media, and diffusion- weighted magnetic resonance imaging (DWI) (b- values: 50, 200, 800). TABLE 1. Magnetic resonance imaging sequences Sequence Orientation Body part T2w-TIRM in transversal orientation transversal neck T1w-TSE (fat suppressed, +/- contrast media) transversal neck T1w-TSE (no fat suppression, with substraction, +/- contrast media) coronal neck DWI (b-values: 50, 200, 800) transversal neck, body T2w-HASTE coronal, sagittal and transversal body T1w-VIBE (with fat suppression) without breath-hold-imaging +/- contrast media transversal body DWI = diffusion-weighted magnetic resonance imaging; HASTE = T2-weighted half-Fourier acquisition single-shot turbo spin-echo; T1w = T1-weighted; T2w = T2-weighted; TIRM = turbo inversion recovery magnitude; TSE = turbo spin echo; VIBE = T1-weighted volumetric interpolated breath-hold examination Body MRI examinations were performed us- ing the following sequences: (a) T2-weighted half-Fourier acquisition single-shot turbo spin- echo (HASTE) in coronal, sagittal and transver- sal orientation, (b) diffusion-weighted magnetic resonance imaging (b-values: 50, 200, 800), and (c) T1-weighted volumetric interpolated breath-hold examination (VIBE) dixon (with fat suppression) in transversal orientation without breath-hold-im- aging, without and with contrast media (Table 1). In PET/CT examinations the mean computed tomography dose index (CTDI) was 2.2 ± 0.8 Milli- Gray (mGy). The mean dose length product (DLP) was 215.1 ± 92.1 mGy*cm (Table 2). Image evaluation Image evaluation was performed by using a con- ventional picture archiving and communication system station (PACS-station, Centricity Universal Viewer, Version 7.0). MRI examinations and (18) F-FDG PET/CT examinations with temporal cor- relation (both examinations within one month) were paired (Figure 2). At MRI, the Hodgkin le- TABLE 2. Radiation exposure and examination time Modality CTDI [mGy] DLP [mGy*cm] Examination time [min] Mean (SD) Range Mean (SD) Range Mean (SD) Range FDG-PET/CT 2.2 (0.8) 1.2–4.1 215.1 (92.1) 93.9–410.2 28 (8:26)1 20–49 MRI Neck 19:45 (3:41) 17:21–24:47 Thorax 09:23 (2:12) 08:01–10:29 Abdomen 09:23 (2:59) 07:32–12:21 CTDI = computed tomography dose index; DLP = dose length product; mGy = milligray; min = minutes; 1 FDG has to distribute for 1h after application. Time given in the table is the scanning time after application Radiol Oncol 2023; 57(2): 150-157. Rosbach N et al. / MRI as surrogate for PET/CT in children with Hodgkin lymphoma 153 sions were identified on DWI and ADC. For each Hodgkin lesion in (18)F-FDG PET/CT with meas- ured SUV two readers (N.R, board-certified ra- diologist with six years of experience and S.B., radiology resident with four years of experience) defined a 2D-ROI in the correlating MRI lesion in ADC maps covering the entire HL lesion and the mean ADC was evaluated. In total 72 ROIs with correlating SUVmax lesions were evaluated. After an initial analysis the 72 ROIs were subdivided into the three different examination areas. 22 ROIs were selected at neck imaging, 28 ROIs at thora- cal imaging and 22 ROIs at abdominal imaging. Image quality and noise were evaluated by using a 5-point Likert scales (1, unacceptable; 5, excellent). Statistical analysis Statistical analyses were performed using RStudio 2021.09.2 (Posit PBC). The nonparametric Kolmogorov-Smirnov test was applied to assess the normality of the data. Variables were expressed as means ± standard deviation and analyzed with the Wilcoxon test. A p < .05 (two-tailed) was consid- ered statistically significant. Correlation between SUVmax and meanADC was calculated using the Pearson’s Product Moment Correlation Coefficient. The difference between the correlations of neck, thoracal and abdominal meanADC was calculated using the Fisher Z-Transformation with Z Test sta- tistic (Z-Score) and probability (p).19,20 According to Landis and Koch, weighted κ statistics was used evaluating the interrater agreement.21 Results Between April 2015 and November 2021 39 pedi- atric patients underwent treatment for Hodgkin lymphoma at the University Hospital Frankfurt am Main and received as part of routine diagnos- tics a PET/CT examination. Out of these, seventeen patients (median age: 16 years, range: 12–20 years; six females [median age: 17 range: 12–18 years] and FIGURE 2. MRI and FDG-PET/CT imaging of two Hodgkin lymphoma (HL) patients. Left side (A, B): 12yo patient with newly diagnosed HL, (A) MRI examination with thoracal apparent diffusion coefficient (ADC) map. (B) correlating FDG-PET/ CT examination with a HL lesion and calculated SUVmax. right side (C, D): 9yo patient with newly diagnosed HL, (C) MRI examination with neck ADC map. (D) correlating FDG-PET/CT examination with a HL lesion and calculated SUVmax. TABLE 3. Patient characteristics and classifications Variable Retrospective cohort of patients diagnosed with Hodgkin lymphoma; baseline features No. of patients 17 Median age (SD), years 15.8 (2.2) Sex Male 11 (65%) Female 6 (35%) Lugano classification 1 3 (18%) 2 6 (35%) 3 4 (23%) 4 4 (23%) Hodgkin lymphoma subtypes (WHO classification) Nodular sclerosis 9 (52%) Mixed cellularity 5 (29%) Lymphocyte rich 2 (12%) Lymphocyte depleted 1 (6%) Unless otherwise indicated, data are the number of patients. WHO = World Health Organization A B C D Radiol Oncol 2023; 57(2): 150-157. Rosbach N et al. / MRI as surrogate for PET/CT in children with Hodgkin lymphoma154 eleven males [median age: 15, range: 12–19 years]) met the inclusion criteria (Table 3). One ROI was defined in each of the 72 evalu- able lesions in MRI examinations of 17 patients (Figure 2). Pretherapeutic mean ADC was 931.17 x 10‒3 mm2/s ± 282.39 x 10‒3 mm2/s (minimum: 373 x 10‒3 mm2/s, maximum: 1658 x 10‒3 mm2/s). Pretherapeutic mean SUVmax was 6.53 ± 2.37 (minimum: 2.92, maximum: 13.4). The meanADC lesions of MRI showed a high inverse correlation of -0.75 (95% CI: -0.84 ‒ -0.63, p = 0.001) with the matched SUVmax (Figure 3) of all 72 ROIs. The in- traclass correlation coefficient (ICC) for the evalua- tion of the mean ADC was 0.98 (95% CI: 0.97–0.99). The 72 ROIs were then subdivided into 22 neck, 28 thoracal and 22 abdominal lesions. At the neck lesions, pretherapeutic meanADC was 919.95 x 10‒3 mm2/s ± 243.77 x 10‒3 mm2/s (minimum: 462 x 10‒3 mm2/s, maximum: 1321 x 10‒3 mm2/s). Pretherapeutic mean SUVmax was 4.26 ± 0.93 (minimum: 2.85, maximum: 6.04). The meanADC lesions of neck MRI showed a high in- verse correlation of -0.83 (95% CI: -0.92 – -0.63, p < 0.001) with the matched SUVmax (Figure 4) of the 22 ROIs. The intraclass correlation coefficient for the evaluation of the neck mean ADC was 0.98 (95% CI: 0.95–0.99). At the thoracal lesions, pretherapeutic mean- ADC was 976.22 x 10‒3 mm2/s ± 355.34 x 10‒3 mm2/s (minimum: 373 x 10‒3 mm2/s, maximum: 1630 x 10‒3 mm2/s). Pretherapeutic mean SUVmax was 4.70 ± 1.30 (minimum: 2.82, maximum: 6.94). The meanADC lesions of thoracal MRI showed a high inverse correlation of -0.82 (95% CI: -0.91 – -0.64, p < 0.001) with the matched SUVmax (Figure 5) of the 28 ROIs. The intraclass correlation coefficient for the evaluation of the thoracal mean ADC was 0.99 (95% CI: 0.98–1.00). At the abdominal lesions, pretherapeutic mean- ADC was 931.68 x 10‒3 mm2/s ± 244.72 x 10‒3 mm2/s (minimum: 529 x 10‒3 mm2/s, maximum: 1658 x 10‒3 mm2/s). Pretherapeutic mean SUVmax was 4.66 ± 1.27 (minimum: 2.69, maximum: 7.44). The meanADC lesions of abdominal MRI showed an inverse correlation of -0.62 (95% CI: -0.83 – -0.28, p = 0.001) with the matched SUVmax (Figure 6) of the 22 ROIs. The intraclass correlation coefficient for the evaluation of the abdominal mean ADC was 0.97 (95% CI: 0.95–0.99). The correlations of neck and thoracal imag- ing differed not significantly (Z-Score = 0.10, p = 0.92). There is no significant difference of the cor- relations of neck and abdominal (Z-Score = 1.42, p = 0.15) and of thoracal and abdominal imaging (Z-Score = 1.42, p = 0.16). FIGURE 4. Neck imaging: correlation of SUVmax and mean apparent diffusion coefficient (ADC). The calculated meanADC of the MRI examinations show a strong inverse correlation with the correlating SUVmax of the FDG-PET/CT examinations. FIGURE 3. Correlation of SUVmax and mean apparent diffusion coefficient (ADC). The calculated meanADC of the MRI examinations show a strong inverse correlation with the correlating SUVmax of the FDG-PET/CT examinations. Radiol Oncol 2023; 57(2): 150-157. Rosbach N et al. / MRI as surrogate for PET/CT in children with Hodgkin lymphoma 155 Image ratings ADC maps were evaluated regarding image noise and image quality. Image noise was rated with mean scores of 4.6 ± 0.7. Image quality was rated with mean scores of 4.4 ± 0.9. The interrater agree- ment was good for image quality (κ = 0.7 ± 0.14) and image noise (κ = 0.64 ± 0.21) (p < 0.0001). Discussion Currently study protocols for HL patients contain PET/CT and MRI for initial staging, early assess- ment, and treatment response. Several studies demonstrated the important role of FDG-PET/CT scans as initial staging and during follow-up in HL patients.22-24 Children are radiation sensitive because of the high cell division rate. Radiation dose induced damages in children are closely ex- amined in several studies.25 The increasing num- ber of examinations with X-rays in patients leads to a lifelong increased risk of radiation induced cancer.26 Paediatric radiology societies point out the necessity of the ALARA (as low as reasonably achievable) principle in radiation exposure at chil- dren.27 On the other hand, assessment of the activ- ity by PET/CT might reduce radiation exposure, as patients with negative PET/CT assessed early point during therapy might not receive radiotherapy. MRI might be beneficial in paediatric patients as there is no radiation exposure. Whole-body MRI (WB-MRI) examinations can play an important role as initial staging and follow-up examination in HL patients.28,29 Spijkers et al. demonstrated a high correlation between WB-MRI with DWI and FDG-PET/CT scans in staging of adult HL pa- tients.30 The results of our feasibility study support that the results of Spijkers et al. also hold in pae- diatric patients, as ADC maps and FDG-PET/CT examinations showed a strong inverse correlation. Our preliminary results in pre-therapeutic imaging suggests that pretherapeutic MRI ADC maps and meanADC demonstrated a strong in- verse correlation with SUVmax of FDG-PET/CT neck and thoracal examinations in paediatric HL patients. However, data must be confirmed in the assessment of therapy response. At abdominal imaging the correlation between meanADC and SUVmax decreased with no sig- nificant difference to neck and thoracal imaging. The inter reader agreement at abdominal MRI meanADC was excellent. Noise and image qual- ity did not influence the evaluation of mean ADC. Pediatric MRI examinations were performed with- out breath-hold imaging. There may be an influ- FIGURE 6. Abdominal imaging: correlation of SUVmax and meanADC. The calculated meanADC of the MRI examinations show a fair inverse correlation with the correlating SUVmax of the FDG-PET/CT examinations. FIGURE 5. Thoracal imaging: correlation of SUVmax and mean apparent diffusion coefficient (ADC). The calculated meanADC of the MRI examinations show a strong inverse correlation with the correlating SUVmax of the FDG-PET/CT examinations. Radiol Oncol 2023; 57(2): 150-157. Rosbach N et al. / MRI as surrogate for PET/CT in children with Hodgkin lymphoma156 ence of breathing artifacts on the acquisition of abdominal DWI sequences. Further examinations in breath-hold imaging are necessary to exclude a potential breathing influence. With this study we shed light on the potential application of MRI instead of PET/CT to assess paediatric patients with HL to reduce radiation exposure. In this study only pretherapeutic FDG-PET/CT and MRI scans were selected to exclude a potential bias due to treatment. MRI scans with ADC maps may play an important role in follow-up examina- tions and assessment of treatment response of HL patients. To evaluate a post therapeutic correlation of meanADC and SUVmax further studies are nec- essary. The examinations of our study were performed with a single MRI scanner, and one single DWI sequence was used at all patients. This is impor- tant as Kivrak et al. and Hoang-Dinh et al. demon- strated a statistically significant difference in the calculated ADC maps of different MRI scanners from different vendors.31,32 The difference in calcu- lated ACD maps may be caused by different DWI sequence settings. Sadinski et al. demonstrated a high reproducibility of ADC maps at a single scanner and Newitt et al. demonstrated a high re- producibility of ADC maps at different scanners from different vendors with the same DWI set- tings.33,34 The evaluation of the robustness of the pretherapeutic correlation between meanADC and SUVmax in different scanners was beyond the scope of our analysis and requires further studies. This study has limitations beyond its retrospec- tive design. Missing MRI, missing pretherapeu- tic MRI and PET/CT examinations and missing temporal correlation between MRI and PET/CT reduced the number of eligible patients, which might has resulted in a selection bias. To exclude inter-scanner noise, we only included examina- tions from the same scanner. This homogenized the signals, but at the same time, limited the num- ber of eligible patients and might limit the general- izability of the results. References 1. The Lancet Haematology. New guidelines for paediatric Hodgkin lymphoma. Lancet Haematol 2020; 7: e851. doi: 10.1016/S2352-3026(20)30371-9 2. Nagpal P, Akl MR, Ayoub NM, Tomiyama T, Cousins T, Tai B, et al. Pediatric Hodgkin lymphoma: biomarkers, drugs, and clinical trials for transla-tional science and medicine. Oncotarget 2016; 7: 67551-73. doi: 10.18632/onco- target.11509 3. Ansell SM. Hodgkin lymphoma: diagnosis and treatment. Mayo Clin Proc 2015; 90: 1574-83. doi: 10.1016/j.mayocp.2015.07.005 4. Ehrhardt MJ, Flerlage JE, Armenian SH, Castellino SM, Hodgson DC, Hudson MM. Integration of pediatric Hodgkin lymphoma treatment and late effects guidelines: seeing the forest beyond the trees. J Natl Compr Canc Netw 2021; 19: 755-64. doi: 10.6004/jnccn.2021.7042 5. Hoppe RT, Advani RH, Ai WZ, Ambinder RF, Armand P, Bello CM, et al. Hodgkin lymphoma, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2020; 18: p. 755-81. doi: 10.6004/ jnccn.2020.0026 6. 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Ultraschall Med 2020; 41: 387-9. doi: 10.1055/a-1010-6018 28. Albano D, Bruno A, Patti C, Micci G, Midiri M, Tarella C, et al. Whole-body magnetic resonance imaging (WB-MRI) in lymphoma: state of the art. Hematol Oncol 2020; 38: 12-21. doi: 10.1002/hon.2676 29. Galia M, Albano D, Tarella C, Patti C, Sconfienza LM, Mulè A, et al. Whole- body magnetic resonance in indolent lymphomas under watchful waiting: the time is now. Eur Radiol 2018; 28: 1187-93. doi: 10.1007/s00330-017- 5071-x 30. Spijkers S, Littooij AS, Kwee TC, Tolboom N, Beishuizen A, Bruin MCA, et al. Whole-body MRI versus an FDG-PET/CT-based reference standard for stag- ing of paediatric Hodgkin lymphoma: a prospective multicentre study. Eur Radiol 2021; 31: 1494-504. doi: 10.1007/s00330-020-07182-0 31. Kıvrak AS, Paksoy Y, Erol C, Koplay M, Özbek S, Kara F. Comparison of ap- parent diffusion coefficient values among different MRI platforms: a multi- center phantom study. 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Abdom Imaging 2015; 40: 2523-8. doi: 10.1007/s00261-015-0396-x Radiol Oncol 2023; 57(2): 158-167. doi: 10.2478/raon-2023-0024 158 research article CT-guided biopsies of unspecified suspect intrahepatic lesions: pre-procedure Lipiodol- marking improves the biopsy success rate Marcel Christian Langenbach1,2, Thomas Joseph Vogl1, Amelie Buchinger1, Katrin Eichler1, Jan-Erik Scholtz1, Renate Hammerstingl1, Tatjana Gruber-Rouh1 1 Institute for Diagnostic and Interventional Radiology, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt, Germany 2 Cardiovascular Imaging Research Center, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Radiol Oncol 2023; 57(2): 158-167. Received 17 February 2023 Accepted 20 April 2023 Correspondence to: Prof. Tatjana Gruber-Rouh, Goethe University Frankfurt, University Hospital Frankfurt, Institute for Diagnostic and Interventional Radiology, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany, E mail: tatjana.gruber-rouh@kgu.de Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. While computed tomography (CT)-guided liver biopsies are commonly performed using unenhanced images, contrast-enhanced images are beneficial for challenging puncture pathways and lesion locations. This study aimed to evaluate the accuracy of CT-guided biopsies for intrahepatic lesions using unenhanced, intravenous (IV)- enhanced, or intra-arterial Lipiodol-marked CT for lesion marking. Patients and methods. Six-hundred-seven patients (men: 358 [59.0%], mean age 61 years; SD ±12.04) with suspect hepatic lesions and CT-guided liver biopsies were retrospectively evaluated. Successful biopsies were histopathologi- cal findings other than typical liver tissue or non-specific findings. Data was ascertained regarding the use of contrast medium for the biopsy-planning CT, unenhanced (group 1) vs. Lipiodol (group 2) vs. IV contrast (group 3). Technical success and influencing factors were insulated. Complications were noted. The results were analyzed using the Wilcoxon-Man-Whitney t-test, Chi-square test, and Spearman-Rho. Results. Overall lesion hitting rate was 73.1%, with significantly better rates using Lipiodol-marked lesions (79.3%) com- pared to group 1 (73.8%) and group 3 (65.2%) (p = 0.037). Smaller lesions (<20 mm diameter) benefited significantly from Lipiodol-marking with 71.2% successful biopsy rate compared to group 1 (65.5%) and group 3 (47.7%) (p = 0.021). Liver cirrhosis (p = 0.94) and entity of parenchymal lesions (p = 0.78) had no impact on the hitting rate between the groups. No major complications occurred during the interventions. Conclusions. Pre-biopsy Lipiodol marking of suspect hepatic lesions significantly increases the lesion-hitting rate and is especially beneficial for biopsy of smaller targets below 20 mm diameter. Further, Lipiodol marking is superior to IV contrast for non-visible lesions in unenhanced CT. Target lesion entity has no impact on the hitting rate. Key words: ethiodized oil; liver; biopsy; X-ray computed tomography Introduction Suspicious hepatic lesions are often detected in- cidentally or during staging and remain suspect despite the use of various diagnostic imaging methods. However, accurate diagnosis is crucial in the majority of suspect cases, especially in pa- tients with known or suspected malignant dis- ease.1,2 Thus, biopsies of suspicious intrahepatic lesions are an established procedure commonly performed in the clinical routine, with several op- tions for image guidance available. Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy 159 Therefore, various guiding procedures have been established for liver biopsies with good suc- cess rates providing reliable histopathological re- sults. Ultrasound-guided biopsy is the primary option for all hepatic lesions, widely available, and allows for a biopsy performed under continuous dynamic guidance.3,4 However, the applicability of ultrasound is limited by the location of the lesion (e.g., infra-diaphragmatic, deep in the liver) and in- terference with other structures, especially ribs or intestinal gas. In challenging cases with uncertain identification of the target lesions by ultrasound, magnetic resonance imaging (MRI)- or computed tomography (CT)-guidance may be beneficial for a safe and reliable biopsy.5-7 MRI guidance is technically feasible but un- common for biopsy hepatic lesions due to high cost, time-consuming, restricted availability, and motion artifacts. The clinical applicability is com- monly limited to other, more steady regions like the prostate or breast.8 In addition to ultrasound-guided biopsy, CT- guided biopsy is an integral part of routine clinical diagnostics. In most cases, the target liver lesions are visible in unenhanced CT images, commonly acquired for biopsy planning. Especially with the knowledge of a previous contrast-enhanced CT or MRI in which the lesions were identified and stat- ed as suspect.9 Nevertheless, some hepatic lesions are challenging to delaminate in an unenhanced CT, particularly in altered liver parenchyma like hepatic cirrhosis. Also, lesions with a deep paren- chymal position or with small size are challeng- ing to detect.10,11 Contrast-enhanced CT images are commonly used to overcome this limitation with two options available; application of intravenous (IV) contrast medium during the examination or a previous lesion marking using Lipiodol as a con- trast agent.12-14 Intravenous contrast is promptly washed out of the lesion and liver parenchyma and may not allow sufficient differentiation during the entire examination.15,16 In comparison, Lipiodol is interventionally administered intra-arterial dur- ing angiography and provides a long-lasting up- take (Lipiodol washout half-life 29 to 55 days17) in the lesion or perilesional altered tissue during the complete intervention.12,18 A novel approach uses fusion imaging, ultra- sound, or real-time fluoroscopy synchronized with previously acquired contrast-enhanced CT or MRI images to delineate the lesion during the intervention. This technique requires the newest equipment accompanied by high costs and is lim- ited to a few centers only.19 This study aimed to evaluate the interventional performance of CT-guided biopsies of suspicious intrahepatic lesions performed using unenhanced CT, intravenous contrast agent, or prior interven- tional lesion marking with Lipiodol for image guidance by analysis of the accuracy and potential influencing factors. Patients and methods Patient series and study setting The institutional review board (IRB) approved this retrospective cohort study with a waiver for written informed consent. Inclusion criteria were as follows: unclear and suspect hepatic lesion, CT- guided biopsy of the hepatic lesion, unenhanced, IV contrast or previous Lipiodol-angiography for lesion marking, age 18 and above. All patients who met the inclusion criteria (Figure 1) were in- cluded. We excluded all patients with a time in- terval between CT-guided biopsy and Lipiodol- angiography of more than two months (n = 18) or an abort of the CT intervention during the exami- nation due to lack of lesion visibility or sufficient biopsy window (n = 23). CT-guided biopsy Data were evaluated for the whole study popula- tion, and a comparison of the three groups, un- enhanced (group 1), Lipiodol enhanced (group 2), and IV contrast medium enhanced (group 3) was performed. Only patients who were not eligible for an ultrasound-guided biopsy or after an unsuc- cessful ultrasound-guided biopsy were referred to the radiology department for a CT-guided inter- vention. The interdisciplinary tumor board made the decision to biopsy. The enhancement protocol was selected at the time of the decision for biop- sy based on several parameters and experience. Patients with lesions expected to be displayed well in unenhanced CT, either by size, location, or based on previous imaging, were planned for an unenhanced biopsy. A contrast protocol was deter- mined in patients with lesions that might be more challenging to display in unenhanced CT. The decision for contrast protocol, Lipiodol (Example given in Figure 2) vs. IV contrast medium (e.g., Figure 3), was also based on the lesions’ location and parameters (e.g., size, architecture with visible changes in the liver parenchyma). Small lesions and lesions with a challenging location were more likely marked with Lipiodol due to a more long- Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy160 lasting uptake which enables a secure identifica- tion during the whole examination, while IV con- trast might have been washed out. In clinical prac- tice, more intravenous contrast applications were performed in the early years of the study period. In the last years, more patients were examined using a previous intra-arterial enhancement by Lipiodol based on good local experience. The study popula- tion and the three groups were subdivided by le- sion size and occurrence of liver cirrhosis. All patients received a transcutaneous CT- guided biopsy of the hepatic lesion using the same 128-multislice CT scanner [SOMATOM Definition AS, Siemens Healthineers] for image acquisition. The interventions were performed by interven- tional radiologists with more than five years of ex- perience in CT-guided interventions. The obtained core biopsies were histopathologically analyzed by the local Institute for Pathology. Successful biopsy was defined as histopathological findings other than typical liver tissue or non-specific findings. Based on our experience, Lipiodol does not affect the analysis of the specimen. Before the examination, the coagulation profile was checked. Patients who were planned for an ap- plication of IV contrast medium were screened for contraindications, e.g., reduced renal function or hyperthyroidism. The use of oral anticoagulants was prohibited based on national guidelines. No changes to institutional guidelines (standard op- erating procedures) were made. If patients were planned for IV contrast, 60 ml of contrast agent [Imeron® 350, Bracco] were administered with an application rate of 3-5 mL/s. We used a fixed delay of 60 s until the first planning scan was initiated. After the patients received the first plan- ning scan, the access path was planned using a standard 3D-CT workstation [syngo.via, Siemens Healthineers] without software modifications. In the case of multiple lesions with a similar sus- pect appearance, the lesion with the best reach- ability was defined as the target. Based on the biopsy planning, the cutaneous entry point was marked. After introducing the coaxial needle, the lesion approach was performed under im- age guidance. A coaxial approach was used for CT-guided intervention. This approach is char- acterized by combining two needles: a puncture sheath [17 G, Puncture Sheath, Somatex® Medical Technologies, Germany] and a biopsy handy [18 G, Biopsy Handy, Somatex® Medical Technologies, Germany]. The sheath - a thicker, shorter needle (5cm, 10 cm, or 15 cm) is inserted down to the ante- rior edge of the lesion. Then, the biopsy handy - a thinner, longer needle (10 cm, 15 cm, or 20 cm) is introduced through the sheath. Finally, the biopsy needle is inserted 2 cm into the edge of the lesion for tissue sampling. Three or four samples can be FIGURE 1. The Standards for the Reporting of Diagnostic Accuracy studies (STARD) flow diagram for a study of 607 patients undergoing CT-guided liver biopsy. Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy 161 taken using the thinner biopsy needle. After the examination, additional CT single shots were per- formed to rule out immediate complications like bleeding or pneumothorax. Afterward, the pa- tients had bed rest for 2 hours, a laboratory check of the blood parameters, and an ultrasound follow- up after 6 hours to rule out potential complications such as capsular hematoma or any other signs of bleeding. Angiography In patients with a prior Lipiodol-marking, the an- giography was performed at a minimum interval of 12 hours before the biopsy. The angiographic ap- proach was according to our standard procedure for hepatic interventions (Figure 2A).12 The same team of interventional radiologists with over five years of experience conducted all interventions. The angiographic approach was through the fem- oral artery in all cases. After the introduction of the catheter [Boston Scientific], contrast medium iomeprol [Imeron®, Bracco] was used to display the abdominal and hepatic vessels. Following this, an overview angiography was performed. The superior mesenteric artery and celiac trunk were selectively catheterized, including an indirect por- tography to demonstrate vascular anatomy, ensure the portal vein’s patency, and rule out portal vein thrombosis. Then, super-selective catheteriza- tion of the segmental and subsegmental hepatic branches was performed using a microcath- eter [Progreat®, Terumo] for approach and pre- cise placement. Lipiodol [Lipiodol Ultra-Fluid®, Guerbet] was administered as a tumor visualizer and transient embolic agent until stasis with a maximum amount of 15 ml. After the procedure, all patients were transferred to our ward for a min- imum of 24-hour surveillance. Data analysis Patient data were retrieved from the electronic pa- tient record (Orbis, Dedalus Healthcare), imaging data from the local radiology information system (RIS, GE Healthcare), and picture archiving and communication system (PACS, GE Healthcare). We included patients who matched the inclusion criteria and received a CT-guided biopsy between 01/2010 and 12/2018. Demographic data, underlying hepatic diseases, and liver cirrhosis were analyzed. Based on the CT images, lesion diameter, number of lesions, and lesion localization were noted. In patients with a prior Lipiodol angiography, the Lipiodol enhance- ment of the lesions was evaluated (visible or non- visible). During the CT-guided intervention, the puncture path (lateral-intercostal, anterolateral, anterior-subcostal, dorsolateral), length cutis-to- lesion, and liver capsule-to-lesion were measured. Dose-length-product (DLP) and the number of images of the entire examination, which includes the planning scan and the post-procedure scan, as well as for puncture sequence, including scans for needle placement, positioning, and sample acqui- sition, were noted and compared. This differentia- tion was made to achieve optimal comparability, as the differences in radiation exposure are caused FIGURE 2. The decision for a biopsy of the suspicious hepatic lesion was made for the same male patient. Because of the small lesion size and isodensity in the unenhanced CT, a prior intra-arterial Lipiodol-marking (A) was performed. Directly after the intervention (B) and during the CT intervention (C) (white arrow) was well displayable, and the biopsy was successful, revealing a hepatocellular carcinoma. A B C Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy162 by the required placement and positioning scans. For the angiography, the dose-area-product (DAB) was noted. The major endpoint of this study was the evaluation of the hitting rate, defined by histo- pathological findings other than healthy liver tis- sue. Successful biopsy findings were defined as fol- lows: hemangioma, focal nodular hyperplasia, re- generative nodule, granuloma, adenoma, necrosis, and all types of malignancies. Unsuccessful biop- sies were defined as healthy liver tissue, steatosis, inflammatory disease, fibrosis, and toxic damage. Statistics IBM SPSS statistics version 24 (IBM) was used for statistical analysis. For continuous data, median, mean, standard deviation, minimum, maximum, and range were calculated. For all continuous data, results were calculated for the whole study group and the three cohorts. The Chi-square test was used to test for differences in the successful biop- sies between the three cohorts. In the first step, the whole study population was analyzed. In the sec- ond step, different subgroups (e.g., lesions < 20 mm, liver cirrhosis) were compared in detail. Further, the Chi-square test was used to check for differences in the distribution of attributes within the groups. Correlations were calculated according to Spearman-Rho. P-values less than 0.05 was considered statistically significant. A lo- gistic regression analysis was performed to screen for potential factors within the demographic fac- tors influencing the lesion hitting rate. Complications Patient records were also screened for complica- tions, during the intervention or within the 24-hour surveillance. Complications were rated according to the severity score based on SIR’s latest classifica- tion: 0 = no complications, 1 = mild adverse events (no/non-substantial therapy required), 2 = moder- ate adverse event (substantial treatment required), 3 = severe adverse events (escalation of care), 4 = life-threatening or disabling event, 5 = patient death. Complication score was evaluated for the whole study population and the subgroups.20 Results Study population Six hundred-seven patients, 358 men (59%) and 249 women (41%) (mean age 61 years; SD ± 12.04; range 22–86 years) with unclear suspect liver le- sions, were retrospectively evaluated. In 321 pa- tients (52.9%), the biopsy was obtained with un- enhanced imaging, 145 lesions were prior marked with Lipiodol (23.9%), and 141 patients received IV contrast for the intervention (23.2%) (Figure 1). TABLE 1. Overview of patient characteristics. P-values based on the Chi-square test indicate only a difference in the distribution of liver cirrhosis in comparison of the three enhancement groups Characteristic Overall Unenhanced biopsy Lipiodol-enhanced i.v. contrast-enhanced p-value Age, y (SD) 61.0 ± 12.0 61.9 ± 11.95 60.4 ± 11.12 59.6 ± 13.04 0.228 Gender, n (%) Male Female 358 (59.0) 249 (41.0) 177 (55.1) 144 (44.9) 108 (74.5) 37 (25.5) 73 (51.8) 68 (48.2) 0.134 Hepatic disease, n (%) HCV HBV HCV+HBV NASH Toxic damage Others No disease 101 (16.6) 28 (4.6) 8 (1.3) 34 (5.6) 48 (7.9) 53 (8.7) 335 (55.2) 34 (10.6) 6 (1.9) 3 (0.9) 12 (3.7) 20 (6.2) 26 (8.1) 220 (68.5) 53 (36.6) 17 (11.7) 3 (2.1) 17 (11.7) 16 (11.0) 19 (13.1) 20 (13.8) 14 (9.9) 5 (3.5) 2 (1.4) 5 (3.5) 12 (8.5) 8 (5.7) 95 (67.4) 0.354 Cirrhosis 188 (33.0) 59 (19.2) 100 (71.9) 29 (3.6) 0.013 Intrahepatic lesions, n (%) 1 lesion 2 lesions 3 or more lesions 297 (48.9) 116 (19.1) 194 (32.0) 164 (51.1) 45 (14.0) 112 (34.9) 68 (46.9) 37 (25.5) 40 (27.6) 65 (46.1) 34 (24.1) 42 (29.8) 0.541 Lesion size, mm (SD) 29.3 ± 18.0 30.1 ± 18.1 25.9 ± 17.4 29.8 ± 18.7 0.182 Results are presented as mean and standard deviation or number (%) HBV = Hepatitis-B-Virus; HCV = Hepatitis-C-Virus; i.v. – intravenous; NASH – Non-alcoholic steato hepatitis; SD = Standard deviation Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy 163 Liver cirrhosis was histopathologically diagnosed in 188 patients (33.0%). Patient characteristics are displayed in detail in Table 1. Most patients had a known hepatic disease (n = 310, 51.1%). The most common disease was hepati- tis C in 101 cases (16.6%). Other more frequent pre- existing hepatic diseases were toxic liver damage (n = 48, 7.9%), non-alcoholic steatohepatitis (NASH) (n = 34, 5.6%), and hepatitis B infection (n = 28, 4.6%). No pre-existing hepatic disease was report- ed in 297 patients (48.9%). Lesion characteristics Based on patient medical records and the planning CT scan for the biopsy, 297 patients (48.9%) had a solitary suspect hepatic lesion, while 310 (51.1%) had two or more lesions. The target lesions were localized in all liver segments, with the majority found in liver segments 4 and 6 (each n = 112, 18.5%), and no differences according to the Chi-square test were seen between the three groups (p = 0.052). The mean lesion size was 29 ± 18 mm (range 4–116 mm). Target lesion smaller than 20 mm was seen in 229 patients (31.5%), and lesion smaller than 10 mm was seen in 38 patients (6.3%) (Table 1). Biopsy Access path was in 335 cases (55.2%) from lateral- intercostal, in 224 patients (36.9%) from anterior- subcostal, in 44 cases (7.2%) from anterior-lateral subcostal and in 4 cases (0.7%) from dorsal-lateral intercostal. The mean access path length from cu- tis to the lesion was 83 mm (± 26 mm), and within the liver from capsule to lesion, 43 mm (± 26 mm). Per patient, 2.6 probes were obtained in mean (± 1.3), equal across all groups assessed by the Chi- square test (p = 0.307) (Table 2). Regarding the visual aspect, all lesions previously marked in the angiography were seen in the biopsy-CT, either by lesion enhancement or circular enhancement of the surrounding tissue. Lesion hitting rate The target lesion was successfully biopsied in 444 patients (73.1%). In 163 patients (26.9%), the biopsy probe revealed healthy liver tissue defined as non- hitting of the target lesion. For group 1 with unen- hanced CT, the biopsy was successful in 73.8% (n = 232). Group 2, with a prior Lipiodol-marking of the target lesion, showed the significantly highest hit- ting rate with 79.3% (n = 115) (Figure 2). The lowest hitting rate was seen in group 3 with IV contrast medium with 65.2% (n = 92) (Figure 3). The differ- ence between all three groups was stated as signif- icant (p = 0.037). In patients with liver target lesions smaller than 20 mm, the successful hitting rate was significantly higher after Lipiodol-marking compared to unenhanced or intravenous contrast- enhanced images (71.2%, 65.5%, and 47.7%, respec- tively) (p = 0.021). The location within the liver based on the liver segment model had no impact on the lesion hitting rate (p = 0.107) (Figure 4). The presence of liver cirrhosis showed no sig- nificant impact on the overall success of liver bi- opsy in all three groups (p = 0.94). In the subset of patients without liver cirrhosis, a successful biopsy was highest in the Lipiodol group, 74.4%, compared to 71.5% in the unenhanced group and 58.5% using IV contrast (p = 0.049). FIGURE 3. A male patient with a suspect and unclear hepatic lesion with 6 mm in segment 7. While not visible in the unenhanced CT (A), the lesion impressed arterial hypervascularized after intravenous contrast medium application (B) (white arrow). FIGURE 4. Presentation of histopathologically correlated puncture successes for the overall cohort and individual groups individually. A B Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy164 Patients with a known malignant disease also showed a significant difference comparing the three procedures. A successful biopsy was pos- sible in 83.0% (n = 39) using Lipiodol-marking, in 68.2% (n=131) unenhanced, and in 58.3% (n = 35) with IV contrast medium (p = 0.024). The entity of the biopsied lesion did not impact the hitting rate by showing no significant difference between the protocols. Based on the performed logistic regression analysis, the lesion diameter (regression coeffi- cient 0.023, p = 0.048) and the number of obtained probes (r = 0.199, p = 0.047) significantly impacted the hitting rate. Age and length of the puncture path, from cutis or liver capsule to lesion, revealed no significant impact. Histopathological results Histopathological results revealed that the major malignant disease was HCC in 155 cases (25.5%). Inflammatory changes in the liver tissue were found to lead the non-malignant changes in 134 patients (22.1%). Appendix 1 shows the high vari- ability of the diagnosed diseases. Radiation exposure Overall DLP was 498.31 mGycm² in mean (± 429.53 mGycm²) for the complete examination, includ- ing control and post-procedure scan. In the unen- hanced group 1, 361.00 mGycm² (± 166.55 mGycm²), in the Lipiodol-marking group 2, 411.27 mGycm² (± 517.87 mGycm²), and in the IV contrast-medium group 3 900.27 mGycm² (± 495.55 mGycm²), reach- ing a significant difference with higher exposure in group 3 (p = 0.019). Only counting the applied radiation during the biopsy scans, which contain the scans for needle placement, positioning, and sample acquisition, overall DLP was 80.07 mGycm² in mean (± 57.97 mGycm²), in group 1 with 79.69 mGycm² in mean (± 59.98 mGycm²), in group 2 with 88.19 mGycm² in mean (± 56.33 mGycm²) and group 3 with 72.59 mGycm² in mean (± 56.37 mGycm²), showing no significant differences (p = 0.053). The total amount of the intervention (i)-sequences was n = 34.4 (± 24.0), in group 1 n = 34.6 (± 23.8), in group 2 n = 37.7 (± 23.4), and in group 3 n = 30.6 (± 24.7) equal across all groups (p = 0.182) (Table 2). TABLE 2. Biopsy characteristics overall and for the three enhancement groups. A Chi-square test was performed to test for differences between the groups. Significance is indicated in bold Characteristic Overall Unenhanced biopsy Lipiodol- enhanced i.v. contrast- enhanced p-value Biopsy access path Lateral-intercostal Anterior-subcostal Anterior-lateral Dorsal-lateral 335 (55.2) 224 (36.9) 44 (7.2) 4 (0.7) 162 (50.5) 25 (7.8) 132 (41.1) 2 (0.6) 94 (64.8) 9 (6.2) 42 (29.0) 0 79 (56.0) 10 (7.1) 50 (35.5) 2 (1.4) 0.718 Segment localization 1 2 3 4 5 6 7 8 6 (1.0) 54 (8.9) 41 (6.8) 112 (18.5) 95 (15.7) 112 (18.5) 82 (13.5) 105 (17.3) 2 (0.6) 29 (9.0) 23 (7.2) 66 (20.6) 57 (17.8) 69 (21.5) 31 (9.7) 44 (13.7) 2 (1.4) 14 (9.7) 8 (5.5) 23 (15.9) 21 (14.5) 21 (14.5) 22 (15.2) 34 (23.4) 2 (1.4) 11 (7.8) 10 (7.1) 23 (16.3) 17 (12.1) 22 (15.6) 29 (20.6) 27 (19.1) 0.052 Access path length [mm] (SD) Cutis to lesion Liver capsule to lesion 83 ±26 43 ±26 82 ±27 44 ±21 85 ±27 41 ±21 83 ±26 43 ±21 0.527 0.473 Obtained probes (SD) 2.6 ±1.3 2.7 ±1.5 2.5 ±0.9 2.4 ±0.9 0.158 Radiation exposure (SD) Whole examination [mGycm²] Biopsy scans [mGycm²] Intervention (i)-images [n] 498.3 ±429.5 80.1 ±58.0 34.4 ±24.0 361.0 ±166.6 79.7 ±60.0 34.6 ±23.8 411.3 ±517.9 88.2 ±56.3 37.7 ±23.4 900.3 ±495.6 72.6 ±56.4 30.6 ±24.7 0.019 0.053 0.182 Complications Sub-capsular liver bleeding Fluid edge around liver Bleeding along the access path Circulatory problems 3 (0.5) 2 (0.3) 1 (0.2) 1 (0.2) 1 (0.3) 1 (0.3) 0 1 (0.3) 1 (0.7) 0 1 (0.7) 0 1 (0.7) 1 (0.7) 0 0 0.870 Values are mean and standard deviation or number (%) i.v. = – intravenous; SD = Standard deviation Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy 165 For the previously performed angiography in group 2 to mark the lesion with Lipiodol, an over- all DAP of 10421 cGycm² in mean (± 10277 cGycm²) was noted. Complications In 599 patients, no treatment-related complication was stated in the patient’s medical record. Eight patients presented with a grade 1 complication: 3 patients with sub-capsular bleeding (0.5%), mini- mal hemorrhagic fluid edge surrounding the liver in 2 patients (0.3%), bleeding in the access path (1 patient, 0.2%), bleeding in the tumor (1 patient, 0.2%), and circulatory problems due to orthostatic problems (1 patient, 0.2%). No complications of grade 2 or higher were seen related to the interven- tion. No significant differences in the occurrence of complications were reported (p = 0.870). Details are given in Table 2. Discussion This study’s purpose was to compare three differ- ent enhancement protocols, unenhanced, with a prior Lipiodol marking and application of IV con- trast, for CT-guided biopsies of unspecified sus- pect intrahepatic lesions. CT-guided biopsies of suspect and unclear he- patic lesions are an established procedure in the diagnostic cascade with high reliability and a de- tection rate based on the study of Haage et al. of 90.5%.21 Several technical adaptions to increase the safety and success rate are possible, for ex- ample, variation of the access path or the needle thickness.22 The histopathological reliability of the obtained tissue is very good, as various stud- ies showed with an accuracy between 80% and 97%.23,24 A previous study by Gerhards et al. investigated the use of Lipiodol for biopsy of HCC-suspect or proven HCC lesions in a very small and selected patient cohort. The lesion hitting rates showed comparable results to our study, with a benefit of Lipiodol confirming our presented results. This study goes beyond the results of Gerhards et al. by investigating various lesion entities, different bi- opsy groups, with and without contrast enhance- ment, and a larger study cohort.25 The detectability of HCC lesions by the addi- tional use of Lipiodol compared to conventional contrast medium was proven by Rizvi et al. With prior Lipiodol-marking, the detection rate of HCC lesions was perfect (sensitivity 1.0, specificity 0.6, accuracy 0.91), while conventional contrast medi- um (sensitivity 0.65, specificity 1.0, accuracy 0.68) showed benefits in discriminating the lesion enti- ty.26 Setting this fact in context to our study, only a reliable lesion marking is essential for a successful CT-guided biopsy. This is the case in most lesions and is supported by our results with a favorable lesion hitting rate due to the usage of Lipiodol. A clear delineation and diagnosis of the lesion entity are optional, as it will be diagnosed based on the histopathological probe. Nevertheless, Lipiodol distribution within the suspected lesion can unveil HCCs, which may direct the patient directly into therapy, avoiding a biopsy.12,17 We also investigated the impact of existing liv- er cirrhosis on the biopsy results. Cirrhotic liver might have been a potential influencing factor making a successful biopsy more difficult as the lesion is camouflaged by reticular liver tissue. This was refuted by the results of our study showing no difference between the three study groups. Possible reasons for the results are the stronger tis- sue response during the intervention allowing for a bit easier needle control or the better visibility of the lesions in unenhanced imaging based on the altered tissue type, even in reticular liver tissue. Another important point is the impact of lesion size on technical success. Smaller lesions are much more difficult for a secure biopsy.27,28 With a previ- ous Lipiodol marking, especially lesions below 20 mm were easier to detect securely and to puncture successfully. Previous studies mainly investigat- ed only HCC lesions with this question, but our study showed a benefit across all entities. Further, small lesions are defined as very variable in the literature with a size below 20 to 50 mm making the existing data inconclusive and problematic to compare with our results. The sensitivity of Lipiodol-CT for this small lesion ranged between 58% and 98% based on previous studies.29-32 For ex- ample, our results should be considered in clinical practice for patients with a small unclear lesion in MRI imaging, which will be difficultly detectable in unenhanced CT. These patients benefit the most from a prior marking, with Lipiodol offering the best reliable biopsy method. Interestingly, the patient group with an intra- venous contrast medium application during the intervention showed the lowest lesion hitting rate. We identified two potential biases causing these results. First, these patients were primarily sched- uled for interventions without contrast medium. In the CT scan directly before the examination Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy166 for the biopsy planning, the lesion was not detect- able. Reasons for this might have been a small size or a challenging access path. Then the decision to administer IV contrast to visualize the lesion was reached. Second, the time of visibility is lim- ited, and especially small lesions are challenging to detect after wash-out of the contrast medium. Compared to the unenhanced group, the IV con- trast group might contain more technically com- plex patients. Patients presenting small lesions or a challenging access path in pre-interventional im- aging might benefit from a prior Lipiodol-marking of the target lesion. Especially lesions that may be isodense in unenhanced, enhanced CT and ob- scured in delayed phase CT should be marked be- fore the intervention. As mentioned, most studies investigate the role of Lipiodol in the context of HCC, and it is known that the best and strongest enhancement is seen for hypervascularized lesions. But based on previous studies on TACE, it is known that Lipiodol shows an enhancement also in a wide variability of other tumors, e.g., breast cancer or colorectal carcinoma, which are often hypo vascularized. Although not all lesions enhance strong and homogeneity, the wall area or the area of altered liver tissue around the lesions shows a typical enhancement that can be used for biopsy guidance. This underlines the potential for clinical use even if the entity of the lesion is not known before the intervention. Besides CT, CEUS (Contrast-enhanced ultra- sound) and cone beam CT (CBCT) with intra-ar- terial contrast application are useful other imag- ing modalities to assess suspicious intrahepatic lesions interventionally.33 These techniques also allow for the accurate detection and localiza- tion of hepatic lesions. However, both modalities may not be widely available in all medical facili- ties, limiting their accessibility for some patients. Additionally, CEUS requires highly skilled opera- tors with specific training and expertise. The qual- ity and accuracy of the results may depend on the operator’s skill and experience. CBCT fusion imag- ing with ultrasound for transcutaneous hepatic in- terventions is a new approach with promising first results, but it still requires further investigation.34 The overall complication rate was very low, with no major complications. This is according to the prevalence of complications reported in other studies21-23, stating that a CT-guided biopsy is an approved and secure intervention well established in the clinical practice. The contrast medium did not influence the complication rate, even in chal- lenging lesion locations. Besides all benefits of the biopsy, the applica- tion of Lipiodol is an interventional procedure with the potential occurrence of complications and increased radiation exposure. Although a tran- sarterial embolization is established in the clinical routine, the rate of major complications is low at under 0.9%.35 Additionally, the increased radia- tion exposure comes with the risk of stochastic ra- diation damage and radiation-related late effects. Consequently, the application should be reserved for patients who benefit the most. Based on the re- sults, prior Lipiodol-marking can be beneficial in patients with a lesion below 20 mm or a potentially challenging access path. We acknowledge that this retrospective study had several limitations. This study intended to compare different enhancement protocols for CT- guided biopsies of various intrahepatic lesions. Therefore, we used a retrospective design and a relatively small cohort in a few tumor entities. A study including a prospective design and multiple centers, probably with a closer look at selected le- sion entities, is required to prove the results. The histopathological results are problematic in a few cases as the pathologists described several pat- terns—further, the decision process for the en- hancement protocol needed to be randomized. The multidisciplinary tumor board made the decision based on several parameters. This was based on pre-interventional imaging, lesion characteristics, age, or the patient’s individual preference. This de- cision might have led to a selection bias. Also, IV enhancement was favored in the early years, while Lipiodol enhancement was performed based on convincing local experience in the later years. For lesions with the pathological finding of necrosis, this might also have been caused by a diffuse ne- crotic area, which might be considered as a non- successful biopsy. From the pathological results, no differentiation is possible. In conclusion, the use of Lipiodol as a contrast agent and pre-puncture marking in angiography increases the lesion-hitting rate in CT-guided bi- opsies of suspect and unclear hepatic lesions sig- nificantly, especially for small suspect liver lesions with a diameter below 20 mm and might be used as an alternative to unenhanced imaging or IV contrast enhancement. For lesions that cannot be seen in unenhanced CT, Lipiodol marking is supe- rior to IV marking and should be preferred. Radiol Oncol 2023; 57(2): 158-167. Langenbach MC et al. / Lipiodol-marking improves biopsy success in CT-guided liver biopsy 167 Acknowledgment The present study was founded receiving a re- search grant by Guerbet. The authors declare no relationships with any companies whose products or services may be related to the subject matter of the article. The funding sources were not involved in study design, data collection or analysis, manu- script preparation, or the decision to submit the manuscript. References 1. Bravo AA, Sheth SG, Chopra S. Liver biopsy. N Engl J Med 2001; 344: 495- 500. doi: 10.1056/NEJM200102153440706 2. Rockey DC, Caldwell SH, Goodman ZD, Nelson RC, Smith AD. Liver biopsy. Hepatology 2009; 49: 1017-44. doi: 10.1002/hep.22742 3. Gazelle GS, Haaga JR. Guided percutaneous biopsy of intraabdominal le- sions. AJR Am J Roentgenol 1989; 153: 929-35. doi: 10.2214/ajr.153.5.929 4. Otto R. Interventional ultrasound. Eur Radiol 2002; 12: 283-7. doi: 10.1007/ s00330-001-1272-3 5. 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Comput Aided Surg 2011; 16: 181-7. doi: 10.3109/10929088.2011.578367 11. Haaga JR, Reich NE, Havrilla TR, Alfidi RJ, Meaney TF. CT guided biopsy. Cleve Clin Q 1977; 44: 27-33. doi: 10.3949/ccjm.44.1.27 12. Langenbach MC, Vogl TJ, von den Driesch I, Kaltenbach B, Scholtz J-E, Hammerstingl RM, et al. Analysis of Lipiodol uptake in angiography and computed tomography for the diagnosis of malignant versus benign hepatocellular nodules in cirrhotic liver. Eur Radiol 2019; 29: 6539-49. doi: 10.1007/s00330-019-06297-3 13. Sainani NI, Schlett CL, Hahn PF, Gervais DA, Mueller PR, Arellano RS. Computed tomography-guided percutaneous biopsy of isoattenuating focal liver lesions. Abdom Imaging 2014; 39: 633-44. doi: 10.1007/s00261- 014-0089-x 14. Willatt JM, Francis IR, Novelli PM, Vellody R, Pandya A, Krishnamurthy VN. Interventional therapies for hepatocellular carcinoma. Cancer Imaging 2012; 12: 79-88. doi: 10.1102/1470-7330.2012.0011 15. Sun JH, Zhou GH, Zhang YL, Nie CH, Zhou TY, Ai J, et al. Chemoembolization of liver cancer with drug-loading microsphere 50-100μm. Oncotarget 2017; 8: 5392-9. doi: 10.1097/RCT.0000000000000659 16. Martino CR, Haaga JR, Bryan PJ, LiPuma JP, El Yousef SJ, Alfidi RJ. CT-guided liver biopsies: eight years’ experience. Work in progress. Radiology 1984; 152: 755-7. doi: 10.1148/radiology.152.3.6463257 17. Nezami N, VAN Breugel JMM, Konstantinidis M, Chapiro J, Savic LJ, Miszczuk MA, et al. Lipiodol deposition and washout in primary and metastatic liver tumors after chemoembolization. In Vivo 2021; 35: 3261-70. doi: 10.21873/ invivo.12621 18. Chan MKH, Lee V, Chiang CL, Lee FAS, Law G, Sin NY, et al. Lipiodol versus diaphragm in 4D-CBCT-guided stereotactic radiotherapy of hepatocellular carcinomas. Strahlenther Onkol 2016; 192: 92-101. doi: 10.1007/s00066- 015-0929-9 19. Ahn SJ, Lee JM, Chang W, Lee SM, Kang H-J, Yang H-K, et al. Clinical utility of real-time ultrasound-multimodality fusion guidance for percutaneous biopsy of focal liver lesions. Eur J Radiol 2018; 103: 76-83. doi: 10.1016/j. ejrad.2018.04.002 20. 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 Interventional Radiology Standards of Practice Committee. J Vasc Interv Radiol 2017; 28: 1432-7.e3. doi: 10.1016/j.jvir.2017.06.019 21. Haage P, Piroth W, Staatz G, Adam G, Günther RW. [CT-guided percutaneous biopsies for the classification of focal liver lesions: a comparison between 14 G and 18 G puncture biopsy needles]. [German]. Rofo 1999; 171: 44-8. doi: 10.1055/s-1999-9895 22. Pagani JJ. Biopsy of focal hepatic lesions. Comparison of 18 and 22 gauge needles. Radiology 1983; 147: 673-5. doi: 10.1148/radiology.147.3.6844603 23. Lüning M, Schmeisser B, Wolff H, Schöpke W, Hoppe E, Meyer R. [Analysis of the results of 96 CT-guided fine needle biopsies of liver masses]. [German]. Rofo 1984; 141: 267-75. doi: 10.1055/s-2008-1053132 24. Knöpfle E, Bohndorf K, Wagner T. [Does the core biopsy of solid liver lesions permit an exact histological classification? Results of a prospective study under routine clinical conditions]. [German]. Rofo 1997; 167: 406-11. doi: 10.1055/s-2007-1015552 25. Gerhards A, Mildenberger P, Herber S, Lehr HA, Thelen M. [Prospective ben- efit and effect of lipiodol marking in hepatocellular carcinoma]. [German]. Rofo 2005; 177: 1380-6. doi: 10.1055/s-2005-858565 26. Rizvi S, Camci C, Yong Y, Parker G, Shrago S, Stokes K, et al. Is post-Lipiodol CT better than i.v. contrast CT scan for early detection of HCC? A single liver transplant center experience. Transplant Proc 2006; 38: 2993-5. doi: 10.1016/j.transproceed.2006.08.125 27. Frazer C. Imaging of hepatocellular carcinoma. J Gastroenterol Hepatol 1999; 14: 750-6. doi: 10.1046/j.1440-1746.1999.01946.x 28. Schaudt A, Kriener S, Schwarz W, Wullstein C, Zangos S, Vogl T, et al. Role of transarterial chemoembolization for hepatocellular carcinoma before liver transplantation with special consideration of tumor necrosis. Clin Transplant 2009; 23(Suppl 21): 61-7. doi: 10.1111/j.1399-0012.2009.01111.x 29. Choi BI, Park JH, Kim BH, Kim SH, Han MC, Kim CW. Small hepatocellular carcinoma: detection with sonography, computed tomography (CT), angi- ography and Lipiodol-CT. Br J Radiol 1989; 62: 897-903. doi: 10.1259/0007- 1285-62-742-897 30. Taourel PG, Pageaux GP, Coste V, Fabre JM, Pradel JA, Ramos J, et al. Small hepatocellular carcinoma in patients undergoing liver transplantation: de- tection with CT after injection of iodized oil. Radiology 1995; 197: 377-80. doi: 10.1148/radiology.197.2.7480680 31. Takayasu K, Moriyama N, Muramatsu Y, Makuuchi M, Hasegawa H, Okazaki N, et al. The diagnosis of small hepatocellular carcinomas: efficacy of various imaging procedures in 100 patients. AJR Am J Roentgenol 1990; 155: 49-54. doi: 10.2214/ajr.155.1.1693808 32. Spreafico C, Marchianò A, Mazzaferro V, Frigerio LF, Regalia E, Lanocita R, et al. Hepatocellular carcinoma in patients who undergo liver transplantation: sensitivity of CT with iodized oil. Radiology 1997; 203: 457-60. doi: 10.1148/ radiology.203.2.9114104 33. Wilsen CB, Patel MK, Douek ML, Masamed R, Dittmar KM, Lu DSK, et al. Contrast-enhanced ultrasound for abdominal image-guided procedures. Abdom Radiol (NY) 2023; 48: 1438-53. doi: 10.1007/s00261-023-03804-5 34. Monfardini L, Orsi F, Caserta R, Sallemi C, Della Vigna P, Bonomo G, et al. Ultrasound and cone beam CT fusion for liver ablation: technical note. Int J Hyperthermia 2018; 35: 500-4. doi: 10.1080/02656736.2018.1509237 35. Tu J, Jia Z, Ying X, Zhang D, Li S, Tian F, et al. The incidence and outcome of major complication following conventional TAE/TACE for hepatocellular car- cinoma. Medicine 2016; 95: e5606. doi: 10.1097/MD.0000000000005606 Radiol Oncol 2023; 57(2): 168-177. doi: 10.2478/raon-2023-0025 168 research article Radiological assessment of skeletal muscle index and myosteatosis and their impact postoperative outcomes after liver transplantation Miha Petric1,2, Taja Jordan3, Popuri Karteek4, Sabina Licen5, Blaz Trotovsek1,2, Ales Tomazic1,2 1 Department of Abdominal Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 4 Department of Computer Science, Memorial University of Newfoundland, St. John’s, NL, Canada 5 Faculty of Health Sciences, University of Primorska, Izola, Slovenia Radiol Oncol 2023; 57(2): 168-177. Received 12 March 2023 Accepted 16 May 2023 Correspondence to: Miha Petrič; Department of Abdominal Surgery, University Medical Centre Ljubljana, Zaloška cesta 7, SI-1000 Ljubljana, Slovenia. E mail: miha.petric@kclj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Liver transplantation offers curative treatment to patients with acute and chronic end-stage liver disease. The impact of nutritional status on postoperative outcomes after liver transplantation remains poorly under- stood. The present study investigated the predictive value of radiologically assessed skeletal muscle index (SMI) and myosteatosis (MI) on postoperative outcomes. Patients and methods. Data of 138 adult patients who underwent their first orthotopic liver transplantation were retrospectively analysed. SMI and MI in computer tomography (CT) scan at the third lumbar vertebra level were cal- culated. Results were analyzed for the length of hospitalisation and postoperative outcomes. Results. In 63% of male and 28.9% of female recipients, low SMI was found. High MI was found in 45(32.6%) patients. Male patients with high SMI had longer intensive care unit (ICU) stay (P < 0.025). Low SMI had no influence on ICU stay in female patients (P = 0.544), length of hospitalisation (male, P > 0.05; female, P = 0.843), postoperative complication rates (males, P = 0.883; females, P = 0.113), infection rate (males, P = 0.293, females, P = 0.285) and graft rejection (males, P = 0.875; females, P = 0.135). The presence of MI did not influence ICU stay (P = 0.161), hospitalization (P = 0.771), postoperative complication rates (P = 0.467), infection rate (P = 0.173) or graft rejection rate (P = 0.173). Conclusions. In our study, changes in body composition of liver transplant recipients observed with SMI and MI had no impact on postoperative course after liver transplantation. CT body composition analysis of recipients and uni- formly accepted cut-off points are crucial to producing reliable data in the future. Key words: muscle mass; liver transplantation; myosteatosis; skeletal muscle index; GLIM score Introduction Since 1963 when the first liver transplantation (LT) was performed by Starzl1, it has become a standard treatment modality for patients with acute liver failure and chronic liver disease.2 Ninety percent 5-year survival rate and better quality of life are the two most important outcomes of LT.2 Most liv- er transplant centres use the Model for End-stage Liver Disease (MELD) score for organ allocation.3 Patients with MELD score 15 or more, patients with poor quality of life due to chronic liver disease Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis 169 symptoms (diuretic-intractable ascites, variceal bleeding, pruritus, cachexia), and patients with acute liver failure are those who benefit most from LT.3 MELD score can underestimate the severity of liver disease in specific groups of patients (acute on chronic liver disease, presence of sarcopenia, chronic kidney disease, etc.).3 Several modifica- tions of the MELD score have been introduced, but none offers a more reliable and accurate scoring system. Albumin-bilirubin (ALBI) score is mainly used as an objective method to assess liver func- tion and predict postoperative complications, par- ticularly after hepatectomy in patients with hepa- tocellular carcinoma (HCC).4 Its role in determin- ing post-LT outcomes is not yet determined. Two main objective parameters of nutritional status are sarcopenia and myosteatosis (MI). The European Working Group on Sarcopenia defines sarcopenia as the presence of low muscle mass (under the 5th percentile) and low muscle function (strength or performance) in patients with advanced age, can- cer, or other diseases.5,6 Myosteatosis is defined as the abnormal fatty transformation of skeletal muscle. It negatively affects muscle strength and is common in advanced age7,8, diabetes7,8, obesity9,10, chronic9, and malignant diseases.11,12 Overview of the literature shows large number of different methods used for body composition assessment in patients with liver cirrhosis.13 There is still no con- sensus on the best tools for each body component in patients with liver cirrhosis. Most frequently used are computed tomography (CT), bioimped- ance analysis (BIA), dual-energy X-ray absorp- tiometry (DXA) and anthropometry.13 Some of them BIA, DXA and body mass index (BMI)) are not applicable in patients with end-stage liver dis- ease due to frequent water retention.14,15 In the last decade, computed tomography with automated or semiautomated body composition analysis at the third lumbar vertebra has emerged as an objec- tive method of defining the nutritional status of patients with chronic liver disease.15,16 Nutritional assessment with CT is not affected by water reten- tion or presence of ascites. Skeletal muscle volume and myosteatosis can be measured from CT images obtained as a part of routine pre-transplant evalu- ation.14 Skeletal muscle mass index (SMI) is calcu- lated as muscle mass area divided by the square of the height. The Global Leadership Initiative on Malnutrition (GLIM)16 score was introduced as a potential nutritional assessment tool in recent years. It was shown to have good predictive value as a risk assessment tool for postoperative morbid- ity and mortality in patients after colorectal sur- gery.17 However, its role as a predictive factor in LT is not yet established. This study aimed to investigate feasibility of radiological assessment of a nutritional status of a patient and the predictive value of SMI and MI on postoperative complications, length of hospi- talization, liver graft rejection, and mortality. We further compared the predictive value of SMI and MI with MELD, ALBI, and GLIM scores. Patients and methods We retrospectively analyzed 138 adult patients who had first orthotropic LT from brain dead donors be- tween 1.1.2012 and 1.1.2020 in our institution. We excluded patients who had re-LT procedure and those whose abdominal CT scan could not be ob- tained from data base or received reduced size and liver graft from donation after cardiac death (DCD) donor. From the medical database, we collected re- cipient age, gender, body mass index, underlying liver disease, presence of ascites, hepatocellular carcinoma (HCC), and laboratory parameters (se- rum levels of sodium, creatinine, albumin, pro- tein, bilirubin, and International normalized ratio (INR)). We calculated MELD and ALBI scores from laboratory parameters. Among GLIM criteria, we used chronic liver failure and end-stage liver dis- ease as etiologic and SMI or MI as phenotypic cri- teria. Length of intensive care unit (ICU) stay, hos- pitalization, postoperative complications accord- ing to Clavien-Dindo classification18, infections, and 90-day mortality were collected from the da- tabase of liver recipients and analyzed. Diagnosis of liver rejections was confirmed with laboratory tests and histological examination of all liver graft specimens obtained by ultrasound-guided biopsy. Acute rejection was defined with 6 points or more according to the liver allograft fibrosis score.19 The Slovenian National Medical Ethics Committee approved our study design (approval number 0120–230/2018–10) and waived the need to obtain informed consent from participants. CT-body composition analysis Abdominal CT scans were obtained from the hos- pital database system. In case of multiple CT scans, we used last CT scan before LT procedure. A single slice of each patient at the level of the 3rd lumbar vertebrae was selected for automatic segmentation. CT scans were analysed using the “Automated Body Composition Analyzer using Computed to- Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis170 mography image Segmentation” (ABACS)20 soft- ware, which uses predefined Hounsfield units (HU) values to recognize different tissues. ABACS uses HU values from −29 to +150 HU to assess and calculate the total cross-sectional area for mus- cular tissue (SMA – skeletal muscle area). The L3 skeletal muscles included the psoas muscle, the lumbar muscles, the erector spinae, the transver- sus abdominis muscle, the internal and external oblique muscles, and the rectus abdominis. SMI was calculated using the following formula: SMI = SMA (cm2) / height2 (m2)9,21 and patients were di- vided into a group with lower SMI (men < 52.4 cm2/ m2, women < 38 cm2/m2)9,21 and another with nor- mal SMI. MI was determined by the medium value of HU in a skeletal muscle area. We used recently defined threshold parameters for MI in a patient with a chronic liver disease (< 33 HU in patients with a BMI ≥ 25 kg/m2 and < 41 HU in those with a BMI < 25).22-24 Based on previous literature findings and conclusions no adjustment for sex was made for MI.23,24 Statistical analysis Data were analysed using SPSS for macOS, 26th edi- tion. Descriptive statistics such as frequencies, per- centages, mean/median, and standard deviations were used for description and summary. Because the data were not normally distributed, patient characteristics were compared between groups us- ing the Kruskal-Wallis test and the Mann-Whitney U test. In addition, Spearman rank-order correla- tion and multiple linear regression were used to determine the relationship between variables and predict patient outcomes based on their character- istics and condition, as well as the chi-square in- dependence test. A P-value ≤ 0.05 was considered statistically significant. Results Patients characteristics Between 1.1.2012 and 1.1.2020, 138 patients (100 men and 38 women) met the criteria for inclusion in the study. The median age was 57.5 years (22 to 69 years). Table 1 provides general data on the pop- ulation laboratory and clinical variables. Sixty-one (61, 44.2%) patients had LT due to in- herited or metabolic liver disease, 60 patients had alcoholic liver disease (43.5%), and 17 patients had virus-related liver disease (12.3%). At the time of LT, 80 (58%) patients had ascites, and 27 patients had HCC (19.4%). Incidence of low SMI and MI In our study group, 63% of male and 28.9% of fe- male patients had low SMI. MI was present in 45 (32.6%) patients. We found no statistical signifi- cance between the aetiology of underlying liver disease and SMI (P = 0.214). The aetiology of liver disease had a statistically significant influence on the incidence of MI. Patients with alcoholic ae- tiology had more fatty infiltrated muscles than patients with other liver disease aetiologies (P = 0.008) (Table 2). A Spearman’s rank-order correlation was run to determine the relationship between the MI and BMI. There was a moderate, negative correlation between MI and BMI, which was statistically sig- nificant (rs = -0.597, p < 0.000). TABLE 1. Laboratory and clinical data of patients Variable Min Max IQR SD 95% CI Lower Upper BMI 15 38 6 4.775 25.13 26.74 Waiting time for liver transplantation (days) 1 691 166 151.311 108.44 159.38 Sodium 121 147 6 4.701 136.09 137.68 Creatinine 41 696 46 67.082 85.91 108.49 Albumin 10 62 10 7.674 31.28 33.86 Protein 23 89 12 11.494 65.80 69.74 Bilirubin 2 687 56 107.535 51.83 88.04 INR 1 4 1 .502 1.44 1.61 MELD score 7 46 9 6.700 14.30 16.56 ALBI score -4 0 1.13 0.802 -1.87 -1.60 ALBI score = albumin-bilirubin score; BMI = body mass index; INR = international normalized ratio; IQR = Interquartile range; MELD score = model for end-stage liver disease score TABLE 2. Aetiology of liver disease and incidence of myosteatosis Variable Myosteatosis no yes Liver failure Alcohol-related 32 28 Virus-related 14 3 Other 47 14 χ2 9.717 Degrees of freedom (Df) 2 p 0.008 Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis 171 Eighty patients (57.9%) had decompensated liver cirrhosis with the presence of ascites at the time of CT scan. The Mann-Whitney U test indicates that ascites significantly impacted patients’ SMI. The presence of ascites correlated with low SMI (P < 0.05, Figure 1) and had no impact on the incidence of MI (P = 0.244). Presence of HCC in patients with liver disease (27 patients, 19.6%) had no influence on SMI (P = 0.546) or MI (P = 0.174). Influence of low SMI on hospitalization The Mann-Whitney U test indicates that male pa- tients with normal or high SMI (> 52.4 cm2/m2) had a longer ICU length of stay (P < 0.025). Linear re- gression was calculated to predict ICU length of stay based on SMI. A significant regression equa- tion was found (F (1.96) = 6.823, P = 0.010) (Figure 2), with R2 of 0.066. SMI was found to significantly predict length of ICU stay (β = 0.258; 95% CI 0.026, 0.189; P = 0.010). The predicted length of stay equals -1.117+0.107 (SMI) days when SMI is measured in cm2/m2. The ICU length of stay increased by 0.107 days for each cm2/m2 of SMI in the male population. A Spearman’s rank-order correlation showed no difference in hospitalization time between males with low or normal SMI. Mann-Whitney U test in- dicates statistically significant differences between low or normal SMI (P < 0.05) in more extended hos- pitalization in male patients with normal SMI. In addition, the linear regression analysis was per- formed to predict the length of hospital stay based on SMI in male patients; however, no statistically significant regression equation was found (β = -0.031; 95% CI -0.656, 0.480; P > 0.05). In the female population, we found no statistically significant influence of SMI on ICU length of stay (β = -0.123; 95% CI -0.741, 0.463; P = 0.544) and hospitalization time (β = 0.013; 95% CI 0.075, 0.941; P = 0.843). Influence of MI on hospitalisation The Mann-Whitney U test indicates that there were no differences among the groups (MI and no- MI) regarding the ICU length of stay (P = 0.161) or hospitalization (P = 0.771). Influence of SMI and MI on postoperative complications Postoperative complications of stage 2 or more by Clavien-Dindo classification19 were present in 79 patients (57.2%). Infection occurred in 59 patients (42.8%), most commonly as intra-abdominal in- fection (50.8%), respiratory tract infection (23.7%) and urosepsis (6.8%). 5 (3.6%) patients developed critical illness myopathy. Surgical intervention was needed in 38% of patients with postoperative complications. There was no statistically significant differ- ence in the frequency of postoperative compli- cations between males (P = 0.883) and females (P = 0.113) with low or normal SMI. The postopera- tive infection rate was similar in males (P = 0.293) and females (P = 0.285) with low or normal SMI. MI did not show significant influence on postop- erative complications (P = 0.839) and infection rate (P = 0.703). FIGURE 1. Correlation between ascites and skeletal muscle index. FIGURE 2. Scatter diagram showing a positive correlation between the intensive care unit length of stay (days) and skeletal muscle index. Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis172 Influence of SMI and MI on liver graft rejection Rejection was diagnosed in 22 patients (16.1%), 20 in male and 2 in female patients. We found no sta- tistically significant influence of SMI (males, P = 0.875; females, P = 0.135) and MI (P = 0.449) on liver graft rejection. Influence of MELD and ALBI score on postoperative outcomes The median MELD score was 14 (7 – 46). A Spearman’s rank-order correlation was run to de- termine the relationship between the MELD score and ICU length of stay (days). There was a weak, positive correlation between the MELD score and ICU length of stay, which was statistically signifi- cant (rs = 0.261, P < 0.002). There was no significant relationship between the MELD score and compli- cation rate or length of hospitalization (P > 0.05). There is a week, positive correlation between the ALBI score and ICU length of stay and hospi- talisation time, which was statistically significant (rs = 0.279, P < 0.001; rs = 0.197; P = 0.022). There is no correlation between the ALBI score and postopera- tive complications. GLIM score GLIM score using skeletal muscle index as a phenotypic factor We analyzed differences between patients who meets criteria for positive GLIM score (acute or chronic liver disease as etiological factor and skel- etal muscle index as a phenotypic factor) and those with negative GLIM score regarding ICU length of stay, postoperative complications, rate of infec- tions, graft rejections and mortality. Based on the chi-square independence test, no association was found between the groups in post- operative complications (χ2(1) = 0.600; P = 0.438), rate of infections (χ2(1) = 0.918; P = 0.338), graft re- jection (χ2(1) = 1.205; P = 0.272), and mortality (χ2(1) = 0.232; P = 0.630). In addition, there were no statis- tically significant differences in ICU length of stay between groups (χ2(14) = 15.125; P = 0.370), based on the Mann-Whitney test. GLIM score using myosteatosis as a phenotypic factor We analyzed differences between patient who meets criteria for positive GLIM score (acute or chronic liver disease as etiological factor and my- osteatosis as a phenotypic factor) and those with negative GLIM score regarding ICU length of stay, postoperative complications, rate of infections, graft rejections and mortality. Based on the chi-square independence test, there was no association between the groups in terms of postoperative complications (χ2(1) = 1.378; P = 0.242), infection rate (χ2(1) = 2.921; P = 0.089), graft rejection (χ2(1) = 0.873; P = 0.352), and mor- tality (χ2(1) = 0.010; P = 0.922). In addition, there were no statistically significant differences in ICU length of stay between groups (χ2(14) = 16.271; P = 0.297), based on the Mann-Whitney test. Multiple linear regression analysis Multiple linear regression was calculated to pre- dict ICU length of stay on SMI and infection. A sig- nificant regression equation was found (F (2. 95) = 11.192, P < 0.000), with R2 of 0.191. SMI was found to significantly predict length of ICU stay (β = 0.292; 95% CI 0.045, 0.199; P = 0.002). The length of ICU stays increased by 0.122 days per cm2/m2 SMI in male patients with infection. Mortality In our study group, 5 (3.6%) patients died in the first 90 days after liver transplantation. Two pa- tients died immediately after the procedure due to irreversible haemorrhagic shock with dissemi- nated intravascular thrombosis and abdominal organ ischemia. The cause of death in three pa- tients was sepsis with multiorgan failure in one patient (day 66), and severe postoperative bleed- ing, intraabdominal infection with liver abscess, and multiorgan failure in two patients (34 and 53 days). One year survival rate was 95%. Due to the small number of patients, statistical analysis was not performed. Discussion Malnutrition with skeletal muscle mass loss is a frequent complication in patients with chronic or end-stage liver disease.25 The aetiology of malnu- trition is multifactorial.23 Insufficient calorie intake (early satiety, loss of appetite, alcohol consump- tion, diet restriction), metabolic abnormalities, cat- abolic state of metabolism, and malabsorption are the main contributors to muscle loss.25,26 It affects from 30 to 70%27,28 of patients with end-stage liver disease and up to 80%27 of patients with alcohol-re- Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis 173 lated liver disease. Myosteatosis affects more than half of patients with chronic liver disease.9 In our patient population, we used selected parameters for low SMI (men < 52.4 cm2/m2, women < 38 cm2/ m2)9,21 and MI (< 33 HU in patients with a BMI ≥ 25 kg/m2 and < 41 HU in those with a BMI < 25).22-24 These cut-off values were determined specifically for patients on LT waiting list are the most widely used in literature.22-24 Low SMI was present in 63% of males and 28.9% of females. More than one- third of the patients (32.6%) had fatty infiltration of the skeletal muscle. Patients with alcoholic aeti- ology had more fat-infiltrated muscles than other aetiologies for end-stage liver disease (P < 0.008). Alterations in the metabolism of fatty acids, eating habits, and alcohol consumption probably result in fatty infiltration of skeletal muscle fibres. However, due to the small numbers of patients (3 patients) with non-alcoholic steatohepatitis (NASH) or non- alcoholic fatty liver disease (NAFLD) in our study group, we cannot properly assess the relationship between the aetiology of underlying liver disease and the degree of MI. Several studies have shown that sarcopenia and myosteatosis are associated with a higher in- cidence of postoperative complications, infection rate, duration of hospitalization, and mortality in a wide range of gastrointestinal cancers, including tumours of the hepatopancreatobiliary system.29-32 Lack of a strictly defined patient population, dif- ferent methods of measurement of muscle mass and function, and the absence of uniformly ac- cepted cut-off points for patients with chronic or end-stage liver disease are the leading causes for wide ranges of sarcopenia incidence reported in the literature.14,21,33 Therefore, the selected cut-off points for patients with cancer are used for evalua- tion, which may affect the quality of results. Sarcopenia is associated with poorer outcomes in patients with chronic liver disease on the wait- list or after LT.34,35 Patients with end-stage liver disease and sarcopenia have shorter survival than non-sarcopenic patients (22 ± 3 vs. 95 ± 22-month, P < 0.001).9,36 The main cause of death in patients with end-stage liver disease is sepsis.37 Incidence of sep- sis as a cause of death is even higher in presence of sarcopenia (36% vs. 16%, P < 0.001) or myosteato- sis (32% vs. 19%, P = 0.020).9 Lower muscle mass is also associated with a higher incidence of hepatic encephalopathy in patients with end-stage liver disease.38,39 On the other hand, a group from USA40 showed that sarcopenic patients had a higher ten- dency for pulmonary complications than the non- sarcopenic group (38% vs. 18%, P = 0.100); however, there was no significant difference in morbidity and mortality. CT imaging is considered as the gold standard for body composition assessment. SMI calculated on the level of third lumbar vertebra is superior since it correlates best with the actual quantity of the muscles in the body.41 It is not affected by as- cites and is part of preoperative evaluation in pa- tients with hepatocellular carcinoma or the com- plications of portal hypertension. The value of SMI as a predicting factor for postoperative morbidity and mortality is yet to be determined. However, it has been shown in several reports that it has a negative impact on postoperative outcomes. Lower values of SMI are associated with more significant postoperative mortality, higher infection risk, and postoperative complications, more extended in- tensive care unit stay, ventilator dependency, and higher waitlist mortality rates in patients with end- stage liver disease.33,42,43 SMI as a predicting factor for higher mortality rate in patients with end-stage liver disease was superior to other nutritional as- sessment indicators, such as BMI, upper arm mus- cle circumference, and triceps skinfold thickness.44 However, some other reports showed contrasting findings. Using SMI for prespecified definitions of sarcopenia had no impact on mortality or delisting from the transplant waitlist between patients with and without sarcopenia.45 Length of hospitaliza- tion following LT, days of hospitalization during the first year post-LT, survival at one year, or over- all survival was not different between sarcopenic and nonsarcopenic patients.46 Low SMI alone was not associated with graft and patient survival (P = 0.273 and P = 0.278) after LT.47 These conflicting results are probably due to heterogeneity of used specific cut-off points (sex, age, race) and lack of strictly defined parameters representative unique- ly for patients with chronic liver disease. We found a negative correlation between BMI and SMI, probably due to high-volume ascites in the patient population This confirms findings that BMI is not suitable for body assessment in patients with end- stage liver disease. In our study, the presence of as- cites was associated with significantly lower SMI (P < 0.05) in patients with end-stage liver disease but did not influence MI. There is a weak, positive correlation between SMI and ICU length of stay in male patients, which was statistically significant (rs = 0.226, P < 0.025). We speculate that the main reason is a more pronounced systemic inflamma- tory reaction to surgical stress in patients with preserved muscle mass. It is known that muscle mass is mandatory for the normal function of the Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis174 immune system.48 Experience from Japan49 shows a connection between low SMI and decreased incidence of graft rejection in living-donor liver transplantation. Analysis of our results shows no statistically significant difference in postopera- tive complications, rate of postoperative infection, and liver graft rejection rate between males and females with low or normal SMI (P > 0.05). BIA is commonly used technique in body composition analysis in every day clinical practice.50 Its key pa- rameters are resistance, reactance and phase angle (PA). PA is found to be associated with outcomes in different diseases and has been found to be use- ful for monitoring fluid changes and response to interventions.50 The main limitation is the com- plexity of the determinants that requires its ad- justment to the individual phenotypic diagnosis of each patient. Results can be affected by altered water and electrolyte balance, fluid retention and diuretic therapy.50 DXA allows for the quantifica- tion of three body compartments (bone mass, fat mass, and bone fat-free mass (or lean mass)) based upon the differential tissue attenuation of X-ray photons.51 However, it can be affected by presence of ascites.52 Even though DXA can be modified to exclude influence of ascites or tissue oedema the correlation between the lean mass index and SMI was weaker (γ = 0.29, p = 0.035) and falsely high in patients with ascites before liver transplantation.53 Main disadvantages of DXA compared to CT is in- ability to assess muscle mass quality (myosteato- sis).51 Myosteatosis is a more clearly defined factor, and its influence is more uniformly established in literature.54 However, there are many different cut off points (HU < 25 to HU < 39) reported in litera- ture in patients with malignant disease.55 Majority of reports uses a Martin cut-off point (< 33 HU in patients with a BMI ≥ 25 kg/m2 and < 41 HU in those with a BMI < 25)56 for determining presence of myosteatosis. Martin cut-off point is also recent- ly defined threshold parameters for MI in a patient with a chronic liver disease.22-24 It has been shown that severely ill patients with myosteatosis have a lower survival rate than those without fatty infil- tration in muscles.57 Myosteatosis negatively im- pacts the survival of patients with end-stage liver disease (28 ± 5 vs. 95 ± 22-month, P < 0.001)9,36 and is associated with longer hospitalization and higher morbidity.43 Patients with myosteatosis showed a higher mortality rate, most commonly due to res- piratory and septic complication.47 Myosteatosis had no influence on ICU length of stay (P = 0.161), hospitalisation (P = 0.771), postoperative complica- tions (P = 0.839), infection rate (P = 0.703) and graft rejection (P= 0.449) in our patient population. The nutritional status on the waitlist for LT as a possible risk factor is overlooked with the MELD score.58 Another important limiting factor of the MELD score is using serum creatinine levels for score calculation.3 Serum creatinine level may vary significantly and is influenced by chronic kidney disease, ascites, paracentesis, and the in- fluence of gender and liver disease on skeletal muscle mass.59 Variation of serum creatinine levels may affect the MELD score and underestimate the severity of liver disease.58 Thus, several modifica- tions of the MELD score were developed to incor- porate the nutritional parameters. Body composi- tion MELD (BC-MELD)60 has a better predictive value for waiting list mortality than MELD score. MELD-sarcopenia score showed a positive predic- tive value in patients with a lower score (< 15) on the postoperative course; however, it was not use- ful in patients with MELD above 15.61,62 Combining SMI in MELD in multivariate analysis (AUROC= 0.812) is significantly better than MELD alone (AUROC = 0.787) for predicting 5-year mortality (P < 0.001).44 We found a weak, positive correlation between the MELD score and ICU length of stay (rs = 0.261, P < 0.002). ALBI score showed promis- ing results in more accurate prediction of liver dis- ease severity and mortality on waitlist compared to the Child-Pugh score; however, its predictive value was inferior to the MELD score.63 Patients with pre-transplant ALBI grade 3 liver disease had increased mortality after LT.64 We found a weak, positive correlation between the ALBI score and ICU length of stay and hospitalization time, which was statistically significant (rs = 0.279, P < 0.001; rs = 0.197; P = 0.022). We found no correlation between the ALBI score and postoperative complications. The GLIM score16 was established as a potential assessment tool for evaluating patient nutritional status in recent years. Our results showed no dif- ference between the patients who met GLIM cri- teria and those who did not, regarding ICU stay, length of hospitalization, postoperative complica- tions, infection, and mortality. We believe that nutritional status in a patient with end-stage liver disease is an essential aspect of pre-transplant workup. However, its precise role is not yet determined. LT is a complex surgi- cal procedure influenced by numerous factors. In the early phase of LT, donor characteristics and comorbidities, quality of liver graft retrieval, liver graft quality, cold preservation duration, ischemic damage to the liver graft during static cold storage, Radiol Oncol 2023; 57(2): 168-177. Petric M et al. / Radiological assessment of skeletal muscle index and myosteatosis 175 recipient medical conditions and comorbidities, surgical procedure, and early postoperative ther- apy are by our opinion the most essential factors. However, muscle mass status, muscle function and myosteatosis are crucial factors in a period of rehabilitation.65 Patient with cachexia or with high-risk (< 18.5 kg/ m2, Child-Pough C)66 to lose muscle mass should be screened and involved in intensive but person- alized nutritional support therapy. Nutritional re- quirements in end stage liver disease are 35 kcal/ kg per day in non-obese patients (BMI 30 kg/m2) and 1,2g/kg per day intake of proteins.66 It is safe to calculate requirements based on the dry weight of a patients.66 In presence of hepatic encephalopa- thy (HE), animal protein should be substituted with vegetable protein origin. Randomized control study67 showed that substitution of animal protein with vegetable protein for a period of six months (30–35 kcal/kg/d,1.0–1.5 g/kg/d protein) improved neuropsychiatric performance in patients with minimal HE and decrease their risk of develop- ing overt HE compared to no intervention. It is unreal to expect to gain muscle mass in cirrhotic patients, but muscle mass preservation should be focus of such nutritional interventions. There are several strategies to prevent muscle mass loss in patients with end stage liver disease. First strategy is nutritional supplementations. Patients should have frequent small meals to avoid prolong fast- ing period (> 6 h).66,68,69 Enteral supplements with side branched amino acids should be adminis- tered.66,68,69 Snacks rich in carbohydrates should be taken as a late-night snack.66,68,69 Second, physi- cal activity in a form of resistance and endurance exercise, is probably appropriate and beneficial.70 Micronutrition, especially administration of fat- soluble vitamins71 and ammonia lowering thera- py72 is also important. Role of immunonutrition in patient with end stage liver disease is not yet estab- lished.66,73 Post-transplant screening is advised in all patients after liver transplantation.66,73 There are several drawbacks to our study. The two most important are the retrospective nature of data collection and the lack of functional assess- ment of recipients’ muscles. Another factor that may influence results is the relatively small num- ber of cases that may affect some results or may not produce statistical significance due to the com- plexity of the treatment and numerous factors that determine its outcome. In conclusion radiological assessment of a pa- tient’s nutritional status at the third lumbar ver- tebra represents an objective and reproducible method. It should become a standard screening tool in patients with acute or chronic end-stage liver disease. Due to complexity of liver transplant procedure, liver graft and liver recipients’ factors, it is difficult to established impact of a skeletal muscle index and myosteatosis on postoperative outcomes. However, nutritional interventions and physical activity should be part of the clinical pathway in patients with end-stage liver disease waiting for liver transplantation. 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Radiol Oncol 2023; 57(2): 178-183. doi: 10.2478/raon-2023-0022 178 research article ADC values as a biomarker of fetal brain maturation Lucija Kobal1, Katarina Surlan Popovic2, Jernej Avsenik2, Tina Vipotnik Vesnaver2 1 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 2 Clinical Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(2): 178-183. Received 3 February 2023 Accepted 17 April 2023 Correspondence to: Dr. Tina Vipotnik Vesnaver, Institute of Radiology, University Medical Centre Ljubljana, Zaloška 7, SI-1000 Ljubljana, Slovenija. E-mail: tina.vipotnikvesnaver@kclj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. During the period of fetal development, myelination plays a key role and follows specific time and spa- tial sequences. The water content in the brain is inversely proportional to myelination – the more myelinated the brain, the lower the water content in it. The diffusion of water molecules can be quantitatively assessed using the apparent diffusion coefficient (ADC). We were interested in whether, by determining the ADC values, we could quantitatively evaluate the development of the fetal brain. Patients and methods. The study included 42 fetuses with gestational age 25 to 35 weeks. We manually selected 13 regions on diffusion-weighted images. Statistically significant differences between ADC values were checked using one-way analysis of variance and Tukey’s post hoc test. The relationship between the ADC values and the gestational age of the fetuses was then assessed using linear regression. Results. The average gestational age of the fetuses was 29.8 ± 2.4 weeks. ADC values in the thalami, pons and cerebellum differed significantly among each other and from the ADC values in other brain regions. In the thalami, pons and cerebellum, linear regression showed a significant decrease in ADC values with increasing gestational age. Conclusions. ADC values change with the increasing gestational age of the fetus and differ among different brain regions. In the pons, cerebellum and thalami, the ADC coefficient could be used as a biomarker of fetal brain matura- tion since ADC values decrease linearly with increasing gestational age. Key words: myelination; fetal brain maturation; ADC; biomarker; diffusion-weighted imaging; diffusion Introduction Human brain development is a complex process that begins in the third gestational week and con- tinues well after birth, even into adulthood.1 In our study, we were interested in the process of myeli- nation and how it can affect apparent diffusion co- efficient (ADC) values. Myelination is the final stage in the develop- ment of the white matter of the brain. It begins in the second half of pregnancy, after the prolifera- tion and maturation of oligodendrocyte cells. It al- so has a characteristic course, from caudal to ros- tral regions of the brain, from the center outwards, and from dorsal to ventral regions of the brain.2,3 As early as around the 20th week of gestation, mi- croscopic amounts of myelin can be observed, es- pecially in the medulla oblongata and pons. The brainstem is completely myelinated at 29 weeks.4 Between the 37th and 40th gestational week, mature myelin is present in the cerebellum and the internal capsule.4,5 Sensory pathways are myelinated earlier than motor pathways, and occipital areas are my- elinated earlier than parietal, temporal and frontal areas. Myelination of the brain also takes place in the postnatal period ‒ it is completed only after the age of 20 when the areas of the corpus callosum and prefrontal brain become fully myelinated.6-9 Radiol Oncol 2023; 57(2): 178-183. Kobal L et al. / ADC values in fetal brain maturation 179 Knowing the exact course of myelination is cru- cial for detecting pathological changes that affect myelination. In premature infants, hypomyelina- tion may be a predictive factor for motor and cog- nitive impairments. Myelination can also be affect- ed by many genetic and autoimmune diseases and infections.10-12 As the brain matures, the water content changes over time. The amount of water in the brain is in- versely proportional to myelination - the more my- elinated the brain, the less water it contains. The decrease in water content can be attributed to the accumulation of lipids and proteins and changes in the electrolytic composition of tissues. The re- duction in the proportion of water continues even after birth. In addition to the water content, the movement of water molecules in the brain is also affected by the number of cells and the amount of myelin, both of which limit the movement of mol- ecules.13-17 With the development of MRI, especially with the advance of diffusion-weighted magnetic reso- nance imaging (DWI), an opportunity has ap- peared for the non-invasive assessment of myelina- tion in fetuses. Furthermore, the diffusion of water molecules can now be quantitatively evaluated us- ing the ADC values. DWI is an extremely useful technique for detect- ing hyperacute hypoxic-ischemic changes, it can be helpful in other disease processes that affect the movement of water molecules in tissues, e.g., ab- scesses, tumours etc. and it can also be used to mon- itor the normal development of the fetal brain.18-23 This technique is already a part of the standard protocol in fetal MR imaging to diagnose anomalies of the central nervous system but, despite previous studies that have addressed this issue, it is still not completely clear whether a quantitative evaluation of fetal brain development using ADC values can be used in clinical practice. We do not yet have ref- erence ADC values that would allow us to compare healthy fetuses with fetuses suspected of develop- ing anomalies of the central nervous system.24-30 Research is therefore increasingly focused on stud- ying ADC values in different areas of the fetal brain. In our study, we observed how ADC values change during the process of fetal brain matura- tion. We postulated that the values would depend on the gestational age of the fetus and also on the areas of the brain. The determined ADC values could serve as reference values in daily clinical work. They would be useful in assessing the level of fetal brain maturity and early detection of patho- logical changes. The purpose of our research was to determine how ADC values change with the gestational age of the fetus. We were interested in how ADC values differ among different brain areas. We also wanted to discover whether the ADC could be used as a biomarker of fetal brain maturation. Our hypotheses were: 1. ADC is a useful biomarker of fetal brain matura- tion. 2. ADC values depend on the age of the fetus and the area of the brain. Patients and methods The retrospective study was conducted at the Clinical Institute of Radiology of the University Medical Center Ljubljana. The National Medical Ethics Committee of the Republic of Slovenia judged that the research was ethically accept- able and gave consent for its implementation (No. 0120-56/2022/3). Patients We initially selected 59 fetuses that had had an MRI done between 18. 1. 2015 and 4. 3. 2021. Of these, the MRI images of 17 fetuses were excluded since their DWI images had artefacts due to fetal movement. We therefore performed the measure- ments on MRI images of 42 fetuses. We included fetuses that have been referred for MRI due to sus- picious US changes in the central nervous system (CNS), face or neck (wider cisterna magna, sus- pected agenesis of the corpus callosum, ranula etc.) but in which we did not confirm CNS anomalies with MRI. All included pregnant women under- went amniocentesis, which excluded chromosomal abnormalities and infections. After birth, no signs indicating abnormal development of the central nervous system were found in our group. The ges- tational age of the fetuses was from 25 to 35 weeks (mean 29.8 ± 2.4 weeks). Magnetic resonance imaging Examinations were performed on a Siemens Aera 1.5 T MR device. The pregnant women did not eat or drink for four hours before the examination and, during the examination, they were in a supine or left decubitus position. They did not receive any medication before the procedure. An abdominal coil with a small, 24 cm field of view and a 192 × Radiol Oncol 2023; 57(2): 178-183. Kobal L et al. / ADC values in fetal brain maturation180 160 matrix was used. A 3D scout sequence was performed to assess the fetal position. Scout se- quence is an ultrafast T2-weighted sequence, with slice thicknesses of 6–8 mm, gaps of 1–2 mm, and a large field of view. Our protocol consisted of ultra- fast T2-weighted sequences in three planes (axial, coronary, sagittal) with slice thicknesses of 3 and 4 mm and intermediate intervals of 0.3 mm, T1- and T2-gradient sequences in the axial plane (the for- mer with slice thickness 4.5 mm and intermediate intervals 0.5 mm, the latter with slice thickness 3 mm and intermediate intervals 0.3 mm) and DWI- sequence in the axial plane (slice thickness 5 mm with intermediate intervals 2 mm). Diffusion was measured in three directions at values of b = 0 s/ mm2, b = 500 s/mm2, and b = 1000 s/mm2. ADC value measurements ADC measurements were performed by two re- searchers (L.K., T.V.V.). On ADC maps, we manu- ally selected 13 areas (region of interest, ROI) using a Syngo. via program (Siemens Medical Solutions USA, Inc. ©2022): bilaterally in the frontal white matter (FWM), parietal white matter (PWM), tem- poral white matter (TWM) and occipital white mat- ter (OWM), bilaterally in the white matter of the cerebellum, in both thalami and the central part of the pons. The surface area of the ROI was adapted to the age of the fetus and the anatomical area, with values between 15 and 65 mm2. Statistical data analysis Statistical data analysis and graph production were performed using IBM SPSS Statistics (IBM SPSS Statistics for Windows, version 25.0; IBM Corp., Armonk, NY). Interrater reliability was as- sessed using the intraclass correlation coefficient (ICC) with a two-way mixed model for the average of the measurements. ICC values below 0.5 indicate low, between 0.5 and 0.75 moderate, between 0.75 and 0.9 good, and above 0.9 excellent reliability. Since the ICC was 0.75 or higher in all measured brain areas, we av- eraged the measurements of the two researchers. Statistically significant differences between ADC values for different areas of the brain were checked using one-way analysis of variance (ANOVA). Statistically significant differences between ADC values in individual groups were searched for with Tukey’s post hoc test. Before the one-way analysis of variance, we checked the homogeneity of the variances with Levene’s test, whereby we rejected the null hypothesis that the variances are homoge- neous or homoscedastic. The association between the ADC values of the brain regions and the gesta- tional age of the fetuses was then evaluated using linear regression. Before the statistical data analy- sis, we determined the p-value 0.05 as the thresh- old. Results ICC values by individual brain areas are shown in Table 1. The average ADC values for different ROIs are shown in Figure 2. Using one-way analysis of vari- ance, we found that there was a statistically sig- nificant difference between the ADC values for dif- TABLE 1. Intraclass correlation coefficient (ICC) for different regions of interest (ROIs) ROI ICC FWM 0.91 PWM 0.90 TWM 0.75 OWM 0.78 Thalami 0.85 Pons 0.81 Cerebellum 0.92 FWM = frontal white matter; OWM = occipital white matter; PWM = parietal white matter; TWM = temporal white matter FIGURE 1. Position of the ROI in the brain of a fetus with a gestational age of 29 weeks. Region of interests (ROIs) were placed in each brain region bilaterally. Markings are visible only on one side. (A) Frontal white matter, (B) parietal white matter, (C) temporal white matter, (D) occipital white matter, (E) thalamus, (F) pons, (G) hemisphere of the cerebellum. A B C G D E F Radiol Oncol 2023; 57(2): 178-183. Kobal L et al. / ADC values in fetal brain maturation 181 ferent brain areas. The ADC values in the thalami, pons and cerebellum differed significantly from each other and from the ADC values in all other brain areas. There were no significant differences between ADC values in FWM, PWM, TWM and OWM. In the thalami, pons and cerebellum, linear re- gression showed a statistically significant decrease in ADC values with increasing gestational age (Figure 3). ADC values also decreased with in- creasing gestational age in PWM and OWM, but the results were not statistically significant. ADC values increased in FWM and TWM, but the in- crease in values in these areas was also not statisti- cally significant. Discussion Our study confirmed that the average ADC values in fetuses differ among different brain areas. As shown in Figure 2, the lowest values were meas- ured in the pons, thalami and cerebellum, and the highest in the FWM, PWM, TWM and OWM. These differences can be attributed to the charac- teristic course of myelination and the number of neurons in individual areas. The amount of neu- rons in the thalami is greater than in the white mat- ter, so the diffusion of water is more limited. Since myelination proceeds from the caudal to the rostral regions of the brain, from the central regions to the periphery, and from the dorsal to the ventral re- gions, the pons, thalami and cerebellum are myeli- nated earlier than the white matter of the cerebral hemispheres, where the myelination process still takes place in the postnatal period. ADC values in fetuses, therefore cannot be compared with values in newborns and children, nor with values in pre- term infants of the same gestational age, since large changes in the amount and distribution of water in the brain occur after birth.2,3 ADC values in the pons, thalami and cerebellum were much lower than in the white matter of the cerebral hemispheres, which is consistent with pre- vious research. The average values in these areas were comparable with previous studies, while the average ADC values for FWM, PWM, TWM and OWM were slightly lower in our study. The largest deviations can be seen for the FWM area, where in our research we measured an average ADC value of 1.52 × 10-3 mm2/s. Han et al. (1.8 × 10-3 mm2/s), Schneider et al. (1.8 × 10-3 mm2/s), Hoffmann et al. (1.8 × 10-3 mm2/s) and Righini et al. (2.9 × 10-3 mm2/s) all recorded higher values.25,27-31 These differences could be attributed to the slightly lower average age of the fetuses in our study and the possible non-linear changing of ADC values, as described below. OWM is known to be myelinated earlier than PWM, TWM and FWM.8 We were unable to con- firm this with our research. The results of our re- search showed that ADC values did not differ FIGURE 2. Average apparent diffusion coefficient (ADC) values for different regions of interest (ROIs). FIGURE 3. Association between apparent diffusion coefficient (ADC) values in different brain regions and fetal gestational age in days. (A) Frontal white matter, (B) parietal white matter, (C) temporal white matter, (D) occipital white matter, (E) thalamus, (F) pons, (G) cerebellum. A B C D E F G Radiol Oncol 2023; 57(2): 178-183. Kobal L et al. / ADC values in fetal brain maturation182 statistically significantly among these areas. We attribute these results to the fact that the occipital part of the brain in fetuses is thinner than in the rest of the cerebral hemispheres. The ROIs were there- fore placed closer to the cerebrospinal fluid than in other areas, which may have resulted in partial coverage of the cerebrospinal fluid signal in the ROI. As a result, measurements may be affected by averaging the values of cerebrospinal fluid and brain parenchyma. This may also explain why the ICCs between the researchers differed predomi- nantly in the areas of OWM and TWM, where the cerebral mantle is also thinner. In the thalami, pons and cerebellum, linear re- gression showed a statistically significant decrease in ADC values with increasing gestational age (Figure 3). ADC values also decreased with increas- ing gestational age in PWM and OWM, but the re- sults were not statistically significant. In FWM and TWM, ADC values increased in our study, but the increase in values was not statistically significant in these areas either. The FWM is the last of all brain areas to be myelinated, the amount of water there being the highest for the longest time, which could be the reason for the measured values in our re- search. All the studies published so far have confirmed the decline in ADC values with increasing gesta- tional age in the areas of the cerebellum, thalami and pons.25,27-31 The results indicate that in these areas of the brain, the amount of water decreases with age, which affects the reduction of ADC val- ues. In our study, ADC values also declined most rapidly in the cerebellum, pons and thalami, which suggests earlier maturation of these regions and is consistent with other studies. Differences among studies occurred in other brain areas. A decrease in ADC values in PWM and OWM was demonstrated by Han et al., Hoffmann et al. and Righini et al., while Cannie et al. detected an increase in ADC values in OWM.27-30 Similar to our study, some other researchers have also detect- ed an increase in ADC values with gestational age in FWM.48,50,53 Schneider et al, on the other hand, measured increasing ADC values in FWM, PWM, TWM and OWM until the 30th gestational week, but thereafter the ADC values began to decrease.31 The results of Schneider et al. are consistent with the course of fetal brain development. During development, the brain consists of several layers - above the ventricular layer, there is an interme- diate layer, a subplate and a cortical layer. Wide extracellular spaces are present in the intermedi- ate layer and subplate, which allow nerve cells to migrate. In these spaces, water molecules can move freely, which could explain the rise in ADC values. Schneider et al. explained the repeated de- cline in ADC value by a combination of different factors that begin to dominate after the 30th week: the subplate and the intermediate layer slowly dis- appear, the total amount of water decreases and, at the same time, the number of lipids and macro- molecules in the intracellular spaces increases. We therefore allow the possibility that the change in ADC values in FWM, PWM, TWM and OWM with increasing gestational age is not linear, but may be better explained by more complex models, for ex- ample using a quadratic polynomial curve, which means that the values initially increase and then decrease.31 The multilayered structure of the brain is vis- ible on T2-weighted sequences in fetuses of the gestational age of 20–28 weeks but, with increasing gestational age, the boundaries between the layers are blurred. Due to the poor image resolution of the ADC maps, it is very difficult to distinguish the layers from each other, even in younger fetuses. When manually placing the ROIs, due to the poor resolution on the ADC maps we may also capture areas of the subplate, which later develop into the cerebral cortex, and not the white matter, which could affect the results of the measurements. The downside of our research was the relatively small number of subjects. A disadvantage is also the probability of measurement errors due to the movement of the fetus during the examination, which was reduced by preparing the mothers for the MRI examination and eliminating poor-quality images. In fetal MRI, measurements are also affect- ed by the small size of the structures, which may result in the capture of the cerebrospinal fluid or subplate in selected cases. ROI drawing may also be challenging due to the poor resolution of the brain layers on the ADC maps. Another thing to consider before comparing re- sults from different studies is variability in meas- ured ADC values due to the use of different MRI equipment and sequence selections. Conclusions We conclude that ADC values are a reliable indi- cator of brain maturation in the areas of the pons, cerebellum and thalami. The results of our research are concordant with previous research, which has shown that the values in these areas decrease lin- early with increasing gestational age. We also Radiol Oncol 2023; 57(2): 178-183. Kobal L et al. / ADC values in fetal brain maturation 183 confirmed that the ADC values are higher in the FWM, PWM, TWM, and OWM regions than in the pons, cerebellum and thalami. However, it is still unknown how ADC values in FWM, PWM, TWM and OWM change with gestational age, since re- sults vary among studies. Further research is need- ed to define more precisely the variation of ADC values in these areas, which would help us to set reference values. 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European Radiol 2007; 17: 2422-9. doi: 10.1007/s00330-007-0634-x Radiol Oncol 2023; 57(2): 184-190. doi: 10.2478/raon-2023-0020 184 research article Longitudinal monitoring of Apparent Diffusion Coefficient (ADC) in patients with prostate cancer undergoing MR-guided radiotherapy on an MR-Linac at 1.5 T: a prospective feasibility study Haidara Almansour1, Fritz Schick2, Marcel Nachbar3,4, Saif Afat1, Victor Fritz2, Daniela Thorwarth4,5, Daniel Zips3,5,6, Felix Bertram6, Arndt-Christian Müller6,7, Konstantin Nikolaou1,8, Ahmed E Othman1,9, Daniel Wegener6 1 Department of Diagnostic and Interventional Radiology, Eberhard-Karls University, Tuebingen, Germany 2 Section for Experimental Radiology, Department of Radiology, Eberhard-Karls University, Tuebingen, Germany 3 Department of Radiation Oncology, Charité University Medicine Berlin, Berlin, Germany 4 Section for Biomedical Physics, Department of Radiation Oncology, Eberhard-Karls University, Tuebingen, Germany 5 German Cancer Consortium (DKTK), Partner Site Tuebingen and German Cancer Research Center (DKFZ), Heidelberg, Germany 6 Department of Radiation Oncology, Eberhard-Karls University, Tuebingen, Germany 7 Department of Radiation Oncology, RKH Klinikum Ludwigsburg, Ludwigsburg, Germany 8 Cluster of Excellence iFIT (EXC 2180) “Image Guided and Functionally Instructed Tumor Therapies”, University of Tuebingen, Tuebingen, Germany 9 Department of Neuroradiology, University Medical Center Mainz, Mainz, Germany Radiol Oncol 2023; 57(2): 184-190. Received 20 January 2023 Accepted 30 March 2023 Correspondence to: Ahmed E. Othman, Professor and Consultant Radiologist, Department of Neuroradiology, University Medical Center Mainz, 55131 Mainz, Germany. E-mail: ahmed.e.othman@googlemail.com Disclosure: The authors` institution operates a Linac Unity (Elekta, Sweden). The Department of Radiation Oncology Tübingen receives finan- cial and technical support by Elekta, Philips, Siemens, Dr. Sennewald Medizintechnik, Kaiku Health, TheraPanacea, PTW and ITV in the con- text of research cooperation’s. The aforementioned entities had no role in the design of the study; in the collection, analyses, or interpreta- tion of data; in the writing of the manuscript, or in the decision to publish the results. The authors declare no further conflicts of interest. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Hybrid MRI linear accelerators (MR-Linac) might enable individualized online adaptation of radiothera- py using quantitative MRI sequences as diffusion-weighted imaging (DWI). The purpose of this study was to investigate the dynamics of lesion apparent diffusion coefficient (ADC) in patients with prostate cancer undergoing MR-guided radiation therapy (MRgRT) on a 1.5T MR-Linac. The ADC values at a diagnostic 3T MRI scanner were used as the refer- ence standard. Patients and and methods. In this prospective single-center study, patients with biopsy-confirmed prostate cancer who underwent both an MRI exam at a 3T scanner (MRI3T) and an exam at a 1.5T MR-Linac (MRL) at baseline and during radiotherapy were included. Lesion ADC values were measured by a radiologist and a radiation oncologist on the slice with the largest lesion. ADC values were compared before vs. during radiotherapy (during the second week) on both systems via paired t-tests. Furthermore, Pearson correlation coefficient and inter-reader agreement were computed. Results. A total of nine male patients aged 67 ± 6 years [range 60 – 67 years] were included. In seven patients, the cancerous lesion was in the peripheral zone, and in two patients the lesion was in the transition zone. Inter-reader reli- ability regarding lesion ADC measurement was excellent with an intraclass correlation coefficient of (ICC) > 0.90 both at baseline and during radiotherapy. Thus, the results of the first reader will be reported. In both systems, there was a Radiol Oncol 2023; 57(2): 184-190. Almansour H et al. / ADC in patients with prostate cancer undergoing MR-guided radiotherapy 185 Introduction Radiotherapy (RT) is a curative treatment option for patients with localized prostate cancer.1 MR- guided radiotherapy (MRgRT) enables improved soft tissue contrast and enhances accuracy of treatment planning.2 In this context, the hybrid magnetic resonance 1.5T scanner with a linear ac- celerator MR-Linac (MRL) is currently being used in centers around the world to perform high-preci- sion MRgRT with daily plan adaptations based on anatomical MR sequences.3,4 Furthermore, func- tional MRI sequences such as diffusion weighted imaging (DWI) are being additionally taken into account for radiotherapy planning, as they provide valuable “real-time” functional information.5 The apparent diffusion coefficient (ADC) of a tumor le- sion has been shown to function as a biomarker for prostate cancer on diagnostic scanners.6 MRL pre- sents a novel opportunity to integrate ADC-values of a tumor lesion into daily plan adaptations and individualize radiotherapy.7 A prerequisite is the clinical translatability of ADC-measurements on MRL to a “gold standard” 3T diagnostic scanner (MRI3T). In a previous study, it was demonstrated that ADC measurements of a region of interest in in- traprostatic tumor lesions on MRL correlated with corresponding measurements on a diagnostic 3T MRI scanner (MRI3T).8 In that analysis, the MRIs on both scanners were performed prior to treat- ment initiation. However, as an initial step to evaluate, whether ADC measurements on an MRL might function as a biomarker enabling response assessment under RT, the longitudinal stability of ADC data gained on an MRL should be examined. The purpose of this study is to longitudinally investigate the dynamics of lesion ADC in patients with prostate cancer undergoing MR-guided ra- diation therapy on an MR-Linac using the ADC values at a 3T MRI scanner as a reference standard. Patients and methods Participant sample, study design and MRI technique All patients included in this prospective study were recruited in the M-base Pro 1.09 or M-base HyPro 2.0 at our institution (ClinicalTrials.gov Identifiers: NCT02724670; NCT03880851). The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the medical faculty of Tuebingen University (No. 022/2016BO1, 14.03.2016 and No. 920/2018BO1, 10.07.2019). Informed con- sent was obtained from all subjects involved in the study. All patients consented to prospectively un- dergo multiple MRIs on an MRL and additionally on a MRI3T at several points prior to and during RT. The aforementioned studies each examine a novel MR-adaptive concept for radiotherapy of primary localized prostate cancer. Between February 2019 and October 2021, 9 patients with biopsy-confirmed prostate cancer and available MRL and MRI3T data sets prior to RT and under RT were included. All patients were treated daily on a 1.5T MRL (Elekta UnityTM, Philips, Stockholm, Sweden).10 All pa- tients were treated according to national guide- lines with either 39 x 2 Gy per fraction over eight weeks (M-base 1.0 study, n = 3 patients) or 20 x 3 Gy per fraction over four weeks (M-base Hypro 2.0 statistically significant elevation of lesion ADC during radiotherapy (mean MRL-ADC at baseline was 0.97 ± 0.18 x 10-3 mm2/s vs. mean MRL-ADC during radiotherapy 1.38 ± 0.3 x 10-3 mm2/s, yielding a mean lesion ADC elevation of 0.41 ± 0.20 x 10-3 mm2/s, p < 0.001). Mean MRI3T-ADC at baseline was 0.78 ± 0.165 x 10-3 mm2/s vs. mean MRI3T-ADC during radiotherapy 0.99 ± 0.175 x 10-3 mm2/s, yielding a mean lesion ADC elevation of 0.21 ± 0.96 x 10-3 mm2/s p < 0.001). The absolute ADC values from MRL were consistently significantly higher than those from MRI3T at baseline and during radiotherapy (p < = 0.001). However, there was a strong positive correlation between MRL-ADC and MRI3T-ADC at baseline (r = 0.798, p = 0.01) and during radiotherapy (r = 0.863, p = 0.003). Conclusions. Lesion ADC as measured on MRL increased significantly during radiotherapy and ADC measurements of lesions on both systems showed similar dynamics. This indicates that lesion ADC as measured on the MRL may be used as a biomarker for evaluation of treatment response. In contrast, absolute ADC values as calculated by the algorithm of the manufacturer of the MRL showed systematic deviations from values obtained on a diagnostic 3T MRI system. These preliminary findings are promising but need large-scale validation. Once validated, lesion ADC on MRL might be used for real-time assessment of tumor response in patients with prostate cancer undergoing MR-guided radiation therapy. Key words: prostate carcinoma; MRI; adaptive radiotherapy; image guidance; MR-Linac; ADC Radiol Oncol 2023; 57(2): 184-190. Almansour H et al. / ADC in patients with prostate cancer undergoing MR-guided radiotherapy186 study, n = 6 patients) and additional neoadjuvant androgen deprivation therapy (ADT) of six months for intermediate risk patients and 24-36 months for high risk patients.11 The time point of the MRL and MRI3T was during week 2 of RT in both treatment protocols. MRI technique, specifications and ac- quisition parameters of the examinations on both systems have been previously described.8 Most study participants in this study were used in the prior publication8, but only examinations prior to RT were analyzed. No lesion ADC dynamics dur- ing RT were reported in the previous study.8 Lesion ADC evaluation The ADC maps for MRL and MRI3T for each pa- tient, prior to and during radiotherapy, were in- dependently presented to two readers (reader 1, a board certified radiation oncologist with 8 years of experience reader 2, a radiology resident with 4 years of experience). Both readers placed an elliptic region-of-interest (ROI) within the lesions for each patient in MRL and MRI3T image sets. A dedicated workstation (GE Healthcare Centricity™ PACS RA1000, Milwaukee WI, USA) was utilized for im- age analysis using a dedicated software (syngo.via, Siemens Healthcare, Erlangen, Germany). Statistical analysis Continuous variables were reported as mean and standard deviation. Paired t-tests were used for pair-wise pre- vs. during-treatment comparisons, as well as MRL vs. MRI-3T. Intraclass correlation coefficient (ICC, two-way, absolute agreement) was used to compute inter-reader agreement. An ICC of less than 0.4 signalizes poor agreement, of 0.40 to 0.59 indicates fair agreement, of 0.60 to 0.74 good agreement, and an ICC of 0.75 to 1.00 signal- izes excellent agreement.12 Pearson Correlation co- efficient was used to compare lesion ADC between MRL and MRI3T. Level of significance was set at 0.05. Statistical analyses were performed using SPSS (v26.0, IBM-Corp, Armonk, NY, USA). Results A total of nine patients were included. Figure 1 de- lineates the the inclusion/exclusion process. Table 1 summarizes patients’ characteristics. For each patient, the two imaging examinations prior to and during RT were successfully per- formed and evaluated. The mean elapsed time between the baseline MRL exam and the MRL exam during radiothera- py was 26 days ± 14 days. The mean time difference between MRI3T and MRL examinations at baseline was 1.7 days ± 1.7 days. Similarly, the mean time difference between MRI3T and MRL examinations during radiotherapy was was 1.7 days ± 1.3 days. Inter-reader reliability regarding lesion ADC measurement was excellent with ICC > 0.90 both at baseline and during radiotherapy (ICC for MRL Patients with biopsy-confirmed prostate cancer and an indication for curative RT treated within the M-Base Pro 1.0 or M- Base Hypro 2.0 studies and on the 1.5T MRL between 2019-2021 (n=13) Excluded (n=4) Study MRIs not performed per protocol (n=3) MRI sequences/quality not meeting analysis criteria (n=1) Analysed (n=9) Analysis Enrollment RT = radiotherapy; MRL = 1.5 T MR-Linac Unity. ● ● FIGURE 1. Flow diagram illustrating the inclusion/ exclusion process. Radiol Oncol 2023; 57(2): 184-190. Almansour H et al. / ADC in patients with prostate cancer undergoing MR-guided radiotherapy 187 at baseline was 0.927 and during radiotherapy was 0.976; ICC for MRI3T at baseline was 0.978 and dur- ing radiotherapy was 0.998). For reader 1, in both systems, there was a sta- tistically significant elevation of lesion ADC dur- ing radiotherapy (Figure 2). Mean MRL-ADC at baseline was 0.97 ± 0.18 mm2/s vs. mean MRL-ADC during radiotherapy 1.38 ± 0.3 mm2/s, yielding a mean lesion ADC elevation of 0.41 ± 0.20 mm2/s, p <0.001. Mean MRI3T-ADC at baseline was 0.78 ± 0.165 mm2/s vs. mean MRI3T-ADC during radio- therapy 0.99 ± 0.175 mm2/s, yielding a mean lesion ADC elevation of 0.21 ± 0.96 mm2/s p <0.001. The ADC values at MRL were consistently sig- nificantly higher than MRI3T at baseline and dur- ing radiotherapy (p < 0.01). However, there was a strong positive correlation between MRL-ADC and MRI3T-ADC at baseline (r = 0.798, p = 0.01) and during radiotherapy (r = 0.863, p = 0.003) (Figure 3). Similarly, for reader 2, in both systems, there was a statistically significant elevation of lesion ADC during radiotherapy (mean MRL-ADC at baseline was 1.0 ± 0.23 x 10-3 mm2/s vs. mean MRL- ADC during radiotherapy 1.36 ± 0.30 x 10-3 mm2/s, yielding a mean lesion ADC elevation of 0.36 ± 0.17 x 10-3 mm2/s, p <0.001). Mean MRI3T-ADC at base- line was 0.78 ± 0.17 x 10-3 mm2/s vs. mean MRI3T- ADC during radiotherapy 1.0 ± 0.183 x 10-3 mm2/s, yielding a mean lesion ADC elevation of 0.22 ± 0.129 x 10-3 mm2/s p <0.001). The ADC values at MRL were consistently sig- nificantly higher than MRI3T at baseline and dur- ing radiotherapy (p < 0.001). However, there was a strong positive correlation between MRL-ADC and MRI3T-ADC at baseline (r = 0.872, p = 0.002) and during radiotherapy (r = 0.788, p = 0.012). Discussion This prospective study compared lesion ADC val- ues in patients with prostate carcinoma undergo- ing MR-guided radiotherapy on an MRL at 1.5 T to a diagnostic scanner at 3T. Absolute values of lesion ADC measurements differed while dynam- ics in the context of radiation therapy were compa- rable between the scanners. In both systems, there was a statistically significant elevation of lesion ADC during radiotherapy with a strong positive correlation of lesion ADC between the scanners. ADC changes during radiotherapy ADC changes of the intraprostatic tumor are to be expected both during radiotherapy and during ADT and the correlation between ADC and pros- tate cancer aggressiveness has been shown before on diagnostic scanners.17,18 The mean ADC-values calculated in this study on both scanners are similar to values found in the literature: Tamada FIGURE 2. T2-weighted MR images (scan time: 2 minutes) and apparent diffusion coefficient (ADC) maps of a 66-year-old male patient with prostate cancer in the antero-apical region of the transition zone (red arrows) at baseline (left column) and during radiotherapy (right column) as recorded on MR-Linac (MRL) and on standard MRI at 3T (MRI3T). The figure shows similar dynamics of lesion ADC elevation during radiotherapy. Radiol Oncol 2023; 57(2): 184-190. Almansour H et al. / ADC in patients with prostate cancer undergoing MR-guided radiotherapy188 et al. reported mean ADC values of (untreated) tumor regions of 1.02 ± 0.25 x 10-3 mm2/s for the peripheral zone and of 0.94 ± 0.21 x 10-3 mm2/s for the transitional zone of the prostate.13 Van Schie et al. reported median ADC values in the tumor scanned on a diagnostic scanner of 1.08 ± 0.39 x 10-3 mm2/s (mean ± SD) prior to treatment and as- sessed changes of ADC prior to a hypofractionated RT and then weekly during RT in 73 patients in a similar manner as performed in our study. The group found a (non-significant) median increase of the ADC-value in the tumor of 7% for patients with concurrent ADT and a median increase of 20% for patients without ADT.14 ADC as biomarker for response assessment and role of MRL Moreover, ADC values and -changes have been shown to function as biomarkers for RT response in prostate cancer patients.6,15 Radiomics approach- es seem promising in assessing response to RT, as performed by Abdollahi et al. prior to vs. after RT.16 In all of these studies and in prostate cancer di- agnostics, a 3 T MRI scanner has been established as the gold standard for mpMRI.17 MRI3T leads to optimal diagnostic images, often aided by sup- pression of peristalsis via intravenous application of butyl scopolamine or other agents. In contrast, on a 1.5 T MRL, the utilized sequences are opti- mized for fast and geometrically accurate image acquisition in an online workflow without routine administration of peristalisis suppressing medica- tions or contrast agents. These possible limitations, in addition to technical differences of the hybrid system to diagnostic scanners18, pose the question whether an MRL can deliver comparable function- al information during RT of prostate cancer. In principle, the hybrid system offers fertile ground for further plan adaption in prostate carci- noma patients based on mpMRT findings such as ADC values since it offers daily MR-guided plan adaptations. Treatment individualization and plan adaptation under RT are a focus of research in other tumor entities as well.7,19,20 Longitudinal dif- fusion MRI on a 0.35 T hybrid system was already performed in small series for several other tumor entities with promising results (Yang et al.: three head and neck cancer patients and three sarcoma patients21; Shaverdian et al.: three rectal cancer pa- tients22). On the 1.5 T MRL, Lawrence et al. report a high ADC repeatability and comparability to a diagnostic 1.5 T scanner for 59 patients with cen- tral nervous system tumors.23 Habrich et al. used a test-retest approach on 11 patients with head and cancers and showed to a high repeatability of ADC measurements on the 1.5 T MRL.24 In the context of prostate carcinoma, Habrich et al. also examined intravoxel incoherent motion (IVIM) and dynamic contrast enhanced (DCE) MRI changes over the course of a moderately hypofractionated RT in 20 patients, also indicating that longitudinal meas- urements of functional imaging parameters is fea- sible and could be used for response assessment in the future.25 However, concerning intraprostatic tumor le- sions, the verification of longitudinal stability of ADC measurements on an MRL as performed in this study in a comparison to a latest generation 3 T diagnostic scanner has to the best of our knowl- edge not been performed yet. In a previous study, we tested the clinical ap- plicability based on qualitative and quantitative parameters of prostate MR images on an MRL A B FIGURE 3. Scatter plots with a fitted line for ADC values as recorded on MR-Linac and MRI3T for reader 1. (A) at baseline, (B) during radiotherapy (week 2). These plots illustrate the strong positive correlation between MRL-ADC and MRI3T-ADC at both time points. Radiol Oncol 2023; 57(2): 184-190. Almansour H et al. / ADC in patients with prostate cancer undergoing MR-guided radiotherapy 189 against a MRI3T at one point of time prior to start- ing RT. We were able to show a promising and comparable result of T2 weighted image quality, and lesion conspicuity and we reported compara- ble lesion ADC measurements between MRL and MRI3T.8 With the current work, we demonstrate longi- tudinal comparability and reliability between the two system during RT. This represents a neces- sary basis for future analyses of lesion changes over time on the MRL and confirms its potential for individualized treatment adaptations such as dose painting26 and response assessment during treatment. This study has limitations. Firstly, the small sample size of patients who underwent multi- ple prostate imaging at both devices and the fact that only one time-point during radiotherapy was used for analysis. Multiple time-points during the course of radiotherapy should be analyzed to further validate the stability and comparability of ADC measurements. However, logistic challenges hindered further validation with an MRI3T at more than one time point. Secondly, treatment regimens differed in this population (either 20 x 3 Gy oder 39 x 2 Gy, additional neoadjuvant ADT in 3 patients). Thirdly, the DWI acquisition parameters did not fully conform to the published recommendations of the MR-linac consortium, which were published after we had already included the patients in our study and predefinded the technical aspects of the utilized sequences.27 Nonetheless, this study depticts the reality and the challenges of clinical routine and its prelimi- nary findings could be considered novel. Indeed, further prospective studies examining mpMRI data under RT and correlating those with clinical endpoints are desirable to advance individualized radiation treatment. In conclusion, lesion ADC as measured on MRL increased significantly during radiotherapy and le- sion ADC measurements on both systems showed similar dynamics. These preliminary findings are promising but need large-scale validation. Once validated, lesion ADC on MRL might be used as a biomarker for real-time assessment of tumor re- sponse in patients with prostate cancer undergo- ing MR-guided radiation therapy. Acknowledgements This research project was partially financed through a research grant of the DFG (German Research Council, Grant MU 4603/1-1 | OT 534/3- 1, Package No. 997/1). Arndt-Christian Mueller is a recipient of the IIT program (Mbase Pro 1.0 study) of the Medical Faculty of Tuebingen University (AKF 345-1-0). Haidara Almansour is a scholarship recipient of the junior clinician scientist program of the medical faculty of Tuebingen University (no. 461-0-0). The datasets used and analyzed during the current study are available from the corre- sponding author on reasonable request. References 1. Mottet N, van den Bergh RCN, Briers E, van den Broeck T, Cumberbatch MG, de Santis M, et al. EAU-EANM-ESTRO-ESUR-SIOG Guidelines on Prostate Cancer-2020 Update. Part 1: screening, diagnosis, and local treatment with curative intent. Eur Urol 2021; 79: 243-62. doi: 10.1016/j. eururo.2020.09.042 2. Pathmanathan AU, McNair HA, Schmidt MA, Brand DH, Delacroix L, Eccles CL, et al. 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Radiother Oncol 2020; 153: 106-13. doi: 10.1016/j.radonc.2020.09.046 Radiol Oncol 2023; 57(2): 191-200. doi: 10.2478/raon-2022-0052 191 research article Awake craniotomy for operative treatment of brain gliomas – experience from University Medical Centre Ljubljana Tilen Zele1, Tomaz Velnar1, Blaz Koritnik2, Roman Bosnjak1, Jasmina Markovic-Bozic3 1 Department of Neurosurgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Department of Neurophysiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 3 Department of Anaesthesiology and Intensive Care, University Medical Centre Ljubljana, Department of Anesthesiology and Reanimation Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(2): 191-200. Received 09 10 2022 Accepted 02 11 2022 Correspondence to: Prof. assist. Jasmina Markovič-Božič, M.D., Ph.D.; Department of Anaesthesiology and Intensive Care, University Medical Centre Ljubljana, Zaloška cesta 2, SI-Ljubljana, Slovenia. E-mail: jasmina.markovic1@kclj.si. Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Awake craniotomy is a neurosurgical technique that allows neurophysiological testing with patient cooperation during the resection of brain tumour in regional anaesthesia. This allows identification of vital functional (i.e. eloquent) brain areas during surgery and avoidance of their injury. The aim of the study was to present clinical experience with awake craniotomy for the treatment of gliomas at the University Medical Centre Ljubljana from 2015 to 2019. Patients and methods. Awake craniotomy was considered in patients with a gliomas near or within the language brain areas, in all cases of insular lesions and selected patients with lesions near or within primary motor brain cortex. Each patient was assessed before and after surgery. Results. During the 5-year period, 24 awake craniotomies were performed (18 male and 6 female patients; average age 41). The patient’s cooperation, discomfort and perceived pain assessed during the awake craniotomy were in majority of the cases excellent, slight, and moderate, respectively. After surgery, mild neurological worsening was observed in 13% (3/24) of patients. Gross total resection, in cases of malignant gliomas, was feasible in 60% (6/10) and in cases of low-grade gliomas in 29% (4/14). The surgery did not have important negative impact on functional status or quality of life as assessed by Karnofsky score and Short-Form 36 health survey, respectively (p > 0.05). Conclusions. The results suggest that awake craniotomy for treatment of gliomas is feasible and safe neurosurgical technique. The proper selection of patients, preoperative preparation with planning, and cooperation of medical team members are necessary for best treatment outcome. Key words: awake craniotomy; surgery of gliomas; intraoperative neurophysiological testing; primary brain tumours; clinical experiences Introduction Gliomas are one of the most common primary brain tumours and represent 75% of all malignant primary brain tumours in adults.1 The primary treatment consists of surgical resection, followed by radiotherapy and chemotherapy. Despite the modern treatment, gliomas remain incurable le- sions. Glial tumours are classically divided into low- grade gliomas (LGG, WHO grade II) and malignant gliomas (WHO grades III and IV).1,2 LGGs are a het- erogeneous group, histologically classified into low- grade astrocytoma, oligodendroglioma or mixed oligoastrocytoma. The average age at presentation is 35 years, and they typically occur in the frontal lobes. In 50 to 80% of patients, the first symptom is an epileptic seizure.2 On T1-weighted magnetic Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours192 resonance images (MRI), LGGs appear as hypo- to isointense lesions, whereas on T2-weighted im- ages they are hyperintensive.2 Although LGGs are more indolent than their high-grade counterparts, they inevitably progress towards malignancy. This malignant transformation is present in 13 to 86% of cases in LGG recurrences.2,3 With treatment, the median overall survival time is about six to eight years.3,4 On the other hand, malignant gliomas are more sinister. They are histologically classified into anaplastic astrocytomas (AA, WHO grade III) and glioblastomas (GBM, WHO grade IV).1 For AAs, the average age at presentation is 40, and for GBMs 53 years. They typically present with progressive headaches and neurological deficits. On MRI high grade gliomas usually appear as contrast enhanc- ing lesions with central necrosis and surrounding brain oedema. They have a predilection for cerebral hemispheres. With treatment, the median overall survival time for AAs is two to five years and for GBMs less than two years.5,6 For patients with gliomas, overall survival was demonstrated to be related to the histological and molecular subtype of the tumour, the patient’s age, the presence of neurological deficit, the Karnofsky Performance Status (KPS) score, and the size of the tumour at presentation.7,8 In addition, several stud- ies have also clearly demonstrated that the extent of resection (EOR) during surgery has a significant impact on survival.9,10 Namely, in cases of LGGs the resection of more than 90% and less than 90% of the tumour resulted in an 8-year overall surviv- al rate of 91% and 60%, respectively.9 Similarly, in cases of AAs, gross total resection (GTR) and sub- total resection (STR) resulted in a median survival of 58 and 34 months, respectively.10 And in cases of GBM, GTR and STR resulted in a median survival of 13 and 8 months, respectively.10 Gliomas are infiltrative lesions and can arise within or near the functionally the most impor- tant brain regions, such as language areas, motor cortex, etc. Over extensive resection in those vital (i.e. eloquent) areas, that directly control function, would inevitably result in permanent neurologi- cal deficit and significant postoperative morbid- ity, with a negative impact on the patient’s quality of life. In addition, such significant postoperative neurological worsening also independently re- duces the overall survival of those patients.7,8,10,11 Therefore, the goal of the surgical treatment of gliomas is, in addition to maximal, also safe resec- tion - i.e. removal of as much tumour as possible, without causing neurological deficits (“maximal safe resection”). To achieve the goal of maximal safe resection, the intraoperative neurophysiological testing and monitoring is mandatory to identify the precise location of individual brain functions and thus eloquent brain areas during surgery. In cases in which neurophysiological techniques cannot ad- equately assess brain functions under general anaesthesia and the cooperation of the patient during intraoperative testing is needed an awake craniotomy should be used.12,13 Awake craniotomy is a neurosurgical technique that allows removal of brain tumour under regional anaesthesia while the patient is awake. It thus allows cooperation of the patient during intraoperative testing and monitoring in order to avoid the injury of elo- quent brain areas. For awake craniotomy a variety of anaesthetic methods are used: local anaesthe- sia (scalp block, infiltration of dura), monitored anaesthesia care (awake-awake-awake technique; scalp block and sedation) or asleep-awake-asleep technique (general anaesthesia and awakening during testing). In the present article, we presented our pro- tocol, technique and experience with awake cra- niotomy for glioma surgery at the Department of Neurosurgery, Ljubljana University Medical Centre, from years 2015 to 2019. Patients and methods Patients We included all consecutive patients who under- went surgery by awake craniotomy due to glio- mas from years 2015 to 2019 at the Department of Neurosurgery of Ljubljana University Medical Centre. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia. All the procedures were performed in ac- cordance with the Declaration of Helsinki. Awake craniotomy was considered in selected patients with the following preoperative morphological images: I) a tumour near or within the language brain areas (Broca’s and Wernicke’s areas, angular gyrus), II) all cases of insular tumours and III) se- lected patients with lesions near or within primary motor cortex or corticospinal tract. Each patient was assessed preoperatively by the anaesthesiologist, neuropsychologist, and neurosurgeon. A collective decision was made whether the patient was suitable for awake crani- otomy. The inclusion criteria encompassed a good clinical, physical, and affective condition. The contraindications for awake craniotomy were a Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours 193 non-compliant patient (e.g., due to old age or un- favourable psychosocial factors), the potential for breathing problems during surgery (e.g., known sleep apnoea, significant obesity), and important preoperative dysphasia (i.e., the patient names less than 80% of objects presented at four- second in- tervals). Informed consent was obtained from all study participants. Preoperative planning and evaluation The preoperative planning consisted of the acqui- sition and analysis of preoperative morphologi- cal and functional images, the selection of sur- gical approach and trajectory to the lesion, and the planning of the extent of the resection. MRI was performed using a 3-Tesla clinical scanner (Siemens Trio, Siemens). The diagnostic imag- ing included T1- and T2-weighted sequences. For 3D-imaging, we used a post-contrast T1-weighted 3D-fast spoiled gradient recalled (FSPGR) se- quence (a series of 124 images, 1.4 mm thick with a matrix up to 512x512 of 240mm field of view). A fMRI was used to define the primary motor and speech areas in all patients. We used single shot echo planar imaging in a transverse plane (TR 3000/TE 40, a series of 43 images, 3 mm thick with a 64x64 matrix with 200 mm field of view) during which the patient performed a motor task (a self- paced sequential tapping of the thumb against each finger) or a speech task (verbal fluency and verb generation). A general model-based statisti- cal analysis (SPM 12) was used to measure the ex- tent of cortical activation. To plan the surgical procedure, we used comput- er assisted 3D-visualization by neuronavigational software (Stealth Station S7 Surgical Navigation System, Medtronic) and 3D-Slicer software (http:// www.slicer.org).14 The 3D-visualization of the medical images allowed us to perform preopera- tive 3D-planning, i.e., to interactively present rel- evant anatomic structures as 3D-objects in virtual space on the computer screen, to define surgical targets, to perform quantitative measurements, and finally to select the most suitable surgical approach or trajectory to the lesion.15 Interactive 3D-preoperative planning started with defining the tumour model characteristics (size, volume, ex- tension) and their relationship to the models of the cortical surface and functional data. Based on the available data, we planned the position and size of the trepanation opening, the sites of intraoperative electrophysiological testing, and the preliminary EOR. Anaesthetic method We used awake-awake-awake technique of awake craniotomy - i.e. procedural sedation and analge- sia with dexmedetomidine in combination with scalp block. Initially deksmedetomidine infusion (200 µg in 50 ml of 0.9% NaCl; rate 0.2–1.5 4 µg/ kg/h) was started to sedate the patient followed by scalp block. Scalp block was performed by local in- filtration of the scalp nerves (i.e. n. supratrochlear- is, n. supraorbitalis, n. auriculotemporalis, n. zygo- maticotemporalis, n. occipitalis minor and major, n. auricularis major) to enabled effective analgesia for skin incision and craniotomy.16 For local infil- tration of the nerves we used 1–3 ml of the mixture of levobupivacaine (Chirocaine 5 mg/ml solution, Abbvie Pharmacy) and xylocaine with adrenaline (Xylanaest 2% with epinephrin 1: 200000 solution, Kemofarmacija d.d.). Adrenaline causes vasocon- striction and thereby delays absorption of local an- aesthetics, increases duration of anaesthesia and prevents local bleeding after skin incision. In addition, we introduced therapeutic commu- nication with medical hypnosis leaving patients less sedated, more competent during the entire surgical procedure without stress.17 Hypnosis ses- sion was usually carried out 1 to 3 days before sur- gery, to gain patients approval and confidence and to teach the patient how to construct an imaginary place where they can feel safe and protected. Anaesthesia was conducted by two senior an- aesthesiologists. The standard intraoperative mon- itoring was used. All patients had dedicated intra- venous and arterial lines, a Foley catheter, and a nasal oxygen catheter. Hypothermia was prevent- ed with use of warming blankets. Cefazolin 2g was used intravenously for perioperative antibiotic prophylaxis. The patients lay supine with their head rotated approximately 45 degrees to the side. They were encouraged to find the most comfortable posi- tion and were generously padded as necessary. The head was fixed in a Mayfield clamp under a scalp block combined with dexmedetomidine. Additional local anaesthesia was applied along the skin incision line and between the dural sheets before the skin and dura incision. Sedation during the procedure was maintained with dexmedeto- midine, and painful phases were treated with bo- luses of remifentanil. Bispectral Index Scale (BIS) was used for sedation monitoring. After the surgi- cal procedure, all patients were transferred to the intensive care unit for overnight observation. Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours194 The operative procedure and neurophysiological testing Tumour removal was performed in accordance with classical microsurgical techniques. In all cases, microsurgical microscope was employed. During the surgical procedure, frameless neu- ronavigation, intraoperative neurophysiological testing and augmented malignant tumour visu- alization with 5-aminolevulinic acid (5-ALA) were used. For precise intraoperative localization of the tumour, we used neuronavigation with a Stealth Station S7 (Medtronic) navigation station, thus defining the size and position of the trepanation opening and the morphological tumour bounda- ries on the cortical surface. During tumour re- moval, neuronavigation was used for approximate depth orientation, and as a help to assess the EOR intraoperatively. Injury to the eloquent regions was avoided af- ter they had been identified by intraoperative neu- rophysiological testing. For cortical stimulation, a bipolar electrode was used with biphasic electrical current (50 Hz, 0.5 ms, 4 to 10 mA). For subcortical simulation, a monopolar electrode on the tip of as- pirator was used, with a train of five pulses in row (a train of five pulses at 200 Hz, 0.5 ms, from 2 to maximum 20 mA). The patient’s speech was moni- tored by an examiner, who talked to the patient and detected potential speech difficulties. During the stimulation of the cortical surface, the patient named objects on pictures or repeated words after the examiner. When stimulating the speech area, the speech changed or ceased. The stimulation of the primary motor area on the cortical surface re- sulted in motor evoked potentials (MEPs) detected in the corresponding muscles of the patient’s body, and discomfort and/or movements, as reported by the patient. Subcortical stimulation was performed to define the distance from the corticospinal tract in patients where the tumour was in its vicinity. During tumour removal, the subcortical white mat- ter was continuously stimulated with a monopolar electrode, and MEPs were monitored. At the begin- ning of the resection, the stimulation current was set at 20 mA. When MEPs were detected during the progression of the resection, the stimulation current was gradually reduced. When the MEP re- sponse was already induced by a 3–5 mA current, the resection in that direction was stopped, due to the proximity of the corticospinal tract.18 In cases where the preoperative diagnostic im- ages showed the characteristics of malignant tu- mours, 5-ALA was used. The fluorescence of the tumour tissue was observed under the operating microscope (OPMI Pentero 900 Microscope, Carl Zeiss). Assessment The demographic data of the patients were col- lected before surgery. According to the American Society of Anaesthesiologists, their physical status classification system ASA score was noted and KPS was assessed in all patients. In 12 patients, health- related quality of life (HRQoL) was assessed by 36-Item Short Form (SF-36) health survey.19,20 During the surgery, we measured duration of anaesthesia, duration of the operative procedure and duration of the neurophysiological testing. Patient cooperation during surgery was assessed by a neurosurgeon and neurophysiologist and graded on scale from 0 to 10 (modified 11-point numerical rating scale, where 0 represented poor cooperation and 10 excellent cooperation). Patient comfort and pain were assessed by the anaesthesi- ologist. Pain was assessed by visual analogue scale (VAS) from 0–10 (0 meaning no pain, 10 meaning intolerable pain). Comfort was assessed by modi- fied VAS scale from 0–10 (0 being the least and 10 being the most comfortable). Intraoperative com- plications were recorded. After surgery, we monitored postoperative neu- rological status and noted non-neurological com- plications. The level of effort needed for the whole procedure, as reported by the patients, was as- sessed by anaesthesiologist 2–5 days after surgery, by modified VAS scale from 0–10 (0 being the least and 10 being the most difficult). KPS was assessed 1 week after surgery and the patients were asked to describe what was the worst experience during the awake craniotomy. A contrast enhanced brain MRI scan was performed the day following the surgery to assess EOR in all patients. In cases of malignant gliomas (AA, GBM) EOR was assessed on T1 weighted contrast enhanced MRI images. In those cases, gross total resection (GTR) was de- fined as no residual enhancement on postoperative images, near total resection (NTR) was defined as rim enhancement of the resection cavity, and subtotal resection (STR) was defined as residual nodular enhancement. In cases of LGG, EOR was assessed on T2 and FLAIR weighted postoperative MRI images. In those cases, GTR was defined as no hyperintense signal changes on postoperative images, STR was defined as persistence of hyper- intense signal around resection cavity, and biopsy Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours 195 resection (BR) when approximately more than 50% of hyperintense signal remained on postoperative images. Length of hospital stay (LOS) was noted. Three months after the surgery, the KPS score was assessed in all patients and the HRQoL was assessed by SF-36 health survey in the same 12 pa- tients as before surgery. All patients were asked, if they would be willing to undergo awake crani- otomy again if necessary and their answers were noted. Statistical analysis The means or ranges of continuous variables are presented, and categorical data are summarized as counts. The differences between the KPS scores before, after and 3 months after surgery were eval- uated using One Way ANOVA. The differences between the SF-36 health survey subscales before and 3 months after surgery were evaluated us- ing Wilcoxon test. A p-value of less than 0.05 was considered statistically significant. Data were ana- TABLE 1. Demographics and preoperative data No. of patients 24 Age (years) 41 ± 11 Weight (kg) 81 ± 12 Height (cm) 176 ± 8 Gender (M/F) 18/6 ASA (I/II/III) 9/15/0 First operation/reoperation 22/2 Tumour size (cm3) 46 ± 27 Tumour location (side): Insular (left/right) 4/3 Frontal Central-PMC (left/right) 2/2 Frontal- Broca area (left/right) 6/1 Temporo-frontal (left/right) 3/0 Temporal-Wernicke area (left/right) 3/0 The results are expressed as mean ± SD or number of patients. ASA = American Society of Anaesthesiologist; F = female; M = male; PMC = primary motor cortex A2 B2 A3 B3 A4 B4 A5 B5 A1 B1 FIGURE 1. Preoperative magnetic resonance images (MRI) of low-grade glioma located in primary motor cortex (A) and anaplastic astrocytoma located in anterior speech area (Broca area) (B). Preoperative imaging included T1 weighted contrast enhanced (A1, B1), T2 weighted (A2, B2), FLAIR (A3, B3) and functional magnetic resonance (fMRI); (A4, A5, B4, B5) images. The fMRI demonstrated the hand activation area (yellow/red colour) lateral (A4) and leg activation area behind (A5) the lesion in primary motor cortex. In second case fMRI demonstrated speech activation areas posterior to the lesion on the left side (B4, B5). Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours196 we performed awake craniotomies in 24 patients with glial brain tumours. They were 22 to 60 years old. In all cases, the main presenting symptom was epileptic seizure. In addition, in two patients, a mild dysphasia, and in one a disorientation was present. The main comorbidities in patients were well controlled diabetes and arterial hypertension. Four more patients were considered for awake cra- niotomy but were later found to be unsuitable and underwent surgery under general anaesthesia, one due to anxiety, one due to morbid obesity, one due to asthma and one due to psychosis. The preoperative fMRI was successful in all pa- tients and revealed speech dominance on the left side in all but one case in which it was bilateral. In 4 cases with tumour in frontal-central region (i.e., near or within primary motor region) the fMRI was used also to define primary motor regions (Figure 1). Intraoperative and early postoperative results Intraoperative and early postoperative outcomes are presented in Table 2. The longest surgical pro- cedure lasted 5 hours and 20 minutes. Patient co- operation during surgery was in majority of the cases excellent. Discomfort during surgery report- ed by the patients was generally described as just a little uneasy and/or just starting to bother. Pain perceived during surgery was regarded as moder- ate. In two patients, however, we observed signifi- cant fatigue during surgery that started about 2.5 hours after the start of the procedure, and it was so intense that further neurophysiologic testing was not feasible. During cortical stimulation, we ob- served epileptic seizure activity in 8 out of 24 pa- tients (33% of the patients). In one of these patients, further testing was not possible, due to limited compliance after the seizure. Neurophysiological testing by cortical stimulation was therefore suc- cessful in identifying the cortical language ar- eas or primary motor cortex in all but three cases. Subcortical stimulation was performed in cases with insular and frontocentral located tumours. The corticospinal tract upon stimulation with a 20 mA current or less was detected in 8 out of 11 tested patients (72% of the patients). After the surgery, the neurological worsening was observed in 3 out of 24 patients (13% of the pa- tients) and the other postoperative complications were present in 6 out of 24 patients (25% of the patients) (Table 2). Postoperative pain was mild in almost all cases. Level of the effort during surgery TABLE 2. Intraoperative and early postoperative outcomes INTRAOPERATIVE DATA Duration of anaesthesia (minutes) 278 ± 47 Duration of procedure (minutes) 215 ± 48 Duration of testing (minutes) 73 ± 26 Comfort score (0- least; 10- most) 8 ± 2 (5–10) Pain score (0- no pain; 10- intolerable) 4 ± 2 (0–5) Cooperation score (0- poor; 10- excellent) 10 ± 1 (9–10) Complications (none/seizure/incomplete testing) 13/8/3 EARLY POSTOPERATIVE DATA Pain score after procedure (0- no pain; 10- intolerable) 2 ± 1 (0–3) Pain score ICU (0- no pain; 10- intolerable) 2 ± 1 (0–3) Level of effort (0- least; 10- most difficult) 3 ± 2 (0–6) Non-neurological complications Wound infection 1 Pulmonary embolism 1 Nausea 2 Pain 2 Neurological complications Walking disability 1 POCD and dysphasia 1 Mild hemiparesis, POCD, seizures 1 Tumour histology LGG 14 AA 7 GBM 3 Extent of resection LGG (GTR/STR/BR) 4/7/3 AA or GBM (GTR/NTR/STR) 6/3/1 Length of stay (days) 6 ± 5 The results are expressed as mean ± SD (range in brackets) or number of patients. AA = anaplastic astrocytoma; BR = biopsy resection; GBM = glioblastoma; GTR = gross total resection; ICU = intensive care unit; LGG = low grade glioma; NTR = near total resection; POCD = postoperative cognitive decline; STR = subtotal resection lysed by SPSS 13.0 software package (IBM Corp., Armonk, NY, USA). Results Patients and preoperative data The demographic and preoperative data are showed in Table 1. During the 5-year study period, Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours 197 experienced by the patients was assessed and re- ported to be mild to moderate. When asked “what was the worst experience during the awake crani- otomy”, 15 patients said “nothing”, four patients said, “bone cutting”, two patients said, “Mayfield clamp placement”, and the remaining three pa- tients said, “scalp block”, “craniofix bone flap fixa- tion” or “waiting for the end of surgery”. GTR was achieved in 29% (4 out of 14 cases) in cases of LGG, and in 60% (6 out of 10 cases) in cases of GBM and AA (Figure 2). All patents were discharged home. The time of postoperative hospital stay was from 3 to 9 days for most of the patients, and 23 days for one patient. Late postoperative results Three months after surgery all patients were un- der postoperative oncological treatment with ra- diotherapy and/or chemotherapy. All patients were still on a sick leave, and none yet returned to work. The average KPS scores after surgery and 3 months after surgery were slightly lower and are shown in Figure 3A. However, the differences be- tween the KPS scores before, after and 3 months after surgery were not significantly different (p = 0.14). Patient HRQoL was assessed by SF-36 health survey before surgery and 3 months after surgery, the results for each SF-36 subscale are shown in Figure 3B. We found some increase in patient per- ceived problems with work or other daily activities because of physical health (i.e., “role limitations due to physical problems” SF-36 subscale) three months after surgery in comparison to before sur- gery, however, the difference was not statistically significant (p = 0.07). The scores in other SF-36 sub- scales (i.e., physical functioning, bodily pain, gen- eral health, vitality, social functioning, role limi- tations due to emotional problems, mental health) were very similar before and after surgery, and A2 B2 A1 B1 A B FIGURE 2. Preoperative (A) and postoperative (B) T1 weighted contrast enhanced (A1, B1) and T2 weighted (A2, B2) magnetic resonance images (MRI) of glioblastoma located in supramarginal gyrus near Wernicke area. Preoperative images (A) demonstrate rim enhancing central necrotic oval lesion surrounded by oedema. Postoperative images (B) demonstrate post-resection cavity filled with partially haemorrhagic fluid and mild irregular contrast enhancing of resection edge – i.e. near total resection. FIGURE 3. The Karnowski Performance Scale (KPS) scores (A) and 36-Item Short Form (SF-36) health survey subscale scores (B) before surgery, after surgery and 3 months after surgery. The differences between the KPS scores and SF-36 subscale scores before and after surgery were not statistically significantly different (p > 0.05; n = 24 for KPS; n = 12 for SF-36). BP = bodily pain; GH = general health; PF = physical function; MH = mental health; RE = role limitations due to emotional problems; RP – role limitations due to physical problems; SF = social functioning; VT = vitality; 3M = three months Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours198 not significantly different (p > 0.1). Therefore, the surgery did not have important negative impact on functional status or quality of life as assessed by KPS and SF-36 health survey, respectively. Three months after surgery, the patients were asked, “if they would be willing to undergo awake craniot- omy again if needed”. One patient answered “no”, one answered, “only if absolutely necessary” and the other 22 patients answered “yes”. Discussion At our institution, awake craniotomy for surgical treatment of gliomas was performed in 24 patients in the last 5 years. Our experiences with awake craniotomy are good and suggest that awake cra- niotomy can be safely performed with low risk of complications or failure rate. The goal of an awake craniotomy is to provide the surgeon with optimal functional monitoring during the removal of as much of the tumour as possible and thus avoiding damage to critical brain structures. Awake craniotomy is mandatory in pa- tients where neurophysiological testing and moni- toring cannot adequately assess function under general anaesthesia, and when the cooperation of the patient during testing is needed. The functions typically monitored during awake craniotomy are mainly higher mental functions, especially speech. Such monitoring allows safer tumour removal, re- sulting in improved treatment outcome, even for tumours in eloquent brain regions.12,13,18,21,22 On the other hand, awake craniotomy is a tech- nically demanding procedure, with more potential intraoperative complications in comparison to the same procedure performed under general anaes- thesia.21 Namely, patient intolerance or non-coop- eration can result in too early termination of the surgery, and incomplete or suboptimal resection.21 Therefore, it is very important to properly select patients for the procedure. In our series, all the patients performed well, and the selection proto- col for the patients was shown to be good. During preoperative evaluation, four potential candidates were found to be unsuitable for awake craniotomy. In addition to obesity, potential breathing prob- lems and dysphasia, also patient immaturity, hy- pertension and alcohol abuse are also some of the risk factors for sedation failure, which may worsen the surgery and postoperative recovery.23 Anxiety and other psychological problems may also nega- tively influence the patient’s cooperation.24 The selection of patients should therefore be individu- alized, and the decision-making process should involve all team members. A variety of anaesthetic methods are used for awake intracranial procedures. In our institution, we combine scalp block and procedural sedation and analgesia with dexmedetomidine – i.e. awake- awake-awake protocol. This technique proved to be very safe in experienced hands, allows more control with the patient, the anaesthesiologist is by the patients side all the time and can react prompt- ly if something goes wrong, and it usually results in better patient cooperation.25,26 All patients are well-educated regarding all the steps of the surgi- cal procedure to facilitate the intraoperative test- ing and to decrease their anxiety. In our, and also other studies, the most painful and unpleasant part of the procedure, as reported by the patients, is the placement of the cranial fixation device and trepanation.27,28 It is important to note that because of inadequate analgesia during cranial fixation, or later due to positioning, the surgical outcome may be compromised.29 The patient’s communi- cation with the surgical team and addressing the patient’s discomfort are therefore crucial. In the case of the awake-awake-awake protocol, as used in our cases, the duration of the surgery proved important. The cooperation of two of our patients became inadequate after approximately 2.5 hours from the beginning of the surgery. To overcome this problem, we recommend early functional test- ing and analysis of the functions for which patent’s cooperation is needed. In addition, we recommend good preoperative planning and preparation to re- duce operation time. In our study, the patient’s perception of awake craniotomy was generally good. Hypnosis was well accepted in our study. Also, it was previously shown that it prolongs the time of patient coopera- tion during the neurophysiological testing.30 The main challenges for patients undergoing awake craniotomies include anxiety and fears, terrify- ing noises and surroundings, immobility, loss of control, the feeling of helplessness and being left alone. In such situations, psychological support might be very helpful and motivates patients to have a sense of control by active participation dur- ing surgery instead of being lost in anxiety.31,32 The rate of occurrence of epileptic seizures during surgery in our study of 33% was higher compared to other reports, which reported a sei- zure rate of from 15 to 16.7%.21,28 At the beginning of the operation, cortical stimulation, especially when used in excess during neuromonitoring, may sometimes provoke epileptic seizures.21,28 Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours 199 Reported treatment was the iced Ringer solution, which was also successfully used in our patients to stop the seizure. We believe that the higher rate in our series could be attributable to more extensive testing in selected patients. In this regard, it was observed that less stimulation is generally used with increased experience.28 The neurological worsening observed postop- eratively in 13% of our patients is comparable to the meta-analysis data, which report rates of about 30% for early and 7% for late deficits of all severi- ties after intraoperative stimulation mapping.33 GTR in our study was higher in cases of GBM or AA in comparison to LGG (29% vs. 60%). The com- parable GTR rates are also reported in the litera- ture for awake craniotomy glioma surgery.34,35 In this regard, it is important to note, that although 5-ALA and neuronavigation help to delineate the borders of the tumour tissue, the final edge of the resection is defined by the functional borders, as defined by intraoperative neurophysiological test- ing. Consequently, in several cases of awake cra- niotomy total resection of the glioma cannot be achieved due to tumour infiltration of eloquent regions. Based on intraoperative neurophysiologi- cal testing, we believe that further resection in our patients could result in significant postoperative functional deficit. One of the goals of surgical treatment of gliomas is, in addition to maximal resection, preservation of the neurocognitive profile and quality of life.19,20 Namely, gliomas are currently surgically incurable lesions and potential postoperative neurological deficits further reduce survival time and quality of life of those patients. In our study, the HRQoL assessed three months after surgery was not sig- nificantly impaired. We did however observe no- ticeable patient reported problems with work and daily activities three months after surgery. The later could be related to ongoing postoperative on- cological treatment. Also, none of the patients at 3 months after the surgery was yet allowed to return to work, which probably also influenced their per- ception and the results. To conclude, our experience suggests that awake craniotomy using our protocol is a feasible and safe surgical procedure. To achieve the best treatment outcome, proper selection of the patients, preoper- ative preparation with planning, and cooperation of the medical team are necessary. Acknowledgments We are thankful to all colleagues, surgeons, anaesthesiologists, nurses and technicians at the Department of Neurosurgery, Department of Anaesthesiology and Surgical Therapy and Department of Neurophysiology who in any way helped in this work. We are also thankful to all the patients who cooperated in the study. References 1. Lapointe S, Perry A, Butowski NA. Primary brain tumours in adults. Lancet 2018; 392: 432-46. doi: 10.1016/S0140-6736(18)30990-5 2. McCormack BM, Miller DC, Budzilovich GN, Voorhees GJ, Ransohoff J. Treatment and survival of low-grade astrocytoma in adults 1977-1988. Neurosurgery 1992; 31: 636-42. doi: 10.1227/00006123-199210000-00004 3. Bauman G, Lote K, Larson D, Stalpers L, Leighton C, Fisher B, et al. Pretreatment factors predict overall survival for patients with low-grade glioma: recursive partitioning analysis. Int J Radiat Oncol Biol Phys 1999; 45: 923-9. doi: 10.1016/s0360-3016(99)00284-9 4. Johannesen TB, Langmark F, Lote K. Progress in long-term survival in adult patients with supratentorial low-grade gliomas: a population-based study of 993 patients in whom tumours were diagnosed between 1970 and 1993. J Neurosurg 2003; 99: 854-62. doi: 10.3171/jns.2003.99.5.0854 5. 5.DeAngelis LM. Brain tumours. N Engl J Med 2001; 344: 114-23. doi: 10.1056/NEJM200101113440207 6. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008; 359: 492- 507. doi: 10.1056/NEJMra0708126 7. Berger MS, Deliganis AV, Dobbins J, Keles GE. The effect of extent of resec- tion on recurrence in patients with low grade cerebral hemisphere gliomas. Cancer 1994; 74: 1784-91. doi: 10.1016/s0090-3019(99)00103-2 8. Chang EF, Clark A, Smith JS, Polley MY, Chang SM, Barbaro NM, et al. Functional mapping-guided resection of low-grade gliomas in eloquent areas of the brain: improvement of long-term survival. J Neurosurg 2011; 114: 566-73. doi: 10.3171/2010.6.JNS091246 9. Smith JS, Chang EF, Lamborn KR, Chang SM, Prados MD, Cha S, et al. Role of extent of resection in the long-term outcome of low-grade hemispheric gliomas. J Clin Oncol 2008; 26: 1338-45. doi: 10.1200/JCO.2007.13.9337 10. McGirt MJ, Chaichana KL, Gathinji M, Attenello FJ, Than K, Olivi A, et al. Independent association of extent of resection with survival in patients with malignant brain astrocytoma. J Neurosurg 2009; 110: 156-62. doi: 10.3171/2008.4.17536 11. Liang J, Lv X, Lu C, Ye X, Chen X, Fu J, et al. Prognostic factors of patients with gliomas - An analysis on 335 patients with glioblastoma and other forms of gliomas. BMC Cancer 2020; 20: 35. doi: 10.1186/s12885-019-6511-6 12. Sanai N, Berger MS. Intraoperative stimulation techniques for functional pathway preservation and glioma resection. Neurosurg Focus 2010; 28: E1. doi: 10.3171/2009.12.FOCUS09266 13. Hervey-Jumper SL, Li J, Lau D, Molinaro AM, Perry DW, Meng L, Berger MS. Awake craniotomy to maximize glioma resection: methods and techni- cal nuances over a 27-year period. J Neurosurg 2015; 123: 325-39. doi: 10.3171/2014.10.JNS141520 14. Fedorov A, Beichel R, Kalpathy-Cramer J, Finet J, Fillion-Robin JC, Pujol S, et al. 3D Slicer as an image computing platform for the quantitative imaging network. Magn Reson Imaging 2012; 30: 1323-41. doi: 10.1016/j. mri.2012.05.001 15. Žele T, Matos B, Knific J, Bajrović FF, Prestor B. Use of 3D visualisation of medical images for planning and intraoperative localisation of superficial brain tumours: our experience. Br J Neurosurg 2010; 24: 555-60. doi:10.31 09/02688697.2010.496876 Radiol Oncol 2023; 57(2): 191-200. Zele T et al. / Awake craniotomy for glial tumours200 16. Osborn I, Sebeo J. “Scalp block” during craniotomy: a classic technique revisited. J Neurosurg Anesthesiol 2010; 22: 187-94. doi: 10.1097/ ANA.0b013e3181d48846 17. Frati A, Pesce A, Palmieri M, Iasanzaniro M, Familiari P, Angelini A, Salvati M, Rocco M, Raco A. Hypnosis-aided awake surgery for the management of intrinsic brain tumors versus standard awake-asleep-awake protocol: a preliminary, promising experience. World Neurosurg 2019; 121: e882-e891. doi: 10.1016/j.wneu.2018.10.004 18. Raabe A, Beck J, Schucht P, Seidel K. Continuous dynamic mapping of the cortico- spinal tract during surgery of motor eloquent brain tumours: evaluation of a new method. J Neurosurg 2014; 120: 1015-24. doi: 10.3171/2014.1.JNS13909 19. Bunevicius A. Reliability and validity of the SF-36 Health survey question- naire in patients with brain tumours: a cross-sectional study. Halth Qual Life Outcomes 2017; 15: 92. doi: 10.1186/s12955-017-0665-1 20. Ware JE, Jr. Cathy Donald Sherbourne CD. The MOS 36-item short-form health survey (SF-36): I. Conceptual framework and item selection. Medical Care 1992; 30: 473-83. PMID: 1593914 21. Trimble G, McStravick C, Farling P, Megaw K, McKinstry S, Smyth G, et al. Awake craniotomy for glioma resection: technical aspects and initial results in a single institution. Br J Neurosurg 2015; 29: 836-42. doi: 10.3109/02688697.2015.1054354 22. Saito T, Muragaki Y, Tamura M, Maruyama T, Nitta M, Tsuzuki S, et al. Awake craniotomy with transcortical motor evoked potential monitoring for resec- tion of gliomas in the precentral gyrus: utility for predicting motor function. J Neurosurg 2019; 132: 987-97. doi: 10.3171/2018.11.JNS182609 23. Senel FC, Buchanan JM Jr, Senel AC, Obeid G. Evaluation of sedation failure in the outpatient oral and maxillofacial surgery clinic. J Oral Maxillofac Surg 2007; 65: 645-50. doi: 10.1016/j.joms.2006.06.252 24. Palese A, Skrap M, Fachin M, Visioli S, Zannini L. The experience of patients undergoing awake craniotomy: in the patients’ own words. A qualitative study. Cancer Nurs 2008; 31: 166-72. doi: 10.1097/01. NCC.0000305699.97625.dc 25. Kulikov A, Lubnin A. Anesthesia for awake craniotomy. Curr Opin Anaesthesiol 2018; 31: 506-10. doi: 10.1097/ACO.0000000000000625 26. Suero Molina E, Schipmann S, Mueller I, Wölfer J, Ewelt C, Maas M, et al. Conscious sedation with dexmedetomidine compared with asleep- awake-asleep craniotomies in glioma surgery: an analysis of 180 patients. J Neurosurg 2018; 129: 1223-30. doi: 10.3171/2017.7.JNS171312. PMID: 29328000 27. Khu KJ, Doglietto F, Radovanovic I, Taleb F, Mendelsohn D, Zadeh G, et al. Patients’ perceptions of awake and outpatient craniotomy for brain tumour: a qualitative study. J Neurosurg 2010; 112: 1056-60. doi: 10.3171/2009.6.JNS09716 28. Joswig H, Bratelj D, Brunner T, Jacomet A, Hildebrandt G, Surbeck W. Awake craniotomy: first-year experiences and patient perception. World Neurosurg 2016; 90: 588-96. doi: 10.1016/j.wneu.2016.02.051 29. Whittle IR, Midgley S, Georges H, Pringle AM, Taylor R. Patient perceptions of “awake” brain tumour surgery. Acta Neurochir 2005; 147: 275-7. doi: 10.1007/s00701-004-0445-7 30. Pesce A, Palmieri M, Cofano F, Iasanzaniro M, Angelini A, D’Andrea G, et al. Standard awake surgery versus hypnosis aided awake surgery for the man- agement of high grade gliomas: A non-randomized cohort comparison-con- trolled trial. Clinneurosci 2020; 77: 41-8. doi: 10.1016/j.jocn.2020.05.047 31. Hansen E, Seemann M, Zech N, Doenitz C, Luerding R, Brawanski A. Awake craniotomies without any sedation: the awake-awake-awake technique. Acta Neurochir 2013; 155: 1417-24. doi: 10.1007/s00701-013-1801-2 32. Zemmoura I, Fournier E, El-Hage W, Jolly V, Destrieux C and Velut S. Hypnosis for awake surgery of low-grade gliomas: description of the method and psychological assessment. Neurosurgery 2016; 78: 53-61. doi: 10.1227/NEU.0000000000000993 33. De Witt Hamer PC, Robles SG, Zwinderman AH, Duffau H, Berger MS. Impact of intraoperative stimulation brain mapping on glioma surgery outcome: a meta-analysis. J Clin Oncol 2012; 30: 2559-65. doi: 10.1200/ JCO.2011.38.4818 34. Ahmadi R, Dictus C, Hartmann C, Zürn O, Edler L, Hartmann M, et al. Long- term outcome, and survival of surgically treated supratentorial low-grade glioma in adult patients. Acta Neurochir 2009; 151: 1359-65. doi: 10.1007/ s00701-009-0435-x 35. Motomura K, Chalise L, Ohka F, Aoki K, Tanahashi K, Hirano M, et al. Impact of the extent of resection on the survival of patients with grade II and III gliomas using awake brain mapping. J Neurooncol 2021; 153: 361-72.doi: 10.1007/s11060-021-03776-w Radiol Oncol 2023; 57(2): 201-210. doi: 10.2478/raon-2023-0009 201 research article Cognitive functioning in a cohort of high-grade glioma patients Andreja Cirila Skufca Smrdel1,2, Anja Podlesek2, Marija Skoblar Vidmar3,4, Jana Markovic1, Jana Jereb1, Manja Kuzma Okorn5, Uros Smrdel3,4 1 Department of Psycho-Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Psychology, Faculty of Arts, University of Ljubljana, Slovenia 3 Division of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Faculty of Medicine, University of Ljubljana, Slovenia 5 University Medical Centre Ljubljana, Slovenia Radiol Oncol 2023; 57(2): 201-210. Received 29 November 2022 Accepted 31 December 2022 Correspondence to: Assist. Prof. Uroš Smrdel, M.D., Ph.D., Division of Radiotherapy, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: usmrdel@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY li-cense (https://creativecommons.org/licenses/by/4.0/). Background. High grade gliomas are associated with cognitive problems. The aim of the study was to investigate cognitive functioning in a cohort of patients with high grade glioma, according to isocitrate dehydrogenase (IDH) and methyl guanine methyl transferase (MGMT) status and other clinical characteristics. Patients and methods. The patients with the high-grade glioma treated in Slovenia in given period of time were included in study. Postoperatively they completed neuropsychological assessment consisting of Slovenian Verbal Learning Test, Slovenian Controlled Oral Word Association Test, Trail Making Test Part A and B and self-evaluation questionnaire. We analysed results (z-scores and dichotomized results) also according to IDH mutation and MGMT methylation. We examined differences between groups using T-test, Mann-Whitney U, χ2 and Kendall’s Tau tests. Results. Out of 275 patients in the cohort, we included 90. Forty-six percent of patients were unable to participate due to poor performance status and other conditions related to tumour. Patients with the IDH mutation were younger, with better performance status, larger proportions of grade III tumours and MGMT methylation. In this group cognitive functioning is significantly better in the domains of immediate recall, short delayed recall and delayed recall, and in the fields of executive functioning and recognition. There were no differences in cognitive functioning in regard to MGMT status. Grade III tumours were associated with more frequent MGMT methylation. Self-assessment proved week tool, associated only with immediate recall. Conclusions. We found no differences in cognitive functioning according to MGMT status, but cognition was better when IDH mutation was present. In a cohort study of patients with high-grade glioma, almost half were unable to par- ticipate in a study, which points to an overrepresentation of patients with better cognitive functioning in the research. Key words: cognition; high grade glioma; IDH1 mutation; MGMT methylation Introduction Malignant gliomas are group of aggressive brain tumours, comprising anaplastic astrocytoma (Grade III), anaplastic oligodendrogliomas (Grade III), anaplastic astrocytoma (Grade IV) and glio- blastomas (Grade IV).1 Anaplastic gliomas are still some of the most challenging tumours for patient and caregivers, but also for the therapist. Although there are some cautious advances in this field, there is still a grim outlook for the patients. As a number of patients is ill responding to treatment there are efforts for identifying those who respond well and those who would benefit from a change in treatment strategy. Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients202 Genetic and epigenetic markers like isocitrate dehydrogenase (IDH) mutations, loss of heterozy- gosity of 1p/19q(LoH 1p/19q), and methyl guanine methyl transferase (MGMT) promoter methylation have recently helped to stratify patients, removing the mixed histology like anaplastic oligoastrocy- toma and introducing Grade IV astrocytoma. In IDH1 mutated patients’ survival was markedly longer, as is in anaplastic oligodendrogliomas (with LoH 1p/19q). Prior to widespread genetic testing, it was already clear that patients harbour- ing methylation of MGMT gene promoter fare bet- ter comparing to those without.2-4 Brain tumours are also associated with im- paired cognitive functioning, due to tumour alone but also due to treatment. Cognitive impairment can manifest already at the time of diagnosis; the prevalence of cognitive deficits varies from 60 to 85% in different studies.5 The most common are in the fields of verbal memory, executive function- ing, psycho-motor speed, but also attention and language.6-9 Cognitive functioning is also one of the prog- nostic factors for survival. Early findings suggest- ed that cognitive decline is preceding radiologi- cal progression, which was not confirmed by all studies.10-12 Further studies confirmed cognitive impairment as independent prognostic factor in newly diagnosed patients, both at baseline and in the period after surgery.13-16 It was shown that IDH1 mutation (IDH1-mut) is not only an important prognostic factor, but it is also associated with better cognitive functioning. Many studies have shown that cognitive function- ing is better in IDH1-mut patients when compared with IDH1-wildtype (IDH1-wt) patients.17-19 Among possible causes of the better cognitive functioning of patients with IDH1-mut is brain plasticity which could be affected negatively by the greater tumour growth rate in IDH1-wt tumours, while remaining intact in less invasive IDH1-mut tumours. Preserved cognitive functioning might also be related to the tumour microenvironment, with more pronounced lymphocyte infiltration and programmed death-ligand 1 (PD-L1) expres- sion in IDH1-wt tumours, or even to differences at the synaptic level.20 The effect and possible role of the MGMT meth- ylation status in patients’ cognitive functioning is even less clear. Most clinical studies focus on in- vestigating patients with MGMT promoter meth- ylation (MGMT-met), with cognitive function as a secondary outcome.21 According to one study, the absence of MGMT promoter methylation (MGMT- unmet) predicts greater cognitive deficit when pa- tients are treated with radiochemotherapy.22 The MGMT-met therefore can be considered a predic- tive marker for development of cognitive impair- ment, but further research about its role in cogni- tive functioning as well as the prognosis needed. Here the challenge is the frequent overlap of MGMT promoter methylation with the IDH1 mu- tation, which, in conjunction with relatively small number of patients in the high-grade glioma stud- ies, presents difficulties in statistical analysis. In our study, we examined how the expression of IDH1 mutation and MGMT promoter methyla- tion are linked to the cognitive functioning fol- lowing the operative treatment in the cohort of all Slovene Grade III and Grade IV glioma patients. Patients and methods Patients We analysed the cohort of patients with high grade glioma, treated between March 2019 and December 2021. Their diagnoses (anaplastic astrocytoma, an- aplastic oligodendroglioma or glioblastoma) were histological confirmed. Patients were operated in either of the two neurosurgical departments in Slovenia, then they were referred to Institute of Oncology Ljubljana for evaluation regarding the initiation of radiochemotherapy. At the referral, they consented to be enrolled in the study. Exclusion criteria were histology other than gliomas WHO III/IV, Karnoffsky performance status less than 70% and inability to undergo eval- uation (e.g., marked dysphasia). To be included in the study, they had to be 18 years old or older. The following data were obtained from the medical documentation: age, sex, date of diagno- sis, localization of the tumour, type of surgery, ex- tent of surgery, radiotherapy parameters, systemic therapy, use of corticosteroids, histological, genetic and epigenetic characteristics of tumours. All pa- tients also had a molecular and genetic analysis of the tumour tissue performed, so the IDH1 muta- tions and MGMT promoter methylation status were determined. Cognitive functioning To asses cognitive functioning, we used psycho- metric tests in the domains of verbal memory (Slovenian Verbal Learning Test – TBU, measur- ing immediate recall, short recall, delayed recall and recognition of distracters)23, verbal fluency Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients 203 (Slovenian Controlled Oral Word Association Test –SCOWA)24, psycho-motor speed (Trail Making Test, Part A – TMT A), executive functions (Trail Making Test, Part B – TMT B)25, in accordance with the recommendations for use in the studies con- cerning cognitive functioning of cancer patients.26 The patients also self-evaluated their cognitive functioning on a 0–10 scale (0 = without problems, 10 = extremely intensive problems present). Statistical analysis and ethical consideration We used descriptive statistics with the means val- ues and standard deviation for the demographic data. The correlation between variables was tested with Pearson’s t test or Spearman’s rho test. For each cognitive test, the test scores were analysed either as standardized (z-scores) or as a dichotomized variable: no impairment present (z > –1.5 below the mean of the control group) vs. im- pairment observed (the patient had a z-score lower than –1.5 or was unable to perform the test at all). At the individual level, we analysed the percent- age of impaired patient̀ s results. We next compared groups of patients with dif- ferent IDH1 statuses and MGMT methylation, using either a t-test or Mann-Whitney U-test (in case that Kolmogorov-Smirnov test of normality showed statistically significant departure from normality) for interval variables, a χ2 test for categorical variables and ordinal variables with Kendall’s Tau test. All hypotheses were tested at a 5-percent alpha error rate. We used the statistical program SPSS, to calcu- late the power of the test we used G*Power 3.1.9.7. Written informed consent was obtained from all the patients before the inclusion in the clinical trial. The study was approved by the Institutional Review Board of the Institute of Oncology Ljubljana and by The National Medical Ethics Committee of the Republic of Slovenia (Approval number 0120- 393/2018/10, date 12/12/2018) and was carried out according to the Declaration of Helsinki. Results Demographics and tumour characteristics At the time the research was performed, 275 pa- tients were diagnosed with glial tumour. Of those, 90 patients were recruited into the study, repre- senting 33% of all patients. Figure 1 shows reasons for patients entering and not entering the study as recorded by oncologists at the time of the first con- sultation. Of the 51% of patients incapable of par- ticipating, in the study the major reason was poor performance status, followed by other tumour and treatment related impairments, representing 46% of ineligible patients, the other common issue was language barrier. Participating patients were 30 to 84-year-old (median [M] = 58.78 years, standard deviation [SD] = 11.31 years). There were more males than females (57 vs. 33). On average, female patients were older (M = 57.67 years, SD = 9.35 years, 39–74 years) than male patients (M = 59.42 years, SD = 12.33 years, 30–84 years), but the age difference was not statis- tically significant (p = 0.481). Of all 90 patients, 78 had Grade IV tumour (all classified as glioblastoma), 8 had anaplastic astro- cytoma, and 4 had anaplastic oligodendroglioma. Fifteen patients had IDH1 mutation (7 anaplastic oligodendroglioma, 4 anaplastic astrocytoma, and 4 glioblastoma). MGMT promoter was methylated in 36 patients (4 anaplastic oligodendroglioma, 5 anaplastic astrocytoma, and 27 glioblastoma), whereas in 1 glioblastoma, 2 anaplastic astrocy- tomas and 1 anaplastic oligodendroglioma we couldn’t determine the methylation status. Patients with Grade III tumours had a statisti- cally better performance status – the Karnoffsky All patients with WHO Grade III and Grade IV gliomas in Slovenia, N = 275 Incapable to enter study (140; 51%) • Poor physical performance (67;24%) • Dysphasia, dysarthria (11; 4%) • Marked cognitive impairment, disoriented (22; 8%) • Treatment related complication (25; 9%) • Language barrier (15; 5%) Eligible patients N =135 (49%) Declined (22; 8%) • Oncological treatment (11; 4%) • To enter study (11; 4%) Unknown (23; 8%) Agreed and participated in the study N = 90 (33%) FIGURE 1. Recruitment protocol, N = 275 Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients204 performance status was 70 in 2 (17%) patients, 80 in 2 (17%) patients, 90 in 8 (67%) patients with Grade III tumours – when compared with patients with Grade IV tumours (in these group, the Karnoffsky performance status was 70 in 30 patients, 80 in 35 patients, 90 in 10 patients, and 100 in 3 patients; U = 251.000, z = -2.75, p = 0.006). Compared to the group of patients with Grade III tumours, the group with Grade IV tumours had a statistically significantly higher percentage of IDH1-mut tumours (73% vs. 5%, χ2(1) = 56.077, p < 0.001, V = 0.789, 1–b = 0.987) and MGMT promoter methylations (60% vs. 36%, χ2(1) = 7.067, p = 0.008, V = 0.280, 1–b = 0.756). Table 2 shows the structure of the sample ac- cording to the expression of IDH1 mutation and MGMT methylation. Five patients had both ge- netic markers expressed, two thirds of patients with IDH1-mut also had MGMT-met expressed, and among patients with expressed methylation, IDH1-mut tumours were present in 27% of pa- TABLE 1. Demographic and medical data Variable Levels f (%) Descriptive statistics Gender Male Female 57 (63%) 33 (37%) Age < 50 years 50–70years > 70 years 17 (19%) 60 (67%) 13 (14%) M = 58.78, SD = 11.31 min = 30, max = 84 Education ≤ 9 years 10–13 years 14–19 years ≥ 20 years 14 (16%) 48 (53%) 26 (29%) 2 (2%) Tumour grade Grade III Grade IV 12 (13%) 78 (87%) Tumour location Frontal Parietal Temporal Occipital central Diffuse 34 (38%) 21 (23%) 26 (29%) 4 (4%) 3 (3%) 2 (2%) Hemisphere Right Left Both 38 (42%) 44 (49%) 8 (9%) Surgery type Biopsy Reduction Gross tumour resection 11 (12%) 49 (54%) 30 (33%) Karnoffsky performance status 70 80 90 100 32 (36%) 37 (41%) 18 (20%) 3 (3%) Corticosteroids (Yes/no, mg) Yes No 66 (73%) 24 (27%) M = 5.55, SD = 4.73 min 0, max 24 Radio-chemotherapy (intention to treat) Yes No 90 (100%) 0 (0%) Time to beginning of adjuvant treatment (in weeks) ≤ 6 weeks ≥ 7 weeks 60 (67%) 30 (33%) M = 5.98, SD = 2.25 min 3, max 15 Epilepsy Yes No 27 (30%) 63 (70%) IDH1 mutation IDH1 mutation IDH1 wild type 15 (17%) 75 (83%) MGMT methylation* Yes No 36 (42%) 50 (58%) IDH1 = isocitrate dehydrogenase 1; M = median; MGMT = methyl guanine methyl transferase; SD = standard deviation Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients 205 tients. The difference in the proportion of MGMT methylation in the IDH1-mut and IDH1-wt groups was statistically significant, χ2(1) = 5.333, p = 0.021, V = 0.243, 1–b = 0,82). Cognitive functioning The overview of cognitive evaluation is presented in Table 3. The achievements were impaired (z <–1.5) in a large proportion of the patients, espe- cially in the field of short recall, executive func- tions and psycho-motor speed. The impairment was least frequent in the field of recognition and verbal fluency. The results remained similar, re- gardless of accounting only those who were ca- pable of completing a specific test or the sample as whole – the biggest difference between these two analysis methods is on the TMT A and TMT B tests. We examined on how many out of 7 tests the pa- tients had an impaired test score (z < –1.5 or unable to finish the test) and found that 11 patients (12%) had 0–1 impaired test score, 26 (29%) patients had 2–4 impaired test scores, and 53 (60%) patients had more than 5 impaired test scores. On the 10-point self-evaluation scale of cognitive functioning the mean rating was 3.66, SD = 2.81, min = 0, max = 10; 48 (53%) patients selected rating 0–3, 23 (26%) selected rating 4–6, and 19 (21%) se- lected a rating higher than 7. Correlations between self-assessment and individual tests of cognitive functioning show that self-assessment is weakly but statistically significantly related only to imme- diate recall (r = -0.280, df = 79, t = 2,57, p = 0.012). The cognitive functioning in any of the meas- ured fields was not statistically significantly af- fected by sex, surgery type and the presence or ab- sence of seizures. The test scores did, however, dif- fer with regard to age, education, and performance status. Age was statistically significantly related to participant’s results in the field of verbal fluency (r = -0.278, t = -2.55, df = 79, p = 0.012), immediate recall (r = -0.409, t = -3.96, df = 79, p < 0.001), short delayed recall (r = -0.388, t = 3.72, df = 79, p < 0.001) and de- layed recall (r = -0.333, t = 3.12, df = 79, p = 0.003). Education was significantly related to results in the fields of verbal fluency (rs = 0.381, t = 3.66, df = 79, p < 0.001), immediate recall (rs= 0.334, t = 3.13, df = 79, p = 0.002), short delayed recall (rs = 0.285, t = 2.63, df = 79, p = 0.010), delayed recall (rs = 0.265, t = 2.42, df = 80, p = 0.017) and recognition (rs = 0.264, t = 2.42, df = 79, p = 0.018). Performance status was significantly related to immediate recall (rs = 0.280, t = 2.57, df = 79, p = 0.012) and delayed recall (rs = 0.296, t = 2.74, df = 79, p = 0.008). We analysed the disease and demographic data and the results of psychological tests with regard to IDH1 mutation. Compared to IDH1-wt patients, patients with tumours harbouring IDH1-mut were statistically significantly younger, had better per- formance status and were more likely to have Grade III tumour and MGMT promoter methyla- tion (Table 4). They functioned better in the field of verbal memory (had a better performance in immediate recall, short delayed recall and delayed recall, measured either with z-scores or as dichot- omised test scores) and in the field of executive TABLE 3. Descriptive statistics for standardized test scores (z–values) and proportion of impaired patients in psychological cognitive functioning tests Domain Test % impaired / all N % impaired / capable Mean z score SD of z scores Visual – motor speed TMT A 68 68 57 2.89 3.57 Executive function TMT B 78 46 59 2.80 2.94 Verbal fluency SCOWA 47 81 41 –1.21 0.88 Memory immediate recall short delayed recall delayed recall recognition SVLT-ir SVLT-sr SVLT-dr SVLT-recog 64 79 63 60 80 60 76 58 52 –1.83 –2.05 –1.92 –2.73 1.11 1.31 1.35 3.36 SCOWA = Slovenian Controlled Oral Word Association Test; SVLT = Shiraz Verbal LearningTest; TMT A = Trail Making Test, Part A; TMT B = Trail Making Test, Part B TABLE 2. Number of patients with different combinations of IDH1 mutation expression and methyl guanine methyl transferase (MGMT) methylation MGMT-met MGMT -unmet Total IDH1-mut 5 10 15 IDH1-wt 49 26 75 Total 54 36 90 IDH1-mut = isocitrate dehydrogenase 1 mutation; IDH1-wt = isocitrate dehydrogenase 1 wildtype; MGMT-met = methyl guanine methyl transferase promoter methylation; MGMT- unmet = methyl guanine methyl transferase absence of promoter methylation Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients206 functioning (measured with dichotomised test scores) (Table 5). Patients with IDH1-mut had on average a sta- tistically significantly lower number of impaired tests results than patients with IDH1-wt (M = 2.93, SD = 2.25vs. M = 4.93, SD = 1.99; U = 286.000, z = -3.04, p = 0.002). 5 (33%) patients with IDH1-mut tu- mours and 6 (8%) patients without IDH1-mut had at most one impaired result on cognitive tests; im- paired results on 2–4 tests had 5 (33%) vs. 21 (28%). Impaired scores on more than 5 tests had 5 (33%) vs. 48 (64%). TABLE 4. Patient characteristics, regarding isocitrate dehydrogenase 1 (IDH1) mutation IDH1-wt (N = 75) IDH1-mut (N=15) Result of the statistical test and effect size Age mean (min/max/SD) 61.50 (31 / 84 / 9.21) 38.75 (30 / 67 / 3.86) t = -5.97, df = 88, p< 0.001 Sex (female / male) 29 / 46 4 / 11 χ2(1) = 0.775, f = -0.93, p = 0.379 Education level (≤ 9 years /10–13 years / 14–19 years / ≥ 20 years) 11 / 40 / 22 / 2 3 / 8 / 4 / 0 τb(3) = -0.06, z = -0.83, p = 0.547 KPS (70/80/90/100) 31 / 34 / 7 / 3 1 / 3 / 11 / 0 τb(3) = 0.403, z = 18.95, p < 0.001 WHO grade (III / IV) 1 /74 11 /4 χ2(1) = 56.08, f = -0.789, p < 0.001 Corticosteroids mg (min/max/SD) 2 (0 / 16 / 6) 5 (0 / 24 / 4.5) t = -1.16, df = 88, p = 0.251 biopsy/reduction/gross tumour resection 9 / 44 / 22 2 / 5 / 8 χ2(2) = 3.65, V = 0.210, p = 0.161 Tumour location (frontal / temporal / parietal / occipital / diffuse / central) 26 / 20 / 21 / 4 / 2 / 2 8 / 1 / 5 / 0 / 1 / 0 NA Hemisphere (right / left / both) 21 / 27 / 6 17 / 17 / 2 χ2(2) = 1.14, V = 0.113, p = 0.566 MGMT (yes / no) 10 / 5 26 /49 χ2(1) = 5.33,f = 0.24, p = 0.021 IDH1-mut = isocitrate dehydrogenase 1 mutation; IDH1-wt = isocitrate dehydrogenase 1 wild type; KPS = Karnoffsky performance status; MGMT = methyl guanine methyl transferase; NA = not available; SD = standard deviation TABLE 5. Cognitive functioning regarding to isocitrate dehydrogenase 1 (IDH1) mutation IDH1–wt IDH-mut Result of the statistical test IDH1–wt IDH1-mut Result of the statistical test N Mean Z score (SD) N Mean Z score (SD) % of impaired % of impaired TMT A 53 3.12 1 (3.84) 15 2.06 (2.23) U = 345.000, z = -0.77, df = 66, p= 0.437 72 46 χ2(1) = 3.67, V = 0.20, p = 0.055 TMT B 33 2.82 1 (3.11) 13 2.76 (2.57) U = 221.500, z = -0.17, df = 66, p = 0.864 82 60 χ2(1) = 3.87, V = 0.21, p = 0.050 SCOWA 66 -1.28 (0.87) 15 -0.86 (0.84) t = -1.69, df = 79, p = 0.095 51 27 χ2(1) = 2.89, V = 0.18, p = 0.089 SVLT-ir 65 -1.98 (1.06) 15 -1.15 (1.12) t = -2.729, df = 78, p = 0.008 71 33 χ2(1) = 7.60, V = 0.29, p = 0.006 SVLT-sr 65 -2.20 (1.29) 15 -1.37 (1.22) t = -2.25, df = 78, p = 0.027 84 53 χ2(1) = 7.06, V = 0.28, p = 0.008 SVLT-dr 65 -2.14 (1.27) 15 -0.98 (1.34) t = -3.13, df = 78, p =0.002 69 33 χ2(1) = 6.98, V = 0.28, p = 0.008 SVLT-recog 65 -3.061 (3.54) 15 -1.24 (1.91) U = 611.000, z = -2.25, df = 78, p = 0.023 29 26 χ2(1) = 3.00, V = 0.18, p = 0.083 1 the distribution is significantly non-normal IDH1-mut = isocitrate dehydrogenase 1 mutation; IDH1-wt = isocitrate dehydrogenase 1 wildtype;SCOWA = Slovenian Controlled Oral Word Association Test; SD = standard deviation; SVLT = Shiraz Verbal Learning Test; SVLT-dr = SVLT delayed recall; SVLT-ir = SVLT immediate recall; SVLT-recog = SVLT recognition; SVLT-sr = SVLT short delayed recall; TMT A = Trail Making Test, Part A; TMT B = Trail Making Test, Part B Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients 207 We found no differences between groups with regard to self-evaluation of cognitive functioning problems; the mean rating was 3.60,SD = 2.81, in patients with IDH1-mut tumours vs. 3.67, SD = 2.95 in patients with IDH1-wt (U = 579.000, z = 0.18, p = 0.857); 40 (53%) patients with IDH1-wt tumour gave a self-assessment of 0–3 vs. 8 (53%) patients with IDH1-mut, score 4–6 was given by 18 (24%) vs. 5 (33%) patients and a score above 7 17 (23%) vs. 2 patients (13%). We also compared demographic characteristics in patients with and without MGMT promoter TABLE 6. Patient characteristics regarding methyl guanine methyl transferase (MGMT) methylation MGMT-unmet (N=54) MGMT-met (N = 36) Result of the statistical test and effect size Age mean (min/max/SD) 58.94 (31 / 84 / 10.42) 1 58,53 (30 / 78 / 12.67) t = -0.17, df = 88, p = 0.86 Sex (female / male) 19 / 35 14 / 22 χ2(1) = 0.13, f = 0.04, p = 0.721 Education level (≤9 years /10–13 years / 14–19 years / ≥ 20 years) 8 / 19 / 11 / 0 9 / 29 / 15 / 2 τb (3) = -0.03, z = -1.41, p = 0.776 KPS (70/80/90/100) 20 / 23 / 9 / 2 12 / 14 / 9 /1 τb (3) =0.06, z = 2.82, p = 0.541 WHO grade (III / IV) 3 / 51 9 / 27 χ2(1) = 7.07, f= -0.28, p = 0.008 Corticosteroids mg (min/max/SD) 5.67 (0 /24 / 5,04) 5.48 (0 / 16 / 4.22) t = 0.18, df = 88, p = 0.857 Biopsy/reduction/gross tumour resection 9 / 31 / 14 2 / 18 / 16 χ2(2) = 4.62, V = 0.23, p = 0.099 Tumour location (frontal / temporal / parietal / occipital / diffuse / central) 26 / 20 / 21 / 4 / 2 / 2 8 / 1 / 5 / 0 / 1 / 0 NA Hemisphere (right / left / both) 28 / 40 / 7 10 / 4 / 1 χ2(2) = 4.46, V = 0.223, p = 0.107 IDH1 (yes / no) 5 / 49 10 / 26 χ2(1) = 5.33, f = 0.24, p = 0.021 IDH1 = isocitrate dehydrogenase 1; MGMT-met = methyl guanine methyl transferase promoter methylation; MGMT-unmet = methyl guanine methyl transferase absence of promoter methylation; KPS = Karnoffsky performance status; SD = standard deviation TABLE 7. Cognitive functioning regarding methyl guanine methyl transferase (MGMT) methylation MGMT-met MGMT-unmet Result of the statistical test MGMT-met MGMT –unmet Result of the statistical test N Mean Z score (SD) N Mean Z score (SD) % of impaired % of impaired TMT A 26 2.67 (3.01) 42 3.031 (3.90) U = 530.000, z = -0.20, df = 67, p = 0.840 69 66 χ2(1) = 0.08, V = 0.03, p = 0.782 TMT B 20 3.22 (2.30) 26 2.481 (3.37) U = 334.000, z = 1.64, df = 45, p = 0.101 86 74 χ2(1) = 1.88, V = 0.14, p = 0.170 SCOWA 33 -1.25 (0.87) 48 -1.181 (0.89) U = 756.000, z = -0.35, df = 70, p = 0.729 50 44 χ2(1) = 0.27, V = 0.05, p = .605 SVLT-ir 32 -1.85 (1.27) 48 -1.821 (1.01) U = 771.000, z = 0.03, df = 78, p = 0.975 64 67 χ2(1) = 0.01, V = 0.01, p = 0.928 SVLT-sr 32 -2.03 (1.32) 48 -2.06 (1.32) t = 0.09, df = 78, p = 0.926 75 81 χ2(1) = 0.54, V = 0.08, p = 0.460 SVLT-dr 32 -1.98 (1.52) 48 -1.89 (1.25) t = -0.30, df = 78, p = 0.763 66 61 χ2(1) = 0.29, V = 0.06, p = 0.592 SVLT- recog 32 -2.731 (4.24) 48 -2.721 (2.71) U = 784.000, z = 0.828, df = 78, p = 0.407 58 61 χ2(1) = 0.07, V = 0.03, p = 0.792 1the distribution is significantly non-normal MGMT-met = methyl guanine methyl transferase promoter methylation; MGMT-unmet = methyl guanine methyl transferase absence of promoter methylation; SD = standard deviation; SVLT = Shiraz Verbal Learning Test; SVLT-dr = SVLT delayed recall; SVLT-ir = SVLT immediate recall; SVLT-recog = SVLT recognition; SVLT-sr = SVLT short delayed recall; TMT A = Trail Making Test, Part A; TMT B = Trail Making Test, Part B Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients208 methylation. The patients differed in the tumour grade. Despite the predominance of Grade IV tu- mours in our sample, the methylated phenotype was more prevalent in Grade III patients (25% vs. 5%, χ2(1) = 7.067, V = 0.28, 1 – ß = 0.757, p = 0.008). In all other demographic characteristics, the groups were comparable. In the cognitive functioning, there were no dif- ferences in mean z-scores or dichotomized test scores between patients with methylated and un- methylated promoter MGMT (Table 7). There were no statistically significant differenc- es in self-evaluation of cognitive functioning prob- lems; the mean number of impaired results were 4.50 (SD = 1.90) in patients with MGMT un-meth- ylated tumours vs. 4.29 (SD = 1.75), U = 1100.000, z = 1.51, p = 0.251. There were also no differences in the number of tests in which patients achieved an impaired result (U = 1052.500, z = 0.67, p = 0.501). With the mean 4.69 (SD = 2.31) and 4.54 (SD = 2.07) had 5 (14%) patients with MGMT methylated tumours and 6 (11%) pa- tients MGMT unmethylated tumours at most one impaired result, 2–4 impaired results had 9 (25%) vs. 17 (31%) patients, and on more than 5 tests the results were impaired in 22 (61%) vs. 31 (57%) pa- tients. Discussion High-grade glioma patients are experiencing a number of cognitive functioning problems. In our study we focused on the period following the sur- gical treatment and before commencement of sys- temic treatment. The majority of cognitive problems we found were in the fields of executive functions, visual- motor speed and verbal memory, especially im- mediate and short delayed recall, as well delayed recall. There were the least problems in the field of verbal fluency, but even here more than 40% of pa- tients had an impaired result. Among participat- ing patients, only 12% had an impaired result in up to one measured field, while 60% had impaired results in the majority of the measured domains. The analysis of cognitive test scores expressed as z-values gave conclusions comparable to the ones obtained with the analysis of dichotomized scores. The use of dichotomized scores enabled us to also include in the analyses the results of patients who were unable to complete some tests and so avoiding the overrepresentation of patients with better cognitive functioning in the analyses. These results are in accordance with other stud- ies examining cognitive functions in high-grade glioma patients, but it is noticeable that in our study the proportion of patients presenting with the “impaired” result is higher, possibly due to the fact that our study included the entire cohort of high-grade glioma patients. When comparing the results of different studies, it is necessary to con- sider the use of different criteria for impairment, with the otherwise dominant criterion z< -1.5.5 In comparison with IDH1-wt patients, patients with IDH1-mut (17%), were significantly younger, had better performance status and more often they had Grade III tumour. This is in line with previous studies.27 Additionally, the cognitive functioning of pa- tients with IDH1-mut was statistical significantly better in verbal memory and executive functions. Immediate recall, short-delayed and long-delayed recall differ statistically significantly in the analy- sis of interval variables as well as in the analysis of dichotomized variables. Executive functions measured with the TMT B test only in the analysis of dichotomized variables, which may be the re- sult of the fact that a larger proportion of patients were unable to complete this test, therefore, they are not included in the analysis of interval vari- ables. Patients with IDH1-mut tumours achieved impaired results on significantly lower number of tests. These findings are in line with findings of the previous studies.8,19 According to the MGMT promoter methylation status, the groups did not differ statistically sig- nificantly in demographic data. A statistically sig- nificant difference was found in the expression of MGMT methylation according to the grade of the tumour (75% patients with grade III vs. 34% with grade IV).28 We did not find differences in any of the analysed fields of cognitive functioning and also not in the number of tests in which patients achieved an impaired result. We intended to include all patients with the di- agnosis of high-grade glioma in the observed pe- riod in Slovenia. Given that, after surgery in one of the two centres in Slovenia, all newly diagnosed patients with gliomas are referred to our institu- tion for evaluation regarding further treatment; it gives us an insight into the entire population of pa- tients with glioma. Data collected on the entire co- hort of patients revealed that a large proportion of high-grade glioma patients is unable to participate in the studies of cognitive functioning. In our case 46% of patients were unable to participate due to poor performance status or other somatic factors. Radiol Oncol 2023; 57(2): 201-210. Skufca Smrdel AC et al. / Cognitive functioning in high grade glioma patients 209 The finding that a large proportion of patients are unable at all to participate in cognitive func- tioning studies additionally indicates an over- representation of patients with better cognitive functioning in research. From this point of view the cohort study design corresponds better to the everyday clinical practice with the patients with high grade glioma. Patients’ self-assessments on a 1–10 scale did not correlate with the results of the tests used and probably should not be used for any assessment of cognitive functioning; we only found a weak correlation between self-assessment on a 10-point scale and objective assessment. With otherwise different methodology, foreign studies also came to similar results - there is no or weak correlation between subjective assessment and psychological tests.29 The limitation of our study is lack of data on cognitive functioning prior to surgical treatment. Thus, in the study we did not include eventual dif- ferences between patients with IDH1 mutated and wildtype tumours, which may be present even be- fore surgery19,30, which would also be important in the light of research findings regarding the differ- ent dynamics of cognitive decline after surgery.31 Another point worth mentioning is that several papers showed that epilepsy and the use of antie- pileptics is an important factor of neurocognitive functioning. But in our sample, the use of antie- pileptics could not be analysed as virtually every patient has received them following surgery even those without history of seizures; though in these cases they were weaned from antiepileptics at the beginning of oncological treatment. Our study took place during the coronavirus pandemics. Despite this, oncological treatment was not interrupted nor delayed, but in our study, it was connected with the increase of patients re- fusing to participate and with longer time from surgery to the start of treatment due to infections. It is worth to mention that targeting this popula- tion is beyond single institution capabilities. While the cohort study corresponds better to the clinical practice, on the other hand the low number of the mutations, especially in IDH1, is hampering the statistical analysis. When conducting our study, we noted a distinctive lack of prospective data re- garding patients in suboptimal performance sta- tus, thus overestimating cognitive functioning of high-grade glioma patients. Even as we observed the patients in WHO performance status of 2 the number of cognitive patients rose markedly. 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J Neurooncol 2020; 146: 97-109. doi: 10.1007/ s11060-019-03341-6 Radiol Oncol 2023; 57(2): 211-219. doi: 10.2478/raon-2023-0019 211 research article Changes in the quality of life of early breast cancer patients and comparison with the normative Slovenian population Cvetka Grasic Kuhar1,2, Tjasa Gortnar Cepeda3, Christian Kurzeder4, Marcus Vetter5 1 Institute of Oncology Ljubljana, Department Medical Oncology, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Jesenice General Hospital, Jesenice, Slovenia 4 Breast Center, University Hospital Basel, Basel, Switzerland 5 Cancer Center Baselland, Cantonal Hospital Baselland, Liestal, Switzerland Radiol Oncol 2023; 57(2): 211-219. Received 27 November 2022 Accepted 26 December 2022 Correspondence to: Assist. Prof. Cvetka Grašič Kuhar, M.D., Ph.D., Institute of Oncology Ljubljana, 1000 Ljubljana, Slovenia. E-mail: cgrasic@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0). Background. We aimed to identify changes in quality of life after breast cancer treatment and compare them with the normative population data for the Slovenian population. Patients and methods. A prospective, single-group, cohort design was used. A total of 102 early breast cancer patients treated with chemotherapy at the Institute of Oncology Ljubljana were included. Of those, 71% returned the questionnaires after one-year post-chemotherapy. The Slovenian versions of the European Organisation for Research and Treatment of Cancer (EORTC) QLQ C30 and BR23 questionnaires were used. Primary outcomes were a com- parison of global health status/quality of life (GHS) and C30 Summary Score (C30-SumSc) at baseline and one-year post-chemotherapy with the normative Slovenian population. The exploratory analysis evaluated the differences in symptoms and functional scales of QLQ C-30 and QLQ BR-23 between baseline and one-year post-chemotherapy. Results. At baseline and one-year post-chemotherapy, C30-SumSc of patients was lower than the predicted C30-SumSc from the normative Slovenian population by 2.6 points (p = 0.04) and 6.5 points (p < 0.001), resp. On the contrary, GHS was not statistically different from predicted either at baseline or after one year. Exploratory analysis revealed that one-year post-chemotherapy compared to the beginning of chemotherapy, patients had statistically significantly and clinically meaningful lower scores in body image and cognitive functioning, and increased symptom scores for pain, fatigue, and arm symptoms. Conclusions. The C30-SumSc is reduced one- year post-chemotherapy. Early interventions should be directed to- ward the prevention of the decline of cognitive functioning and body image, and to alleviate fatigue, pain, and arm symptoms. Key words: breast cancer; chemotherapy; quality of life; cognitive dysfunction; fatigue Introduction Global cancer statistics for 2020 estimated 2,261,419 new cases of breast cancer worldwide, which rep- resents 11.7% of all cancers. It became the most common cancer in humans, surpassing lung can- cer incidence.1 Breast cancer survivors represent a large group of long-term cancer survivors with different health issues during and after treatment. According to Slovenian Cancer Registry data in 2019, breast cancer survivors (19.455), represent 14.3% of all cancer survivors (136.500).2 Half of the Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population212 Slovenian breast cancer cases are diagnosed in women in the 20–65 age group, which means they are active in their professional careers and family life. Patients with early breast cancer receive multi- modal cancer treatment (surgical and/or systemic treatment including chemotherapy, targeted ther- apy, and endocrine therapy, and/or radiotherapy). The treatment they receive greatly affects their quality of life (QoL). For example, surgery and radiation therapy could cause local side effects, like breast and arm pain, and arm lymphoedema, however, systemic therapy could have numerous acute or long-lasting systemic side effects (nausea, neuropathy, cardiotoxicity, fatigue, cognitive dys- function etc.). Thus, comprehensive cancer therapy affects many functional or symptom scales of QoL. However, families and employers expect patients to recover fully in a short time. Nowadays, QoL becomes also more and more important in terms of drug development. With the validated quality of life questionnaires (QLQ), spe- cifically the European Organisation for Research and Treatment of Cancer (EORTC) core ques- tionnaire (QLQ C30) and breast module (QLQ BR23)3,4, we can monitor the impact of treatment on patient-reported outcomes (PROs), like global health status/QoL and summary score, and com- pare different modules and symptoms over time.5 What is the QoL at the transition from treatment to survivorship where a woman is expected to be back to work? Arm symptoms and fatigue, as well as cognitive and physical dysfunction and work-related variables (e.g., physical demands at work), interfered with the ability to perform work. Schmidt et al. found associations between depres- sive symptoms, arm symptoms, lower education, and younger age with an impaired return to work after one year.6 Self-reported reasons that hinder the return to work were fatigue and cognitive problems.6,7 Additionally, social and personal fac- tors influence the functioning and working ability of individuals. Identifying dysfunctions that disable patients after breast cancer treatment and comparing QoL of patients with the normative population data8,9 could help caregivers provide survivors with more optimal care. They may benefit from specific in- terventions. The aim of our study was to prospectively eval- uate PROs in the cohort of early breast cancer pa- tients at the start of chemotherapy and one year af- ter the end of chemotherapy and to compare them with normative data for the Slovenian population. In our explorative analysis, we aimed to determine which functional and symptom scales appeared different one year after chemotherapy. Patients and methods Participants Our current study cohort consisted of early breast cancer patients including all subtypes who had taken part in our previous prospective non-rand- omized cohort study evaluating the impact of mo- bile app use for symptom management on PROs during chemotherapy treatment. The inclusion criteria in the aforementioned study were patients with early breast cancer, treated with chemothera- py, possessing an Android-based smartphone for symptom reporting, and willing to fill in paper and pencil questionnaires reporting their quality of life while receiving treatment.10 In the current prospective study, we included 102 patients who had signed informed consent for the former study and were willing to fill in the ad- ditional QLQ C30, QLQ BR23, and socioeconomic questionnaires one year after the end of chemo- therapy. Our first aim was to evaluate and com- pare pre-treatment and post-treatment PROs with normative data for the Slovenian population.11 This reference data on QLQ-C30 dimensions was obtained on 1231 healthy Slovenian individuals. Our second aim was to compare the post-treat- ment health-related quality of life (HRQoL) with the pre-treatment one. This data has not been re- ported yet. Study design A prospective, single-group, cohort design was used combining data from a former two-arm trial with new follow-up data collected one year after the end of chemotherapy.10 Patients who were ad- mitted for treatment with chemotherapy at the Institute of Oncology Ljubljana between December 2017 and September 2018 were eligible (Figure 1). Inclusion criteria were breast cancer stage I-III, treatment with neoadjuvant or adjuvant chemo- therapy, and proficiency in using an Android- based smartphone. Exclusion criteria were stage IV breast cancer, a lack of mobile device proficiency or using non-Android-based smartphones, and not understanding Slovenian. In addition to chemo- therapy treatment, patients were treated with anti- HER2 therapy (in case of HER2 positivity), surgery, endocrine therapy in case of hormone-receptor- Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population 213 positive disease, according to ESMO guidelines, and radiation therapy, if indicated.12 The patient’s demographic characteristics and type of treatment were collected from patient charts. Ethical approv- al for this study had been obtained from the Ethics Committee of the Institute of Oncology Ljubljana (ERID-EK-43 and ERID-EK-0080/2019). All patients had given their written informed consent. Instruments We used the Slovenian version of EORTC QLQ C30 and QLQ BR23 questionnaires. QLQ C30 is a core questionnaire which includes 30 items, consisting of 5 functional scales (cognitive, emotional, physi- cal, social and role functioning), 9 symptom scales, and two questions which include the patient’s assessment of global health status/quality of life (GHS). QLQ BR23 has 23 questions comprising four functional scales (body image, sexual functioning, sexual enjoyment, future perspectives) and symp- toms regarding the treatment of breast cancer (sys- temic therapy side effects, arm and breast symp- toms and others). Socio-economic questionnaires, used routinely for surveys at the Slovenian Cancer Registry, included questions about age, gender, so- cial class, employment, marital status, education, and place of residence. Outcome measures Our primary outcomes were GHS and C30 Summary Score (C30-SumSc), derived from EORTC QLQ C30. Symptoms and functional scales of QLQ C30 and QLQ BR23 were used in the ex- ploratory analysis only. The EORTC Quality of Life Approached: 140 patients Excluded: 36 patients -12 not smartphone users -14 unsupportive platform -10 not interested in the study Filled-in baseline questionnaires: 104 patients Excluded: 2 patients (metastatic disease) Cohort at beginning of chemotherapy: 102 Cohort at 1 year after chemotherapy: 72 Did not return questionnaires at 1 year: 25 patients Assessment of baseline global health status/QoL and Summary Score Assessment of global health status/QoL, Summary Score and explorative analysis at 1 year FIGURE 1. Consort diagram. Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population214 Scoring Manual was followed.13 All scales had val- ues from 0 to 100, where 100 represented the best GHS, the best functioning, or the worst symptoms. The C30-SumSc, which ranged from 0 (worst) to 100 (best), was calculated from 13 out of 15 EORTC QLQ C30 scales (the GHS and financial difficulties scale were excluded) in accordance with Giesinger et al. and instructions from the EORTC.5,14 The pa- tient’s assessment of the clinical significance of changes in QLQ C30 and QLQ BR23 scores were interpreted as “slight” change either for better or for worse when the mean change in scores was about 5 to 10 points; “moderate” change for about 10 to 20 points; and “severe” change greater than 20 points. An established threshold for a clinically meaningful difference in QoL was previously set to 10 points.15 Data for GHS and C30-SumSc data for the nor- mative Slovenian population were obtained from recently published work by Velenik et al.11 We com- puted each patient’s predicted normative values for C30-SumSc and GHS (co-primary outcomes) according to this external reference.11 Using the patient’s age (categorized as 18‒39, 49‒59, 60+) and self-rated social class (lower, middle, higher), the predicted normative value represented the pa- tient’s scores if she had not had cancer. Statistical analysis Categorical variables were summarized with fre- quencies and percentages. Numerical variables were described with means and standard devia- tions (or medians and interquartile ranges if dis- tributions were asymmetric). We compared the mean C30-SumSc and GHS of our patients with the normative general Slovenian population.11 The mean C30-SumSc and GHS at the start of chemotherapy and one-year post-chemo- therapy were compared with the corresponding mean of the normative values using two-tailed one-sample t-tests as the variability for the norma- tive values could not be considered (the normative values were computed from the estimates from the article,11 standard errors of the estimates were not reported). As a part of the exploratory analysis, we per- formed a comparison of the GHS, C30-SumSc and QLQ C30 and QLQ BR23 scales between inclusion (start of chemotherapy) and after one year in a smaller group (72 patients) that had available data on both times. For statistical comparison of scales based on at least two questions, we calculated the average difference and 95% confidence intervals ([After 1 year] ‒ [At inclusion]) with two-tailed paired t-tests and 95% confidence intervals (CIs). For scales based on one question, we performed Wilcoxon’s test of predicted ranks. For all scales but C30-SumSc and GHS (pri- mary outcomes), the corresponding p-values were adjusted using the Holm method to control the family-wise error rate as so many hypotheses were tested. Corrected p-values allow a conclusion per population, but uncorrected ones do not. An (adjusted) p-value smaller than 0.05 was considered statistically significant. Analyses were performed using R statistical software (version 3.6.3)16 and SPSS v.24.0 (IBM Corporation). TABLE 1. Clinical characteristics of participants at beginning of the study and after one-year post-chemotherapy Characteristic At inclusion-before chemotherapy (n = 102) n (%) One year after end of chemotherapy (n = 71) n (%) Tumour stage T1 T2 T3 6 (45.1) 43 (42.2) 13 (12.7) 32 (44.4) 33 (45.8) 7 (9.7) Lymph node stage N0 N1 N2 N3 43 (42.2) 38 (37.3) 12 (11.8) 9 (8.8) 31 (43.1) 30 (41.7) 9 (12.5) 2 (2.8) Tumour subtype Luminal A-like Luminal B-like Luminal B HER2 positive HER2 positive Triple-negative 12 (11.8) 47 (46.1) 19 (18.6) 8 (7.8) 16 (15.7) 7 (9.7) 35 (48.6) 14 (19.4) 6 (8.3) 10 (13.9) Type of surgery Breast-conserving surgery + Sentinel node biopsy Breast-conserving surgery + Axillary dissection Mastectomy + Sentinel node biopsy Mastectomy + Axillary dissection 37 (36.3) 14 (13.7) 23 (22.5) 28 (27.5) 28 (38.9) 11 (15.3) 15 (20.8) 18 (25.0) Breast reconstruction None Deep inferior flap Tissue expander, followed by silicone implant 78 (76.5) 13 (12.7) 11 (10.8) 55 (76.4) 10 (13.9) 7 (9.7) Chemotherapy type Anthracyclines and taxanes Anthracyclines only Taxanes only CMF 70 (68.7) 19 (18.6) 10 (9.8) 3 (2.9) 51 (70.8) 13 (18.1) 6 (8.3) 2 (2.8) Anti-HER2 therapy 27 (26.4) 20 (27.8) Adjuvant endocrine therapy 78 (76.5) 57 (79.2) Radiotherapy 81 (79.4) 59 (81.9) CMF = cyclophosphamide, methotrexate, fluorouracil; HER2 = human epidermal growth factor receptor 2 Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population 215 Results Participants At the beginning of chemotherapy, we included 102 patients (Table 1, left column). These patients were compared with the normative Slovenian pop- ulation regarding baseline GHS and C30-SumSc. Seventy-two patients (71%) returned question- naires at one-year post-chemotherapy (Table 1, right column). The median age of this cohort was 51.5 years. 44% of patients were T1 and 43% were node-negative. Regarding subtype, 49% were lu- minal B-like, 19% luminal HER2+, 8% HER2+ non- luminal, 14% were triple negative and 10% were luminal A-like. The most common type of surgery was breast-conserving surgery with sentinel node biopsy (39%). All patients were treated with chem- otherapy (adjuvant (69%) or neoadjuvant (31%)), 28% had anti-HER2 treatment, and 79% had adju- vant endocrine therapy. 82% of patients received adjuvant radiation therapy. Patients’ socioeconom- ic characteristics are available in Supplementary Table 1. For the calculation of predicted norma- tive values of GHS and C30-SumSc, gender, age, and social class were used, according to Velenik et al.11 Their model was based on 1231 persons, of them 612 (49.7%) were females. The age distribu- tion of females was: 30.7% in cohort 18-39 years, 42.8% in cohort 40-59 years, and 26.5% in cohort 60-90 years. Self-rated social status of females was: 30.9% belonged to the lower, 57.4% to the middle, and 11.8% to the upper social class.11 Primary outcomes At inclusion (before the start of chemotherapy), C30-SumSc of our patients was statistically sig- nificantly lower than the predicted C30-SumSc in the general Slovenian population, namely by 2.6 points (p = 0.04). After one year, compared to the start of chemotherapy, patients’ mean C30-SumSc decreased by 6.5 points, which was statistically significant (p < 0.001), from the patient perspec- tive as a slight change for worse, but not clinically meaningful change (Table 2, Figure 2 - first line). On the contrary, GHS was not statistically or clini- cally significantly different from predicted either at inclusion or after 1 year (Table 2, Figure 2 - sec- ond line). TABLE 2. Patient-reported outcomes presented by EORTC C30 Summary Score (C-30 SumSc) and global health status/quality of life (GHS) at inclusion (beginning of chemotherapy), one-year post-chemotherapy, and the difference among both times Predicted At inclusion At 1 year [1 year] - [At inclusion] n mean Mean (95% CI) p Mean (95% CI) p Mean (95% CI) p C-30 SumSc 70 90.9 88.5 (86.1, 90.8) 0.04 82 (78.4, 85.5) < 0.001 -6.5 (-9.6, -3.4) < 0.001 GHS 71 72.7 71.1 (66.7, 75.6) 0.50 69.6 (65, 74.2) 0.19 -1.5 (-6.6, 3.5) 0.55 EORTC = European Organisation for Research and Treatment of Cancer BR-23 = breast module 23 questionnaire; EORTC: European Organisation for Research and Treatment of Cancer *Modules whose p-value is statistically significant (p < 0.05); FIGURE 2. Difference of the global health status/quality of life (GHS), C-30 Summary Score (C-30 SumSc), and functional and symptoms scales of EORTC C-30 and BR-23 questionnaires. Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population216 Exploratory analysis The exploratory analysis (Figure 2) revealed that patients had significantly lower functioning one year after chemotherapy compared to the begin- ning of chemotherapy in 3 functional scales: in body image and cognitive functioning, the dif- ference in our sample was > 10 points (clinically meaningful change), and in physical function- ing under 10 points (clinically not meaningful). Similarly, pain, fatigue, and arm symptoms score significantly increased by more than 10 points (clinically meaningful), and systemic therapy side effects increased by less than 10 points (clinically not meaningful). Discussion At the start of chemotherapy, early breast cancer patients had the same mean GHS as predicted from the general Slovenian population. C30-SumSc was statistically significantly lower, although this was not clinically significant since the difference was less than 10 points. GHS in breast cancer patients one year after the end of chemotherapy was on average as good as before the beginning of chemotherapy. C30-SumSc was statistically significantly worse, from the pa- tient’s perspective as a slight change for worse, al- though deterioration of 6.5 points is not considered clinically important. In the exploratory analysis, we found significant deterioration in some functional scales and increased symptoms in breast cancer patients one-year post-chemotherapy compared to the pre-chemotherapy state. Cognitive function- ing, body image, and physical functioning signifi- cantly deteriorated. Among symptoms, increased arm symptoms, pain, fatigue, and systemic therapy side effects were self-reported (Figure 2). Patients in our study did not perceive dete- rioration of GHS by a cancer diagnosis or cancer treatment. That means that patients perceived good overall well-being, as they had not been ill. A similar finding for GHS in patients 1–15 years post-diagnosis was found by others.7-9,17 On the other hand, our patients reported a slight deterio- ration of C30-SumCs one-year post-chemotherapy, probably due to the toxicities of multimodality treatment. Ferreira et al. noted similar persistent deterioration of C30-SumSc after two years in pre- menopausal patients treated with chemotherapy and postmenopausal patients treated with endo- crine therapy.18 Deterioration of C30-SumSc is not only due to treatment but could be also due to the progression of cancer. C30 Sum-Sc has recently been shown as an independent prognostic factor for overall survival in several cancers.19 In our explorative analysis, we found a detri- mental effect of cancer treatments (either chemo- therapy, endocrine therapy, surgery, or radiother- apy) on specific functional and symptom scales, evaluated with QLQ C30 and QLQ BR23. Patients reported clinically meaningful deterioration in cognitive functioning and body image from base- line to one-year post-chemotherapy. Deficits in cognitive, role, social, and emotional functioning, particularly in young patients, were also reported by others 1–10 years after surgery.8,20,21 Compared to the general population, researchers found signifi- cantly lower mean scores for cognitive and social functioning, role functioning and emotional func- tioning, physical functioning and body image, and future perspective between 5–15 years post-thera- py.7,9,17,22 That means that the consequences of treat- ment could be life-long. Interestingly, some serum markers of systemic inflammation were found to be statistically significantly higher in cancer survi- vors treated with chemotherapy even 20 years af- ter chemotherapy and were associated with lower cognitive performance.23 Breast reconstruction, however, improved physical functioning and body image compared to breast-conserving surgery; the same applies to social functioning and future per- spective.24,25 In addition to detrimental effects on functional scales, our exploratory analysis showed signifi- cantly increased symptom scores after one year compared to baseline: fatigue, arm symptoms, pain, and, to a lesser degree, the systemic therapy side effects. In addition to these symptoms, other researchers reported insomnia or sleep distur- bances, breast symptoms and financial difficulties, dyspnoea, hot flashes, sexual problems, and poly- neuropathy.7-9,17,20 Some differences in symptoms, however, were described only as trivial, with small clinical relevance.17 We suppose that pain and arm symptoms are related to higher nodal burden and consequently performed axillary dissection and irradiation. However, perceived fatigue, pain, and arm symp- toms could also reflect less personal engagement in avoiding or managing these symptoms. Sixty- nine percent of our patients had primary surgery, followed by adjuvant chemotherapy, and 31% had the opposite sequence of treatment. In view of this information, we would expect that arm symptoms (from the BR-23 questionnaire) will be greater Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population 217 at the beginning (at inclusion in the study) than 1-year post-chemotherapy. But it turned out the op- posite. We can explain this finding by the fact that arm symptoms are scores from three items, name- ly pain (not only pain in the arm, but also pain in the shoulder), swelling of the arm, and difficulties in the mobility of the arm. The swelling of the arm usually occurs with a delay. Swelling is more com- mon when axillary dissection is performed (in our case 35.3% of patients) than when removing only sentinel lymph nodes. Radiotherapy, which was delivered to 81.9% of our patients, could contribute to swelling and pain as well. Symptom pain of the C30 questionnaire is about pain anywhere and is made up of two questions, whether the pain is present and the question if it affects every day functioning. Generally, chemo- therapy, especially taxanes, received by 79.1% of our patients, also contributes to the pain. Sensory polyneuropathy, not only hurts but often im- pedes normal functioning (walking, fine motoric). Additional pain could be contributed to adjuvant endocrine therapy (tamoxifen mainly affects large joints, and aromatase inhibitors affect small joints). Yoon et al. found variation in symptom reporting influenced by race/ethnicity and other sociodemo- graphic characteristics, and several comorbid con- ditions.26 Returning to work is a significant milestone for breast cancer survivors.7 Von Ah et al. found that everyday cognition correlates with work engage- ment. What do these findings mean for cancer sur- vivors in the setting of clinical practice? Cognitive dysfunction and fatigue are the most important is- sues for patients, especially if they are employed. Cognitive dysfunction after chemotherapy (“chemo brain”) is described as the impairment of memory, attention, executive functions, and pro- cessing speed.27,28 Recently it has also been report- ed for hormonal therapy (tamoxifen and nonster- oid aromatase inhibitors), targeted therapy, immu- notherapy, and due to cancer itself, combined in terminus “cancer-related cognitive dysfunction”.28 Subjective cognitive problems were reported by half of breast cancer patients after chemotherapy, but only 15–25% had an objective cognitive de- cline.28 Despite the mild-to-moderate severity of cognitive dysfunction, it represents an important issue for patients.27 It is especially true for pa- tients who are employed.7,8,26 Impaired cognitive functioning in our patients one-year post-chemo- therapy could be related to treatment with chemo- therapy and surgery (general anesthesia) as well as adjuvant endocrine treatment (85% of patients). Among symptoms, fatigue is reported most regularly in all studies. Fatigue is a subjective feel- ing of lack of energy, of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer and/or cancer treatment, and interferes with usual functioning.29 It is a multidimensional symptom that accompanies patients while receiving chem- otherapy and can last many years after chemo- therapy.27 As with cognitive dysfunction, it could be associated with the type of treatment (surgery, chemotherapy, endocrine therapy, targeted thera- py, or radiotherapy). The rehabilitation of cancer survivors should be diverse, according to the needs of the individual patient. For example, physical functioning and fa- tigue could be improved with regular exercise.30 Arm symptoms could be managed with physical rehabilitation and more specifically lymphoedema treatment, elastic compressive gloves, and pain- killers. Pain (in the breast, arm, joints, peripheral polyneuropathy) should be appropriately man- aged by a pain specialist. Cognitive rehabilitation could include cognitive rehabilitation programs, physical activity, or relaxation programs.27 Hot flashes and sexual issues could be managed by a gynecologist. With fewer symptoms and bet- ter symptom scales patients would probably have better cognitive and role functioning and a better body image. In order to improve the QoL of cancer survivors, a pilot study on the comprehensive re- habilitation of breast cancer patients is underway at our institute. Identifying problems early proba- bly allows an earlier targeted approach, thus lead- ing to better patient functioning, an earlier return to work, and less absenteeism in the workplace. Evaluation of the results of comprehensive reha- bilitation on improving functional and symptom scales is eagerly awaited. Strengths of the study Firstly, this is a prospective cohort study of health- related quality of life, using validated question- naires and tools, such as GHS and C30-SumSc, recommended by the EORTC. Many studies per- form only cross-sectional data analysis. Secondly, we performed a comparison with our normative population to obtain information about what pa- tients’ scores would be without cancer. Limitations of the study The first limitation is the small sample size and that we did not have a baseline value of items in Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population218 the questionnaires before any cancer treatment. Secondly, we included only patients that were proficient in using smartphones, and thus prob- ably inadvertently chose a subset of the popula- tion that is highly motivated, healthier, and with middle social status. However, those patients were supported by a mobile app for coping with symp- toms, which would probably be even heavier with- out the app. Thirdly, we included only Android- based smartphone users, which represented 80% of smartphones in Slovenia at that time. However, the app for IOS had not yet been made available. An additional weakness of our study is that the as- pects of depression and anxiety, which affect cog- nitive functioning and fatigue, were not involved. Comorbidities were also not assessed – these are also significant predictors of symptoms, especially amongst those receiving chemotherapy. Conclusions Patients with early breast cancer had similar GHS before chemotherapy as the normative Slovenian population, and it did not deteriorate with treat- ment. One year after chemotherapy, C30-SumSc deteriorated compared to that before chemothera- py. Early interventions should be directed toward the prevention of the decline of cognitive function- ing and body image, and to alleviate fatigue, pain, and arm symptoms. Acknowledgments We thank Nina Ružić Gorenjec, who performed a statistical analysis but could not contribute as a co- author of the manuscript. This research was partly supported by the Slovenian Research Agency (Prognostic and pre- dictive factors for response in the treatment of breast cancer and other cancers, P3-0321). References 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mor- tality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71: 209-249. doi: 10.3322/caac.21660 2. Institute of Oncology Ljubljana. Cancer registry. [Internet]. [cited 2023 Jan 22]. Available at: http://www.slora.si/en/stevilo-zivih-bolnikov 3. Aaronson NK, Ahmedzai S, Bergman B, Bullinger M, Cull A, Duez NJ, et al. 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Int J Psychiatry Med 2010; 40: 163-181. doi:10.2190/PM.40.2.c Radiol Oncol 2023; 57(2): 211-219. Grasic Kuhar C et al. / Comparison of the quality of life between breast cancer patients and the Slovenian population 219 22. de Ligt KM, Heins M, Verloop J, Ezendam NPM, Smorenburg CH, Korevaar JC, et al. The impact of health symptoms on health-related quality of life in early-stage breast cancer survivors. Breast Cancer Res Treat 2019; 178: 703- 11. doi: 10.1007/s10549-019-05433-3 23. van der Willik KD, Koppelmans V, Hauptmann M, Compter A, Ikram MA, Schagen SB. Inflammation markers and cognitive performance in breast cancer survivors 20 years after completion of chemotherapy: a cohort study. Breast Cancer Res 2018; 20: 135. doi: 10.1186/s13058-018-1062-3 24. Zehra S, Doyle F, Barry M, Walsh S, Kell MR. Health-related quality of life following breast reconstruction compared to total mastectomy and breast-conserving surgery among breast cancer survivors: a systematic review and meta-analysis. Breast Cancer 2020; 27: 534-66. doi: 10.1007/ s12282-020-01076-1 25. Yfantis A, Sarafis P, Moisoglou I, Tolia M, Intas G, Tiniakou I, et al. How breast cancer treatments affect the quality of life of women with non-metastatic breast cancer one year after surgical treatment: a cross-sectional study in Greece. BMC Surg 2020; 20: 210. doi: 10.1186/s12893-020-00871-z 26. Yoon J, Malin JL, Tao ML, Tisnado DM, Adams JL, Timmer MJ, et al. Symptoms after breast cancer treatment: are they influenced by patient characteristics? Breast Cancer Res Treat 2008; 108: 153-165. doi: 10.1007/ s10549-007-9599-3 27. Joly F, Lange M, Dos Santos M, Vaz-Luis I, Di Meglio A. Long-term fatigue and cognitive disorders in breast cancer survivors. Cancers 2019; 11: 1896. doi: 10.3390/cancers11121896 28. 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Am J Prev Med 2012; 43: e1-24. doi: 10.1016/j.amepre.2012.04.027 Radiol Oncol 2023; 57(2): 220-228. doi: 10.2478/raon-2023-0027 220 research article Association between PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma treated with adjuvant systemic therapy Domen Ribnikar1,2, Valentina Jeric Horvat1, Ivica Ratosa2,3, Zachary W Veitch4, Biljana Grcar Kuzmanov5, Srdjan Novakovic6, Erik Langerholc7, Eitan Amir8, Bostjan Seruga1,2 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine Ljubljana, University of Ljubljana, Ljubljana, Slovenia 3 Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Division of Medical Oncology and Hematology, Royal Victoria Hospital, Bariie, Ontario, USA 5 Department of Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 6 Department of Molecular Diagnostics, Institute of Oncology Ljubljana, Ljubljana, Slovenia 7 Institute of Biostatistics and informatics, Faculty of Medicine Ljubljana, University of Ljubljana, Ljubljana, Slovenia 8 Division of Medical Oncology and Hematology, University of Toronto and Princess Margaret Cancer Centre, Toronto, Canada Radiol Oncol 2023; 57(2): 220-228. Received 13 February 2023 Accepted 18 May 2023 Correspondence to: Assoc. Prof. Boštjan Šeruga, M.D., Ph.D., Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: bseruga@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. The aim of the study was to evaluate the independent prognostic role of PIK3CA activating mutations and an association between PIK3CA activating mutations and efficacy of adjuvant endocrine therapy (ET) in patients with operable invasive lobular carcinoma (ILC). Patients and methods. A single institution study of patients with early-stage ILC treated between 2003 and 2008 was performed. Clinicopathological parameters, systemic therapy exposure and outcomes (distant metastasis-free sur- vival [DMFS] and overall survival [OS]) were collected based on presence or absence of PIK3CA activating mutation in the primary tumor determined using a quantitative polymerase chain reaction (PCR)-based assay. An association between PIK3CA mutation status and prognosis in all patient cohort was analyzed by Kaplan-Meier survival analysis, whereas an association between PIK3CA mutation and ET was analyzed in estrogen receptors (ER) and/or progester- one receptors (PR)-positive group of our patients by the Cox proportional hazards model. Results. Median age at diagnosis of all patients was 62.8 years and median follow-up time was 10.8 years. Among 365 patients, PIK3CA activating mutations were identified in 45%. PIK3CA activating mutations were not associated with differential DMFS and OS (p = 0.36 and p = 0.42, respectively). In patients with PIK3CA mutation each year of tamoxifen (TAM) or aromatase inhibitor (AI) decreased the risk of death by 27% and 21% in comparison to no ET, re- spectively. The type and duration of ET did not have significant impact on DMFS, however longer duration of ET had a favourable impact on OS. Conclusions. PIK3CA activating mutations are not associated with an impact on DMFS and OS in early-stage ILC. Patients with PIK3CA mutation had a statistically significantly decreased risk of death irrespective of whether they received TAM or an AI. Key words: invasive lobular carcinoma; PIK3CA mutation; endocrine therapy; genomics of invasive lobular carcinoma Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma 221 Introduction Breast cancer represents a spectrum of heterogene- ous diseases with different clinical behaviour and response to specific systemic therapy. Invasive lob- ular carcinoma (ILC) is the second most frequent subtype of breast cancer, representing around 10% of all breast cancer cases.1 It has been demonstrated that ILC is a special disease entity that differs from the more common invasive breast carcinomas such as invasive ductal carcinoma (IDC). These differences include risk factors, histological and clinical characteristics, transcriptional signatures and genomic profiles.2-4 ILCs usually arise in postmenopausal women, are larger in size at the time of diagnosis due to insidi- ous nature of its growth, but are typically of lower histological grade. The majority of ILC tumors ex- press hormone receptors such as estrogen recep- tors (ER), progesterone receptors (PR) but they less commonly exhibit human epidermal growth factor receptor-2 (HER-2) overexpression or ampli- fication.5 Data about prognosis of ILC vary sub- stantially; studies have demonstrated better6,7, the same8, and worse long-term overall survival (OS) in comparison to unselected invasive breast can- cers.9 Additionally, it has been reported that ILC is less responsive to chemotherapy (ChT).10-12 However, these tumors respond well to endocrine therapy (ET) given their biological profile.13 Results of an important retrospective study clearly showed an OS benefit of adjuvant ET for patients with ILC.14 Desmedt et al. conducted the largest genomic study of ILC and found that mutations in PIK3CA (phosphatidylinositol, catalytic unit), HER and ESR1 (estrogen receptor 1) genes are more fre- quently present in ILC tumors that in other inva- sive breast cancers. Furthermore, in their study, approximately 50% of ILC tumors harboured PIK3CA-PTEN-AKT1 signaling alterations. Of note, PIK3CA mutation was associated with lower histological grade and lower Ki-67 (proliferating) index.15 Mutations of PIK3CA gene are the most common somatic genetic alterations in ER-positive breast cancer and are associated with favorable breast cancer characteristics such as smaller tumor size, lower grade, ER positivity and increasing age.16 Investigators of the BIG 1-98 study which com- pared letrozole monotherapy and letrozole switch- ing strategy to tamoxifen (TAM) monotherapy reported that postmenopausal patient population with ILC may derive greater benefit from letrozole (AIs) than patients with IBC, NOS (invasive breast carcinoma, no otherwise specified).17 Furthermore, additional post-hoc analysis of the BIG 1-98 study showed that irrespective of the histological sub- type of breast cancer tumors with PIK3CA muta- tion derive greater benefit from letrozole than from tamoxifen.18 There are no data about the exact num- ber of ILC patients with present PIK3CA mutation in the BIG 1-98 study; however in the large meta- analysis by Zardavas et al. there were 366 out of 951 (39%) patients who had PIK3CA mutated ILC.16 In this study, we aimed to evaluate the inde- pendent prognostic role of PIK3CA activating mutations and an association between PIK3CA ac- tivating mutations and efficacy of ET in patients with operable ILC. We hypothesized that the pres- ence of PIK3CA activating mutations in primary ILC is associated with longer distant metastasis- free survival and overall survival and greater ben- efit of AIs and extended ET as compared to stand- ard 5-year treatment with tamoxifen. Patients and methods Patients After obtaining approval from the institutional review committee and ethical approval of the Ministry of Health of the Slovenian Republic (#0120-323/2019), we performed a retrospective analysis of a cohort of patients with early-stage ILC identified from a pathology database at the Institute of Oncology Ljubljana. Eligible pa- tients were treated between January 1st 2003 and December 31st 2008, thereby allowing at least 10 years of follow-up at the time of data analysis. Clinicopathological characteristics and determination of the PIK3CA mutation Formalin-fixed and paraffin embedded (FFPE) and hematoxylin and eosin stained tumor slides of all included patients were reviewed by an ex- perienced breast pathologist (BGK) to ensure that the diagnosis of ILC and its subtype(s) were cor- rect. We collected the following clinicopathologi- cal parameters from each patient′s from the exist- ing pathology reports: age at diagnosis, subtype of ILC, tumor size, nodal status, grade, mitotic count, ER and PR expression, HER-2 overexpression or amplification, lymphovascular invasion (LVI) and perineural invasion (PNVI). Ki-67 labeling index and tumor infiltrating lymphocytes (TILs) were additionally determined retrospectively by a sin- Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma222 gle breast cancer pathologist as these biomarkers were not assessed routinely in years of the cohort inception. Tumor grading was classified according to the Bloom-Richardson-Elston classification.19 ER and PR expression and HER-2 status were de- termined using the American Society of Clinical Oncology/College of American Pathologists guide- lines.20,21 Proliferation index Ki-67 was estimated with DAKO, Glostrup antibody MIB1 accord- ing to recommendations from the International Ki-67 in Breast Cancer Working Group.22 For the evaluation of TILs the recommendations of the 2014 International TILs Working Group were fol- lowed.23 PIK3CA status (wild-type or mutated) was determined for each patient. DNA isolation from the FFPE tumor samples was performed by macrodissection of tumor tissue from tumor slices containing at least 70% tumor cells. Isolation was performed using the MagMAX FFPE DNA/RNA Ultra Kit (Applied Biosystems, Thermo Fisher TABLE 1. Characteristics of the included patients and their tumors Characteristic All patients(n = 365; 100%) PIK3CA mutated (n = 164; 45%) PIK3CA non-mutated (n = 201; 55%) Median age (range) (yrs) 62.8 (33−90) 63.1 62.4 Median tumor size (mm) 21 19 21 Nodal status N0 N1 N2 N3 209 (57) 84 (23) 26 (7) 46 (13) 100 (61) 37 (22) 1 (7) 16 (10) 109 (54) 47 (23) 15 (8) 30 (15) Tumor grade G1 G2 G3 52 (14) 270 (74) 43 (12) 20 (12) 134 (82) 10 (6) 32 (16) 136 (68) 33 (16) IHC* subtype ER+/PR+/HER2- ER+/PR-/HER2- HER2 + ER-/PR-/HER2- 299 (82) 39 (11) 20 (6) 7 (2) 136 (46) 14 (36) 9 (45) 2 (29) 159 (54) 25 (64) 11 (55) 5 (71) Median Ki-67 (range) (%) 3 (1−50) 2.5 (1−50) 3 (1−40) Mitotic score M1 M2 M3 284 (78) 55 (15) 26 (7) 138 (84) 19 (12) 7 (4) 146 (73) 36 (18) 19 (9) Presence of LVI 24 (7) 4 (2) 20 (10) Median TILs (range) (%) 3 (1−50) 3 3 Median follow-up time (range) (yrs) 10.8 (0.1−18.6) 10.8 10.7 ER = estrogen receptor; HER = 2–human epidermal growth factor receptor-2; *IHC = immunohistochemically defined subtype; PR = progesterone receptor; LVI = lymphovascular invasion; TILs = tumor-infiltrating lymphocytes 78 patients had PIK3CA mutation on exon 9, 76 patients on exon 20; five patients had dual PIK3CA mutation on exon 9 and five patients had both, exon 9 and exon 20 PIK3CA mutations TABLE 2. Distribution of systemic therapy in all patients and according to PIK3CA mutation status Systemic therapy All pts N (%) PIK3CA mutated pts N (%) PIK3CA non-mutated pts N (%) None ET only ChT only ET and ChT 12 (3) 249 (68) 13 (4) 91 (25) 5 (3) 115 (70) 5 (3) 39 (24) 7 (3) 134 (67) 8 (4) 52 (26) None ET 25 (7) 10 (6) 15 (7) ET with TAM 106 (29) 45 (27) 61 (30) ET with AIs 127 (35) 54 (33) 73 (37) Sequence TAM-AI 107 (29) 55 (34) 52 (26) Median duration (range) of ET 5 (0.2−16.7) 5 (0.4−16.7) 5 (0.2−11.2) AIs = aromatase inhibitors; ChT = chemotherapy; ET = endocrine therapy; pts = patients, TAM = tamoxifen (TAM) Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma 223 Scientific, Austin, Tx, USA) according to the manu- facturer’s instructions. Isolated DNA was used to identify five common mutations in the PIK3CA gene using the PIK3CA Mutation Analysis Kit (EntroGen, Inc. Woodland Hills, CA, USA) ac- cording to the manufacturer’s protocol. This is a quantitative polymerase chain reaction (PCR) based assay that detects five hot spot mutations in the PIK3CA gene (NM_006218.2): c.1624G > A p.(Glu542Lys), c.1633G > A p.(Glu545Lys), c.1633G > C p.(Glu545Gln), c.3140A > G p.(His1047Arg) and c.3140A > T p.(His1047Leu). These five hot spot mu- tations represent around 80% of all PIK3CA muta- tions.24 Systemic treatment We collected all data about systemic therapy for each patient included into the study such as whether the patient received ChT, ET and HER- 2-targeted therapy. We also collected data about the type of ChT (cyclophosphamide – metotrex- ate-5-fluorouracil regimen (CMF), anthracycline- based chemotherapy, anthracyclines and taxa- nes, taxanes without anthracyclines), type of ET (tamoxifen monotherapy, AI monotherapy or a switching approach [TAM-AI]) and duration of ET (up to 5 years or more than 5 years). Statistical analysis The outcomes of interest were distant metastasis- free survival (DMFS) and overall survival (OS) defined as time from diagnosis (date of surgery) to distant relapse or death (whichever occurred first) and as time from diagnosis to death from any cause, respectively. The last date of a follow-up was October 31st 2021. Data were analyzed using R version 4.1.2 (Vienna, Austria). Descriptive statis- tics were used to describe patient characteristics. Age and duration of therapy were measured in years, tumor size was measured in millimeters, Ki- 67 index was measured as a proportion of positive tumor cells and logarithmised and for the number of positive axillary lymph nodes the square root was taken of in order to normalize the distribution of the variables and improve model fit. DMFS and OS were estimated using Kaplan-Meier analysis. Log-rank tests were performed to compare the survival of PIK3CA mutated and PIK3CA non-mu- tated cohorts of patients. Four Cox proportional hazard models were used to assess the impact of ET on survival of ER and/or PR-positive PIK3CA mutated patients: a simple and an advanced model for each of DMFS and OS. The simple Cox model regressed on only three variables; age at diagnosis, prior duration of tamoxifen and prior duration of AI therapy. The advanced model regressed on ad- ditional variables which are considered known or possible prognostic factors: tumor size, nodal sta- tus, histological grade, Ki-67 index, PR expression and location of PIK3CA mutation (exon 9 or exon 20), respectively. To avoid immortal time bias, any prior duration of tamoxifen and any prior duration of AI therapy were considered as time-dependent variables, while all other variables were known at a patients′ entry in the study (at the time of sur- gery). Finally, adjustment for multiple significance testing was performed using Holm’s method. An adjusted p-value ≤ 0.05 was considered statistically significant. Results Patients and their tumors Among an initial cohort of 428 patients, we ex- cluded 24 patients who were treated in other can- cer centers in Slovenia, nine patients who had de novo metastatic lobular cancer, eight patients who received neoadjuvant ChT, five patients with other histological subtypes of breast cancer (IBC, NOS, ILC and IBC, NOS), five patients with very small tumors (less than 5 mm in size) and five patients whose biopsies were from years before 2003. Two patients were lost very early in the follow up as they moved out of country, so they were excluded from the analysis. PIK3CA mutation status could 428 patients identified 365 patients eligible for analysis Excluded: 24 patients from other cancer centers 9 patients with de novo metastatic lobular carcinoma 8 patients who received neoadjuvant chemotherapy 5 patients with other histological type of breast cancer (IDC, IDC and ILC) 5 patients with a very small tumor (less than 5 mm) 5 biopsies from years before 2003 2 patients who moved abroad 5 tumors did not pass RT-PCR for determination of PIK3CA mutation status FIGURE 1. Consort diagram. Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma224 not be determined in five cases due to technical is- sues with the samples. This resulted in an analytic cohort of 365 patients (Figure 1; Consort diagram, for patient selection schema). Characteristics of in- cluded patients and their tumors are presented in Table 1. Among all patients, 164 (45%) patients had PIK3CA mutated ILC. Of these 78 (48%) patients had PIK3CA mutations in the helical domain (exon 9) and 76 (46%) patients had the mutation present in the kinase domain (exon 20). Five patients (1%) had dual PIK3CA mutations on exon 9 and five patients (1%) had PIK3CA mutations on both, exon 9 and 20. As shown in Table 1, PIK3CA mutated and PIK3CA non-mutated cohort of patients were well balanced according to prognostic tumor characteristics. Systemic therapy exposure of all included pa- tients is shown in Table 2. As expected, the major- ity of patients received ET with either TAM (106, 29%), AIs (127, 35%) or a switch approach TAM-AI (107, 29%). Ninety-one (25%) patients received ChT and ET and only 13 (4%) patients received ChT alone. Among 25 patients who did not receive any ET, 10 patients were PIK3CA mutated. ET agents were well balanced between PIK3CA mutated and PIK3CA non-mutated patients as shown in Table 2. Median duration of ET was 5 years in both groups of patients. 191 (52%) patients had extended ET and the longest duration of extended ET was 16.7 years. An association between PIK3CA mutation status and disease outcome Median OS was 15.7 years for patients with PIK3CA mutation and 14.6 years for patients with- out PIK3CA mutation. Furthermore, median DMFS for PIK3CA mutated and for PIK3CA non-mutated ILC patients was 15.6 and 13.4 years, respectively. The presence of PIK3CA mutation in early-stage ILC patients was not associated with differential OS (p = 0.42, Figure 2) or DMFS (p = 0.36, Figure 3). Overall, 81 (22%) patients developed distant me- tastases, of these 48 (24%) were PIK3CA non-mu- tated and 33 (16%) were PIK3CA mutated. Twenty (26%) patients of those who developed distant me- tastases had PIK3CA mutation on exon 20, 12 (15%) had PIK3CA mutation on exon 9 and only one (5%) patient had both, exon 9 and 20 PIK3CA mutation. An association between type and duration of ET and disease outcome in patients with PIK3CA mutation The effect of the type of systemic ET and its duration on DMFS Patients who received AIs in comparison to those who received tamoxifen were on average 10 years older at the time of diagnosis, therefore a Cox proportional hazards model was used for adjust- ment. In a simple model, neither type of ET nor its duration and patient age at the time of diagnosis were associated with the risk of relapse (Table 3). In advanced Cox proportional hazards model only a higher number of positive axillary lymph nodes increased the risk of distant relapse (HR 1.64, adj.p < 0.001; see Table 4). The effect of the type of systemic ET and its duration on OS We found that each year of aging increased the risk of death by 5%. Each year of treatment with tamoxifen decreased the risk of death by 27% in comparison to no ET for a patient of the same age (Table 5). Similarly, each year of treatment with AIs decreased the risk of death by 21% compared to no ET for a patient of the same age (Table 5). TABLE 3. Simple Cox proportional hazards model for DMFS in the PIK3CA mutated patient cohort Characteristic HR 95% CI Adj p Age 0.99 0.96−1.02 1.0 Duration of TAM 0.92 0.75−1.14 1.0 Duration of AIs 1.07 0.90−1.28 1.0 Adj p = adjusted p value; AIs = aromatase inhibitors; CI = confidence interval, HR = hazard ratio; TAM = tamoxifen, FIGURE 2. Kaplan-Meier graph showing association between PIK3CA mutation and overall survival. Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma 225 In the advanced Cox proportional hazards mod- el age at the time of diagnosis, grade 3 and higher number of positive axillary lymph nodes were all associated with higher risk of death (HR 1.05, adj. p = 0.014, HR 5.5, adj. p = 0.040 and HR 1.58, adj. p < 0.001, respectively). We also found that longer duration of ET with either tamoxifen or an AI had a favorable impact on OS (p = 0.010 for tamoxifen and p = 0.010 for an AI, respectively). In a time-de- pendent Cox analysis each year of prior treatment with tamoxifen decreased the risk of death by 32% in comparison to no prior ET for a patient with the same characteristics. Similarly, to tamoxifen, each year of prior AI therapy decreased the risk of death by 27% compared to no prior ET for a patient with the same characteristics (Table 6). Discussion ILC of the breast is a distinct entity with unique clinical, histological and molecular characteristics that differ from more common invasive breast can- cer subtypes. The majority of older studies demon- strated that the outcome of ILC patients was better, however more recent data have suggested ILC has worse outcome compared to invasive ductal car- cinoma, especially in the long term. It seems that delay and difficulty in early diagnosis, acquired resistance to conventional therapy and risk of late relapse pose challenges in management of patients with ILC.25 In this retrospective study our main goal was to evaluate the association between the PIK3CA mutational status and prognosis in patients with operable ILC. We found that PIK3CA mutation status has no association with either DMFS or OS. The median OS was 15.7 years for patients with PIK3CA mutation and 14.6 years for patients with PIK3CA wild type ILC. PIK3CA mutations are the most frequent somat- ic genetic alterations in ER+/HER-2– breast cancer.18 Prior data in early-stage disease have demonstrat- ed a favorable disease outcome in patients harbor- ing PIK3CA mutations.16 At least to some extent this could be explained by a relative mutual exclu- sion of other somatic genetic alterations which are associated with higher proliferation and increased risk of distant relapse such as TP53 mutations and amplifications of MYC (MYC proto - oncogene ) gene.18 Furthermore, recent work has shown that ultra-low risk breast cancers have higher expres- sion scores for the PIK3CA-mutation-associated genes.26 Previous data exploring the prognostic effect of specific somatic mutations in ER+/HER-2– early- stage breast cancer have shown that PIK3CA and MAP3K1 (mitogen-activated protein kinase 1) mu- tations co-associate and patients with tumors har- boring both mutations have a more favorable clini- cal course than those with only one gene mutation or without any of these two mutations.27 This may suggest that PIK3CA mutation alone is not prog- nostic in general breast cancer patient population, nor is it in ILC specifically. The majority of first- and second-generation gene-expression profiles that have been used for molecular prognostication in early-stage breast cancer were developed initially in invasive ductal carcinomas and therefore their use in ILC is uncer- tain. However recent data have shown they could also be informative for prognostication in ILC pa- tients. ILC tumors are quite homogenous clinically TABLE 4. Advanced Cox proportional hazards model for DMFS in the PIK3CA mutated patient cohort Characteristic HR 95% CI Adj p Age 1.01 0.97−1.05 1.0 Tumor size 0.99 0.57−1.71 1.0 Grade G1 G2 G3 Ref 0.91 1.03 0.26−3.22 0.16−6.83 1.0 1.0 Ki-67 1.35 0.93−1.95 1.0 PR 1 0.99−1.02 1.0 PIK3CA status Other than exon 20 Only exon 20 Ref 2.19 1.01−4.80 0.582 No. of positive axillary LNs 1.64 1.30−2.06 < 0.001 Duration of TAM 1.02 0.81−1.29 1.0 Duration of AIs 1.06 0.87−1.28 1.0 Adj p = adjusted p value; AIs = aromatase inhibitors; CI = confidence interval; HR = hazard ratio; LNs = lymph nodes; No. = number; PR = progesterone receptor; TAM = tamoxifen TABLE 5. Simple Cox proportional hazards model for OS in the PIK3CA mutated patient cohort Characteristic HR 95% CI Adj p Age 1.05 1.02−1.07 < 0.001 Duration of TAM 0.73 0.61−0.88 0.002 Duration of AIs 0.79 0.67−0.93 0.005 Adj p = adjusted p value; AIs = aromatase inhibitors; CI = confidence interval; HR = hazard ratio; TAM = tamoxifen Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma226 especially relating to classical prognostic features. The majority are grade 2, ER and PR positive, HER- 2 negative and have a relatively low proliferative activity.28 In contrast, it has been shown recently that a 194-gene signature called LobSig may be the best in prognosticating ILC tumors. LobSig out- performed Nottingham Prognostic Index (NPI), Prosigna ROR, Oncotype Dx and Genomic Grade Index (GGI) in a multivariate Cox proportional hazards model, especially in grade 2 ILC moder- ate NPI cases.29 The authors reported that ILCs that were associated with a high-risk score were enriched for mutations in HER-2, HER-3, TP53 (TP53 tumor suppressor gene), ROS1 (ROS proto- oncogene 1, receptor tyrosine kinase) and AKT1 (AKT serin - threonin protein kinase 1) genes. In contrast, those ILCs that had a good outcome (a low-risk score) had relatively few genomic altera- tions and interestingly, PIK3CA mutations were more common in the latter group than in the high risk ILC group. Based on all data discussed above it seems that PIK3CA mutation alone may not be an optimal genetic alteration to stratify ILC pa- tients into better and worse prognostic group but rather an integration of several concurrent genetic alterations might outperform PIK3CA mutation in an individual tumor. We also aimed to explore the predictive effect of PIK3CA mutation on benefit from ET. Efficacy of AIs as compared to tamoxifen have previously been explored and reported.18 In the analysis from the randomized BIG 1-98 trial, the authors demon- strated a greater magnitude of benefit with an AI letrozole in comparison to tamoxifen for patients with PIK3CA mutated early-stage breast cancer (HR 0.18; 95% CI 0.06 – 0.50). Additionally, the same study demonstrated that letrozole was as- sociated with a significantly reduced risk of dis- ease - free survival event (DFS) in patients with luminal A and luminal B ILC concluding the mag- nitude of benefit from AI was greater in patients with ILC versus invasive ductal carcinomas.30 In our study we did not find any substantial differ- ences between the effect of tamoxifen and AIs in PIK3CA mutated early-stage ILC patients when compared to patients who did not receive any ad- juvant therapy. We found that each year of treat- ment with tamoxifen decreased the risk of death by 27% in comparison to no ET for a patient of the same age. Similarly, each year of AI therapy de- creased the risk of death by 21% compared to no ET for a patient of the same age. In addition, in an advanced Cox model we found similar results. The duration of any ET, either with tamoxifen or an AI, was statistically significantly associated with OS. In patients with PIK3CA mutation each year of ta- moxifen and AIs treatment decreased the risk of death by 32% and 27% in comparison to a patient with the same tumor characteristics who did not receive any ET, respectively. However, we did not find any associations between type and duration of ET and DMFS. Only a higher number of positive axillary lymph nodes increased the risk of distant relapse of ILC in our cohort. An interesting study evaluating the relation- ship of PIK3CA mutations and response to short- term neoadjuvant AI therapy demonstrated that the presence of PIK3CA mutation did not pre- TABLE 6. Advanced Cox proportional hazards model for OS in the PIK3CA mutated patient cohort Characteristic HR 95% CI Adj p Age 1.05 1.02−1.08 0.014 Tumor size 1.38 0.90−2.10 0.84 Grade G1 G2 G3 Ref 1.41 5.52 0.59−3.38 1.67−18.18 1.0 0.04 Ki-67 0.94 0.72−1.22 1.0 PR 1 0.99−1.01 1.0 PIK3CA status Other than exon 20 Only exon 20 Ref 1.32 0.79−2.21 1.0 No. of positive axillary LNs 1.58 1.32−1.88 < 0.001 Duration of TAM 0.68 0.55−0.86 0.01 Duration of AI 0.73 0.60−0.88 0.01 Adj p = adjusted p value; AIs = aromatase inhibitors; CI = confidence interval; HR = hazard ratio; No. = number; PR = progesterone receptor; TAM = tamoxifen FIGURE 3. Kaplan-Meier graph showing association between PIK3CA mutation and distant metastasis-free survival Radiol Oncol 2023; 57(2): 220-228. Ribnikar D et al. / PIK3CA activating mutations and outcomes in early-stage invasive lobular breast carcinoma 227 clude a response to AIs. In this study suppression of Ki-67 proliferating index, which is a validated intermediate endpoint biomarker for endocrine sensitivity was used as a surrogate marker of re- sponse.31 Furthermore, another study showed that patients with PIK3CA mutated early-stage breast cancer either treated with tamoxifen or untreated had improved outcome in comparison to PIK3CA non-mutated patients.32 These data were concord- ant with previous findings from in vitro study showing that PIK3CA mutated cell lines were more sensitive to tamoxifen compared to PIK3CA wild type cell lines.33 Our study has several limitations. First, due to the retrospective nature of this study our findings need to be interpreted with caution and possibly validated in the prospective setting. Second, our cohort of ILC patients is very likely molecularly heterogeneous which limits the generalizability of our findings. Third, longer duration of ET in this study is associated with significantly improved OS but not DMFS. This indicates that the impact of immortal time bias may persist despite the use of time-dependent Cox model in our analysis. Fourth, we used a PCR based assay which detects five hot spot mutations in the PIK3CA gene representing about 80% of all PIK3CA activating mutations. Results could be different if we used an alternative method for determination of all PIK3CA mutations such as next generation sequencing (NGS). Finally, the biggest limitation of our study is that despite the use of Cox models confounding by indication for treatment might still have an impact on our re- sults. In conclusion, the presence of PIK3CA mutation was not associated with differential DMFS or OS in patients with operable ILC. Adjuvant ET with either tamoxifen or an AI significantly decreased the risk of death in PIK3CA mutated ILC cohort. Furthermore, there was no significant difference in efficacy between the two classes of endocrine agents. Longer duration of ET with either tamox- ifen or AIs decreases the risk of death in ILC pa- tients with PIK3CA mutation. Future prospective studies based on molecular analyses will hopeful- ly give us more exact answer about optimal endo- crine agent for specific subpopulations of patients with early-stage ILC. Acknowledgment The authors acknowledge the financial support from the Slovenian Research agency (research core funding No P3-0321) and unrestricted donation to the Institute of Oncology Ljubljana for research. References 1. Martinez V, Azzopardi JG. Invasive lobular carcinoma of the breast: inci- dence and variants. Histopathology 1979; 3: 467-88. doi: 10.1111/j.1365- 2559.1979.tb03029.x 2. Yoder BJ, Wilkinson EJ, Massoli NA. 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Biochem Biophys Res Commun 2006; 340: 469-75. doi: 10.1016/j.bbrc.2005.12.025 Radiol Oncol 2023; 57(2): 229-238. doi: 10.2478/raon-2023-0018 229 research article Subpleural fibrotic interstitial lung abnormalities are implicated in non-small cell lung cancer radiotherapy outcomes Makoto Ito1, Takuma Katano2, Hiroaki Okada1, Ami Sakuragi3, Yoshitaka Minami3, Souichiro Abe1, Sou Adachi1, Yukihiko Oshima1, Wataru Ohashi4, Akihito Kubo2, Takayuki Fukui5, Satoru Ito2, Kojiro Suzuki1 1 Department of Radiology, Aichi Medical University, Aichi, Japan 2 Department of Respiratory Medicine and Allergology, Aichi Medical University, Aichi, Japan 3 Department of Central Radiology, Aichi Medical University, Aichi, Japan 4 Department of Biostatistics, Clinical Research Center, Aichi Medical University, Aichi, Japan 5 Division of Chest Surgery, Department of Surgery, Aichi Medical University, Aichi, Japan Radiol Oncol 2023; 57(2): 229-238. Received 19 February 2023 Accepted 02 April 2023 Correspondence to: Dr. Makoto Ito, Department of Radiology, Aichi Medical University, 1-1 Yazako-Karimata, Nagakute, Aichi 480-1195, Japan. E-mail: itou.makoto.292@mail.aichi-med-u.ac.jp Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. The relationship between interstitial lung abnormalities (ILAs) and the outcomes of lung cancer radio- therapy is unclear. This study investigated whether specific ILA subtypes are risk factors for radiation pneumonitis (RP). Patients and methods. This retrospective study analysed patients with non-small cell lung cancer treated with radical-intent or salvage radiotherapy. Patients were categorised into normal (no abnormalities), ILA, and interstitial lung disease (ILD) groups. The ILA group was further subclassified into non-subpleural (NS), subpleural non-fibrotic (SNF), and subpleural fibrotic (SF) types. The Kaplan–Meier and Cox regression methods were used to determine RP and survival rates and compare these outcomes between groups, respectively. Results. Overall, 175 patients (normal, n = 105; ILA-NS, n = 5; ILA-SNF, n = 28; ILA-SF, n = 31; ILD, n = 6) were enrolled. Grade ≥2 RP was observed in 71 (41%) patients. ILAs (hazard ratio [HR]: 2.33, p = 0.008), intensity-modulated radio- therapy (HR: 0.38, p = 0.03), and lung volume receiving 20 Gy (HR: 54.8, p = 0.03) contributed to the cumulative inci- dence of RP. Eight patients with grade 5 RP were in the ILA group, seven of whom had ILA-SF. Among radically treated patients, the ILA group had worse 2-year overall survival (OS) than the normal group (35.3% vs 54.6%, p = 0.005). Multivariate analysis revealed that the ILA-SF group contributed to poor OS (HR: 3.07, p =0.02). Conclusions. ILAs, particularly ILA-SF, may be important risk factors for RP, which can worsen prognosis. These findings may aid in making decisions regarding radiotherapy. Key words: interstitial lung abnormality; subpleural fibrosis; non-small cell lung cancer; radiotherapy; radiation pneu- monitis; survival analysis Introduction Lung cancer is one of the most common and dead- liest cancers worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all cases.1,2 Radiotherapy is a typical non-surgical curative treatment for localised NSCLC.3 Recently, the integration of immunotherapy with chemo- radiation has revolutionised the treatment of lo- cally advanced NSCLC. For instance, the PACIFIC trial reported a 5-year overall survival (OS) rate of 42.9% and progression-free survival (PFS) rate of Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy230 who met these criteria, 9 who received stereotac- tic body radiation therapy for salvage and 4 who had a short follow-up duration (< 6 months) were excluded. We collected information from the re- maining 175 patients’ medical records, including age, sex, Eastern Cooperative Oncology Group performance status, smoking history, blood sam- pling, pulmonary function tests, pathology, and imaging data. Patients treated with radical-intent radiotherapy were staged according to the 8th edi- tion of the Union for International Cancer Control tumour–node–metastasis classification. This study was approved by the Ethics Committee of Aichi Medical University (approval no. 2021-545) with an opt-out approach regarding the analysis before this study. All procedures involved in the study adhered to the principles of the Declaration of Helsinki. The requirement for the acquisition of informed consent from patients was waived ow- ing to the retrospective nature of this study. Radiotherapy Patients were immobilised in the supine position using an external vacuum-type body mould and/ or thermoplastic body mask, and a CT scan with a 2-mm slice thickness was conducted for treatment planning. Respiratory motion was confirmed by obtaining CT images in both the expiratory and inspiratory phases or during shallow breathing. Both the target volume and normal organ struc- tures were contoured using treatment planning systems (CMS XiO, Elekta, St Louis, MO, USA; Eclipse, Varian Medical Systems, Palo Alto, CA, USA; or Maestro, MIM Software, Inc., Cleveland, OH, USA). The clinical target volume (CTV) was defined as a 0–5-mm expansion of the primary tumour and metastatic lymph nodes. To account for respiratory migration, a 5–15-mm margin was added to the CTV to define the planning target vol- ume (PTV). Radiotherapy was delivered by 6- or 10-MV x-rays from linear accelerators (Clinac iX or TrueBeam STx, Varian Medical Systems, Palo Alto, CA, USA). Overall, 60 Gy in 30 fractions was de- livered to the PTV, and 119 (68%) patients received prophylactic elective nodal irradiation of 40 Gy in 20 fractions to the lymph node area. In exceptional cases, 11 (6%) patients received dose escalation up to 64–70 Gy. Additionally, six (3%) patients underwent hy- pofractionated radiotherapy of up to 2.7–3 Gy per fraction to shorten the duration of treatment. The irradiation methods used were three-dimensional conformal or intensity-modulated radiotherapy 33.1% among individuals who received treatment with durvalumab, an anti-programmed cell death- ligand 1 antibody, after chemoradiotherapy for stage III NSCLC.4 However, radiation pneumonitis (RP) is a major concern with this treatment strat- egy. Patients with grade ≥ 2 RP (according to the Common Terminology Criteria for Adverse Events [CTCAE]) from prior chemoradiotherapy cannot use durvalumab.5 Additionally, RP causes respira- tory insufficiency that severely affects the quality of life, leading to poor prognosis and even death.6 Risk factors should be identified before treatment to avoid grade 2 or more severe RP. Various risk factors for RP have been reported, such as age, tumour size, and tumour location.7 Interstitial lung disease (ILD) is a significant pre- disposing condition. Radiotherapy for lung cancer complicated by ILD induces a high RP rate and borders on being contraindicated.8 In contrast, the influence of interstitial lung abnormalities (ILAs) on RP risk remains unclear. Studies investigating the relationship between ILAs and RP are limited, and their results are inconsistent; additionally, no consensus has been reached.9,10 ILAs are specific computed tomography (CT) findings that are po- tentially compatible with ILD in patients without clinical indication of the disease. ILAs had been originally described as non-dependent abnor- malities affecting more than 5% of any lung zone; however, they overlapped with other disease con- cepts.11 In 2020, the Fleischner Society clarified the definition of ILAs and further classified them into three subtypes—namely, non-subpleural (NS), subpleural non-fibrotic (SNF), and subpleural fi- brotic (SF).12 Nevertheless, there are no reports de- scribing lung cancer radiotherapy’s effect on these newly defined ILAs. Therefore, in this study, we retrospectively re- viewed the data of patients with NSCLC who un- derwent radiotherapy and conducted a survival analysis to investigate how the novel ILA subtypes relate to RP risk. Patients and methods Patients We retrospectively reviewed the medical records of patients with NSCLC treated with radiotherapy between January 2010 and November 2021. We included patients treated with radical-intent ra- diotherapy for locally advanced NSCLC or with salvage radiotherapy for locoregional recurrence postoperatively. Of the 188 consecutive patients Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy 231 TABLE 1. Patient characteristics Characteristic Normal (n = 105) ILA (n = 64) ILD (n = 6) p Age (years) 71 (41–90) 72 (60–86) 74 (63–86) 0.01 Sex (men) 82 (78%) 52 (81%) 6 (100%) 0.70 Performance status – – – 0.42 0 53 (50%) 37 (58%) 4 (66%) – 1 41 (39%) 19 (30%) 1 (17%) – 2 7 (7%) 7 (11%) 1 (17%) – 3 4 (4%) 1 (1%) 0 (0%) – Pack year 45 (0–171) 47 (0–122) 48.5 (20–84) 0.98 Pulmonary function test %VC (%) 99.1 (41.8–144.2) 103.0 (41.8–147.5) 86.9 (55.7–98.8) 0.08 FEV1 (L) 1.9 (0.6–3.6) 2.2 (0.9–3.5) 2.0 (1.5–2.1) 0.12 FEV1/FVC (%) 69.2 (34.1–98.1) 69.8 (36.0–85.0) 74.8 (62.2–86.8) 0.36 KL-6 (U/mL) 282 (151–3957) 370 (172–896) 393 (204–859) 0.65 Pathology – – – 0.01 Adenocarcinoma 59 (56%) 21 (33%) 2 (33%) – Squamous cell carcinoma 40 (38%) 38 (59%) 4 (67%) – Others 6 (6%) 5 (8%) 0 (0%) – Lower-lobe primary lesion (radical-intent) 19 (22%) 14 (33%) 3 (75%) 0.20 TNM classification (radical-intent) T classification – – – 0.53 1 13 (15%) 3 (7%) 0 (0%) – 2 23 (26%) 12 (28%) 2 (50%) – 3 19 (22%) 13 (30%) 0 (0%) – 4 32 (37%) 15 (35%) 2 (50%) – N classification – – – 0.32 0 7 (9%) 8 (19%) 0 (0%) – 1 16 (18%) 8 (19%) 1 (25%) – 2 42 (48%) 16 (37%) 2 (50%) – 3 22 (25%) 11 (26%) 1 (25%) – Stage – – – 0.40 II A 3 (3%) 1 (2%) 0 (0%) – II B 5 (6%) 6 (14%) 0 (0%) – III A 34 (39%) 15 (35%) 3 (75%) – Characteristic Normal (n = 105) ILA (n = 64) ILD (n = 6) p III B 36 (41%) 14 (33%) 0 (0%) – III C 9 (11%) 7 (16%) 1 (25%) – Concurrent chemotherapy 65 (62%) 33 (52%) 2 (33%) 0.20 Carboplatin + paclitaxel 30 (46%) 18 (55%) 2 (100%) – Cisplatin + docetaxel 22 (34%) 8 (24%) 0 (0%) – Cisplatin + vinorelbine 13 (20%) 7 (21%) 0 (0%) – Durvalumab 17 (16%) 9 (14%) 0 (0%) 0.83 PD-L1 (positive) 36 (34%) 23 (36%) 0 (0%) 0.99 Mutation – – – 0.99 EGFR 8 (7%) 5 (8%) 1 (17%) – ALK 6 (6%) 2 (3%) 1 (17%) – None/unknown 91 (87%) 57 (89%) 4 (66%) – Radiotherapy method – – – 0.08 3DCRT 80 (76%) 40 (63%) 5 (83%) – IMRT 25 (24%) 24 (37%) 1 (17%) – ENI 77 (73%) 38 (59%) 4 (67%) 0.06 Hypofractionated radiotherapy 3 (3%) 3 (5%) 0 (0%) 0.67 Dose escalation 6 (6%) 5 (8%) 0 (0%) 0.75 Lung dose V5 (%) 34.5 (4.3–90.8) 40.3 (5.8–95.7) 34.6 (10.6–66.8) 0.18 V10 (%) 27.1 (2.7–67.7) 32.4 (3.7–81.4) 27.8 (8.8–49.9) 0.19 V20 (%) 19.8 (1.1–44.8) 22.0 (1.0–48.0) 22.6 (7.3–33.0) 0.59 Mean (Gy) 10.8 (1.7–23.5) 11.8 (2.0–19.6) 12.3 (3.8–18.0) 0.53 Median follow-up (years) 1.8 (0.1–12.1) 1.3 (0.1–9.9) 0.9 (2.1–3.9) 0.06 Median follow-up: survivors (years) 2.9 (0.6–12.1) 1.9 (0.6–9.9) 0.9 (0.9–0.9) 0.51 Data are presented as median (range) or number (%). The p-values represent comparisons between the normal and ILA groups. ALK = anaplastic lymphoma kinase; EGFR = epidermal growth factor receptor; ENI = elective nodal irradiation; FEV1 = forced expiratory volume in 1 second; FEV1/FVC = forced expiratory volume % in 1 second; ILAs = interstitial lung abnormalities; ILD = interstitial lung disease; IMRT = intensity-modulated radiotherapy; KL-6 = Krebs von den Lungen-6; n = total number of patients; PD-L1 = programmed cell death-ligand 1; TNM = tumour–node–metastasis; VC = vital capacity percentage; Vx = percentage of lung volume receiving > x Gy; 3DCRT = three-dimensional conformal radiotherapy% Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy232 (IMRT). Two-step IMRT was used for the entire period or only as boost irradiation of 20 Gy in 10 fractions. The dose constraints were as follows: the goals for the global and spinal maximum doses were < 107% (< 125% allowed) and < 50 Gy (< 52 Gy al- lowed), respectively, whereas the goals for the lung volumes were 5 Gy (V5) < 60%, V20 < 25%, and mean lung dose < 12 Gy. Vx refers to the percentage of the lung volume receiving > x Gy. The goal for the mean heart dose was < 20 Gy. Allowance val- ues for lung and heart doses were not set; however, every effort was made to reduce them as much as possible. Chemotherapy and immunotherapy Patient age, general condition, and organ func- tion determined the appropriateness of concur- rent chemotherapy and its regimen. The most commonly used regimen was weekly carboplatin/ paclitaxel, and cisplatin/docetaxel or cisplatin/vi- norelbine was also administered. Durvalumab has been available to patients at our institution since October 2018. We performed CT after concurrent chemoradiation therapy was completed to ensure that there was no disease progression or grade ≥ 2 RP before administration. Durvalumab was intra- venously administered at a dose of 10 mg/kg every 2 weeks for 1 year. If grade 2 RP appeared, the ad- ministration was suspended until the patient re- covered to grade 1. We stopped administration in cases of serious adverse events, such as grade ≥ 3 RP or confirmed disease progression. ILA classification and treatment outcomes ILAs were defined according to the Fleischner Society classification.12 The grouping was primar- ily performed by two physicians: a chest physi- cian engaged in ILDs and a diagnostic radiologist engaged in chest radiology. They reviewed medi- cal records including past history, family history, and reasons for imaging studies before reviewing CT images. Next, they used diagnostic chest CT images taken before treatment for grouping. All patients were classified into the following three groups: normal (no abnormalities), ILA, and ILD. The ILA group was further subdivided into NS, SNF, and SF groups. This process was performed independently by a chest physician and a diagnos- tic radiologist without discussion and was later col- lated. Grouping of any discrepant cases was final- ized by consultation among the two, with another radiologist acting as an intermediary. Details of the CT protocols used for grouping are as follows. SOMATOM Definition AS/AS+/Flash (Siemens Healthcare, München, Germany) and Light Speed VCT VISION (General Electric Healthcare, Milwaukee, Wisconsin, USA) CT scanners were used. Slice thickness was 2–2.5-mm. Tube voltage was 120 kVp. Tube current was auto exposure con- trol. Scan mode was helical acquisition. Pitch factor was 0.8–1.5. Rotation time was 0.3–0.5. We measured the time to the event from the date of radiotherapy commencement. Toxicity was graded according to the CTCAE version 5.0. Grade 2 RP was considered an event with steroid ad- ministration for chest symptoms; administration TABLE 2. Clinical details of eight patients with grade 5 radiation pneumonitis Case Age ILA subcategory Radiotherapy method Concurrent chemotherapy Durvalumab Lung V20 (%) Mean lung dose (Gy) Days to death 1 64 SF IMRT Yes No 19.2 12.8 104 2 73 SF 3DCRT Yes No 21.4 13.9 210 3 73 SF IMRT No No 30.6 15.7 123 4 76 SF IMRT Yes Yes 34.1 17.9 155 5 77 SNF 3DCRT No No 33.2 16.5 155 6 79 SF IMRT Yes No 31.5 17.3 160 7 79 SF 3DCRT No No 19.4 9.7 40 8 80 SF 3DCRT No No 27.6 15.2 99 ILAs = interstitial lung abnormalities; IMRT = intensity-modulated radiotherapy; SF = subpleural fibrotic; SNF = subpleural non-fibrotic; V20 = percentage of lung volume receiving > 20 Gy; 3DCRT = three-dimensional conformal radiotherapy Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy 233 of general cough suppressants was not included. Survival analysis was performed for patients un- der radical treatment. Statistical analyses All statistical analyses were performed using EZR version 1.55 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) based on R and R com- manders.13 Patient characteristics were compared using Fisher’s exact test for categorical variables and Student’s t-test or the Mann–Whitney U test for continuous variables. Additionally, the Kaplan–Meier method was employed to estimate the cumulative incidence of RP and survival rates, and comparisons were performed using Gray’s or log-rank test with post-hoc Bonferroni analy- ses for multiple comparisons. Finally, a Cox pro- portional hazards model was used for univariate and multivariate (stepwise elimination) analyses to determine factors contributing to RP and sur- vival rates. Statistical significance was set at p < 0.05. Factors demonstrating p < 0.2 in the uni- variate analysis were included in the multivariate analysis. Receiver operating characteristic (ROC) curves were used to evaluate the relationship between RP and lung V20. The cut-off point was determined based on the Youden index, and well- balanced sensitivity and specificity values were obtained.14 TABLE 3. Univariate and multivariate analyses of the cumulative incidence of grade ≥ 2 radiation pneumonitis Parameter Univariate analysis Multivariate analysis HR (95% CI) p HR (95% CI) p ILA 2.95 (1.81–4.82) <0.001 2.33 (1.18–4.61) 0.01 Age (years) 1.02 (0.99–1.05) 0.14 0.99 (0.95–1.03) 0.78 Sex (men) 1.02 (0.57–1.84) 0.94 – – Performance status (0,1 vs. 2,3) 1.17 (0.56–2.44) 0.68 – – Pack year 1.01 (0.99–1.01) 0.27 – – %VC (%) 0.99 (0.98–1.01) 0.96 – – FEV1 (L) 1.01 (0.64–1.55) 0.99 – – FEV1/FVC (%) 0.99 (0.97–1.01) 0.67 – – KL-6 (U/mL) 1.00 (0.99–1.01) 0.09 1.00 (0.99–1.01) 0.13 Pathology (adenocarcinoma) 0.58 (0.35–0.96) 0.04 0.85 (0.42–1.72) 0.65 Lower-lobe primary lesion 1.22 (0.69–2.12) 0.49 – – T classification (T4 vs. others) 0.81 (0.47–1.39) 0.44 – – N classification (positive) 1.18 (0.51–2.72) 0.70 – – Concurrent chemotherapy 1.17 (0.72–1.88) 0.53 – – Durvalumab 1.32 (0.72–2.40) 0.37 – – PD-L1 1.11 (0.54–2.25) 0.78 – – Mutation 1.24 (0.62–2.49) 0.54 – – Radiotherapy method (IMRT) 0.69 (0.39–1.19) 0.18 0.38 (0.16–0.91) 0.03 ENI 1.06 (0.65–1.75) 0.81 – – Lung dose V5 (%) 2.14 (0.67–6.82) 0.19 * * V10 (%) 3.93 (0.85–18.2) 0.08 * * V20 (%) 34.8 (3.05–396.4) 0.004 54.8 (1.52–1977.0) 0.03 Mean (Gy) 1.10 (1.04–1.17) <0.001 * * CI = confidence interval; ENI = elective nodal irradiation; FEV1 = forced expiratory volume in 1 second; FEV1/FVC = forced expiratory volume % in 1 second; HR = hazard ratio; ILAs = interstitial lung abnormalities; IMRT = intensity-modulated radiotherapy; KL-6 = Krebs von den Lungen-6; N = node; PD-L1 = programmed cell death-ligand 1; RP = radiation pneumonitis; T = tumour; %VC = vital capacity percentage; Vx = percentage of lung volume receiving > x Gy; vs. = versus * Variables with high multicollinearity were excluded. Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy234 Results Patient characteristics The final analysis included 175 patients, with the normal, ILA, and ILD groups comprising 105, 64, and 6 patients, respectively. Table 1 summarises patient characteristics. The p-values represent comparisons between the normal and ILA groups. The normal group was younger and had more patients with adeno- carcinomas than the ILA group. The median fol- low-up period of the 70 surviving patients was 2.5 (range, 0.6–12.1) years. The 64 patients with ILA were subcategorised into NS, SNF, and SF sub- types comprising 5, 28, and 31 patients, respective- ly. Examples of the ILA subcategories are shown in Figure 1. The two specialists (a chest physician and a diagnostic radiologist) matched on 125 of the 175 patient classifications (72%), and the remain- ing cases were grouped by discussion among the three physicians. Radiation pneumonitis Grade ≥ 2 RP was identified in 71 (41%) patients. Of the 105 patients in the normal group, 21 (20%), 8 (8%), and 1 (1%) exhibited grades 2, 3, and 4, respec- tively. Of the 64 patients in the ILA group, grades 2, 3, 4, and 5 were observed in 19 (30%), 8 (13%), 2 (3%), and 8 (13%) individuals, respectively. Of the six patients in the ILD group, 2 (33%) and 3 (50%) showed grades 2 and 3, respectively. Seven of the eight patients with grade 5 RP were in the ILA-SF group, and one was in the ILA-SNF group (details shown in Table 2). Figure 2 shows the cumulative incidence of grade ≥ 2 RP by group or subgroup. The incidence of RP was significantly higher in the ILA group than in the normal group (p < 0.001), particularly between the SNF (p = 0.014) and SF groups (p < 0.001) and the normal group. Table 3 presents the results of the univariate and multivariate analyses. ILAs (hazard ratio [HR]: 2.33, p = 0.01), IMRT (HR: 0.38, p = 0.03), and lung V20 (HR: 54.8, p = 0.03) were significantly associated with the cumu- lative incidence of grade ≥ 2 RP in the multivariate analyses. ROC analysis using V20 showed intermediate grade ≥ 2 RP predictive accuracy in the normal group; the area under the curve (AUC) was 0.72 (95% confidence interval [CI]: 0.60–0.83). The sen- sitivity and specificity for a cut-off value of 21% for V20 were 76% and 67%, respectively. Conversely, the predictive accuracy in the ILA group was low, with an AUC of 0.59 (95% CI: 0.45–0.75); further- more, the sensitivity and specificity for a cut-off value of 19% were 76% and 56%, respectively. During follow-up after radiotherapy, five patients received molecular-targeted therapies, and two had grade ≥ 2 RP. However, it was not until 2 years and 10 years after radiotherapy that the two pa- tients, respectively, began receiving molecular-tar- geted therapies, which had already cured their RP. Survival The 2-year OS and PFS rates for the 134 radically treated patients were 47.8% (95% CI: 38.5–56.4%) and 21.2% (95% CI: 14.4–28.9%), respectively. Patients in the ILA group had worse 2-year OS than those in the normal group (35.3% vs. 54.6%, p = 0.005); however, the multivariate analysis showed TABLE 4. Characteristics of patients treated with radical-intent radiotherapy Characteristic Normal (n = 87) ILA-SF (n = 24) p Age (years) 69 (41–90) 73 (60–82) 0.09 Performance status – – 0.39 0 41 (47%) 12 (50%) – 1 36 (41%) 8 (33%) – 2 6 (7%) 4 (17%) – 3 4 (5%) 0 (0%) – Pack year 45 (0–171) 53 (0–122) 0.38 KL-6 (U/mL) 294 (151–1607) 399 (208–705) 0.91 Pathology – – 0.09 Adenocarcinoma 46 (53%) 7 (29%) – Squamous cell carcinoma 35 (40%) 14 (58%) – Others 6 (7%) 3 (13%) – Lower-lobe primary lesion 19 (22%) 6 (25%) 0.79 T classification – – 0.36 1 13 (15%) 1 (4%) – 2 23 (26%) 10 (42%) – 3 19 (22%) 5 (21%) – 4 32 (37%) 8 (33%) – Concurrent chemotherapy 58 (67%) 13 (54%) 0.33 Durvalumab 15 (17%) 1 (4%) 0.19 Mutation 8 (13%) 3 (18%) 0.70 Median follow-up: survivors (years) 2.8 (0.6–12.1) 1.5 (0.7–6.0) 0.46 Data are presented as median (range) or number (%). ILA-SF = subpleural fibrotic interstitial lung abnormalities; KL-6 = Krebs von den Lungen-6; n = total number of patients Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy 235 no significant difference. No significant difference was found in the 2-year PFS between the ILA and normal groups (17.3% vs 24.1%, p = 0.26). The characteristics of the patients treated with radical-intent radiotherapy are summarised in Table 4. The parameters to be entered into the mul- tivariate analysis are listed separately for the nor- mal and ILA-SF groups. No significant bias was found between the two groups; however, only one patient in the ILA-SF group received durvalumab. Patients in the ILA- SF group had significantly worse 2-year OS (29.2% vs 54.6%, p < 0.001) and PFS (9.7% vs 24.1%, p = 0.009) rates than those in the normal group. Table 5 shows the results of the univariate and multivari- ate analyses for the OS and PFS rates. The multivariate analysis revealed that ILA-SF was the only independent adverse factor for OS (HR: 3.07, 95% CI: 1.17–8.10, p = 0.02) and that it also tended to influence PFS (HR: 1.95, 95% CI: 0.91– 4.14, p = 0.08). In contrast, younger age, adenocarci- noma, concurrent chemotherapy, and durvalumab treatment contributed to the prolongation of OS and/or PFS. Discussion To the best of our knowledge, this is the first study to classify patients with NSCLC according to the Fleischner Society ILA subtypes and to demon- strate subtype-associated risk with respect to ra- diotherapy outcomes. In our analysis, ILAs were considered risk factors for grade ≥ 2 RP. Among radically treated patients, the ILA group showed significantly worse 2-year OS than the normal group. This was particularly true for the ILA-SF group, which also independently contributed to OS in the multivariate analysis. To date, only a few studies have examined the relationship between ILAs and RP. A previous study retrospectively analysed the association be- tween RP and original ILA scores in 145 patients with NSCLC.9 The ILA scores were rated as fol- lows: 0, no interstitial lung change; 1, ILAs with- out honeycombing (ground-glass attenuation, fine reticular opacity, and microcysts); and 2, honey- combing. They concluded that abnormalities, with or without honeycombing, were predictors of RP. Several reports indicate that ILA is a risk factor for RP in patients with small -cell lung cancer.15,16 Conversely, another study concluded that ILAs did not independently contribute to RP.10 This discrep- ancy may be due to the ambiguous previous defi- nition of ILAs. Moreover, previous studies have FIGURE 1. Examples of interstitial lung abnormality subcategories. (A) non-subpleural, (B) subpleural non-fibrotic, and (C) subpleural fibrotic. White arrows point to lesions. A B C A B FIGURE 2. Cumulative incidence of grade ≥ 2 radiation pneumonitis by group (A) and subgroup (B). ILAs = interstitial lung abnormalities; ILD = interstitial lung disease; NS = non-subpleural; SF = subpleural fibrotic; SNF = subpleural non-fibrotic Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy236 tended to confuse smoking-related centrilobular nodularity and pleuroparenchymal fibroelastosis with ILAs.19,20 These disease concepts are explic- itly excluded from the novel definition of ILAs. Awareness of the improved definition of ILAs is essential for correct diagnosis, which should be made before radiotherapy to guide treatment strat- egy. Grade ≥ 2 RP occurred at a rate as high as 71 (41%) patients in this study. We believe that one of the important factors was the large number of pa- tients with ILA included, 64 (37%), as a character- istic of the institution. If the presence of ILA had been accurately recognised in advance, counter- measures could have been taken. The leading treatment-related factor that pre- dicts RP is lung V20.19,20 V20 should be further reduced when radiotherapy is administered to patients with ILAs. Moreover, the use of IMRT should be considered along with techniques such as virtual lung block and end-inspiration irra- diation.21,22 However, the ROC analysis in the ILA group showed low predictive accuracy for grade ≥ 2 RP using V20 (AUC = 0.59). This finding implies that reducing the lung dose in patients with ILAs may not necessarily preclude severe RP. ILA subtype divisions demonstrated notable differences. The ILA-SF group had the highest cumulative incidence of grade ≥ 2 RP, corroborat- TABLE 5. Univariate and multivariate analyses for overall and progression-free survival rates Parameter Overall survival Progression-free survival Univariate Multivariate Univariate Multivariate HR (95% CI) P HR (95% CI) p HR (95% CI) p HR (95% CI) p ILA-SF 2.59 (1.51–4.43) <0.001 3.07 (1.17–8.10) 0.02 1.88 (1.16–3.05) 0.01 1.95 (0.91–4.14) 0.08 Age (years) 1.02 (0.99–1.04) 0.09 0.95 (0.91–0.99) 0.04 1.01 (0.98–1.03) 0.59 – – Sex (men) 1.21 (0.70–2.10) 0.49 – – 0.91 (0.58–1.45) 0.71 – – Performance status (0,1 vs. 2,3) 1.90 (1.07–3.39) 0.03 0.82 (0.19–3.38) 0.78 2.81 (1.66–4.78) <0.001 1.35 (0.46–3.93) 0.59 Pack year 1.01 (1.00–1.01) 0.04 1.01 (0.99–1.02) 0.14 1.00 (0.99–1.01) 0.67 – – %VC (%) 0.99 (0.98–1.01) 0.56 – – 0.99 (0.98–1.01) 0.51 – – FEV1 (L) 0.84 (0.55–1.29) 0.43 – – 0.92 (0.62–1.36) 0.68 – – FEV1/FVC (%) 0.99 (0.97–1.02) 0.92 – – 1.01 (0.98–1.02) 0.75 – – KL-6 (U/mL) 1.00 (0.99–1.01) 0.11 1.00 (0.99–1.01) 0.34 1.01 (1.00–1.02) 0.03 1.00 (0.99–1.01) 0.40 Pathology (adenocarcinoma) 0.46 (0.31–0.69) <0.001 0.18 (0.08–0.44) <0.001 0.54 (0.36–0.79) 0.002 0.34 (0.18–0.65) 0.001 Lower-lobe primary lesion 1.60 (1.02–2.53) 0.04 1.03 (0.39–2.68) 0.95 1.09 (0.72–1.65) 0.69 – – T classification (T4 vs. others) 1.15 (0.74–1.79) 0.53 – – 1.47 (0.99–2.17) 0.05 1.53 (0.78–2.99) 0.21 N classification (positive) 1.02 (0.49–2.12) 0.95 – – 0.88 (0.48–1.60) 0.67 – – Concurrent chemotherapy 0.51 (0.33–0.80) 0.004 0.19 (0.07–0.56) 0.002 0.60 (0.41–0.89) 0.01 0.65 (0.31–1.30) 0.22 Durvalumab 0.22 (0.08–0.60) 0.003 0.43 (0.11–1.69) 0.22 0.34 (0.18–0.65) 0.001 0.40 (0.17–0.93) 0.03 PD-L1 (positive) 0.77 (0.39–1.50) 0.44 – – 1.03 (0.59–1.79) 0.92 – – Mutation 0.76 (0.47–1.22) 0.26 – – 0.96 (0.64–1.42) 0.83 – – Radiotherapy method (IMRT) 0.75 (0.43–1.30) 0.31 – – 0.86 (0.55–1.34) 0.50 – – ENI 0.83 (0.49–1.38) 0.47 – – 0.99 (0.64–1.56) 0.99 – – Lung dose V5 (%) 0.45 (0.12–1.72) 0.24 – – 0.56 (0.19–1.66) 0.30 – – V10 (%) 0.37 (0.06–2.27) 0.29 – – 0.44 (0.10–1.92) 0.28 – – V20 (%) 0.36 (0.02–5.27) 0.46 – – 0.23 (0.02–2.27) 0.21 – – Mean (Gy) 0.98 (0.93–1.04) 0.48 – – 0.97 (0.93–1.02) 0.29 – – CI = confidence interval; ENI = elective nodal irradiation; FEV1 = forced expiratory volume in 1 second; FEV1/FVC = forced expiratory volume % in 1 second; HR = hazard ratio; ILA-SF = subpleural fibrotic interstitial lung abnormalities; IMRT = intensity-modulated radiotherapy; KL-6 = Krebs von den Lungen-6; PD-L1 = programmed cell death- ligand 1; %VC = vital capacity percentage; Vx = percentage of lung volume receiving > x Gy. Radiol Oncol 2023; 57(2): 229-238. Ito M et al. / Subpleural fibrotic interstitial lung abnormalities and radiotherapy 237 ing previous studies that have shown ILA-SF to be 6.6 times more likely to progress than other sub- types.23 Contrarily, no difference was observed in the cumulative incidence of grade ≥ 2 RP between the ILA-NS and normal groups. The course of ILA-NS is usually non-progressive, suggesting a favourable prognosis.21 ILA-NS may not be a risk factor for RP; nonetheless, we emphasise that there were few patients with ILA-NS in this study; there- fore, we cannot mention its clinical significance. Therefore, this subtype should be investigated in more eligible patients in the future. No consensus exists that patients with ILAs have a poor prognosis after radiotherapy. However, studies have reported that the outcomes of surgery and chemotherapy for patients with lung can- cer and ILAs are poor.24,25 In this study, all eight patients with grade 5 RP were in the ILA group, and seven of these patients were in the ILA-SF cat- egory. We believe that the severity of RP worsened the prognosis of the ILA group, particularly that of patients with the ILA-SF subtype. The advent of durvalumab has greatly improved the prognosis of NSCLC; however, it is difficult to administer in the long term, particularly in patients with ILA- SF. Death sometimes results from severe RP rather than cancer; therefore, the indication for radiother- apy should be carefully considered. Our study had some limitations, particularly its retrospective and single-institution design. Additionally, other factors, such as emphysema, chronic obstructive pulmonary disease, and bio- markers (e.g., transforming growth factor-beta), may contribute to RP.7 These factors, which we did not consider in this study, may be confound- ers. Furthermore, many of our patients were el- derly and frail and were treated with radiother- apy alone. We also included several cases from the period before the emergence of durvalumab. These differ from modern standard treatments and may have implications for survival analysis. Retrospective studies on cases with durvalumab have been reported recently26, and high-quality fu- ture prospective trials are warranted Conclusions The novel definition of ILAs and ILA subtypes may be important in determining risk for RP fol- lowing radiotherapy in patients with NSCLC. RP symptoms can be severe, especially in patients with ILA-SF, and may worsen prognosis. 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Kashihara T, Nakayama Y, Okuma K, Takahashi A, Kaneda T, Katagiri M, et al. Impact of interstitial lung abnormality on survival after adjuvant durvalum- ab with chemoradiotherapy for locally advanced non-small cell lung cancer. Radiother Oncol 2023; 180: 109454. doi: 10.1016/j.radonc.2022.109454 Radiol Oncol 2023; 57(2): 239-248. doi: 10.2478/raon-2023-0017 239 research article The influence of BCL2, BAX, and ABCB1 gene expression on prognosis of adult de novo acute myeloid leukemia with normal karyotype patients Zlatko Pravdic1, Nada Suvajdzic Vukovic1,2, Vladimir Gasic3, Irena Marjanovic3, Teodora Karan-Djurasevic3, Sonja Pavlovic3, Natasa Tosic3 1 Clinic of Hematology, Clinical Center of Serbia, Belgrade, Serbia 2 School of Medicine, University of Belgrade, Serbia 3 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia Radiol Oncol 2023; 57(2): 239-248. Received 12 January 2023 Accepted 30 March 2023 Correspondence to: Prof. Nataša Tosić, Ph.D., Laboratory for Molecular Biomedicine, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11042 Belgrade 152, Serbia. E-mail: natasa.tosic@imgge.bg.ac.rs Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Deregulation of the apoptotic process underlies the pathogenesis of many cancers, including leu- kemia, but is also very important for the success of chemotherapy treatment. Therefore, the gene expression profile of main apoptotic factors, such as anti-apoptotic BCL2 (B-cell lymphoma protein 2) and pro-apoptotic BAX (BCL2- associated X), as well as genes involved in the multi-drug resistance (ABCB1), could have significant impact on the prognosis and could be used as targets for specific therapy. Patients and methods. We analyzed the expression of BCL2, BAX, and ABCB1 in bone-marrow samples collected at diagnosis from 51 adult patients with acute myeloid leukemia with normal karyotype (AML-NK) using real-time poly- merase chain reaction method, and examined their prognostic potential. Results. Increased expression of BCL2 (BCL2+) was associated with the presence of chemoresistance (p = 0.024), while patients with low BAX expression were more prone to relapse (p = 0.047). Analysis of the combined effect of BCL2 and BAX expression showed that 87% of patients with BAX/BCL2low status were resistant to therapy (p = 0.044). High expression of ABCB1 was associated with BCL2+ status (p < 0.001), and with absence FLT3-ITD mutations (p = 0.019). Conclusions. The present analysis of BCL2, BAX, and ABCB1 gene expression profiles is the first study focusing solely on AML-NK patients. Preliminary results showed that patients with high BCL2 expression are likely to experience resistance to chemotherapy, and may benefit from specific anti-BCL2 treatment. Further investigations conducted on a larger number of patients could elucidate actual prognostic significance of these genes in AML-NK patients. Key words: acute myeloid leukemia with normal karyotype; BCL2; BAX; BCL2/BAX ratio; ABCB1; prognosis Introduction Acute myeloid leukemia (AML) is a malignant he- matological disease that occurs as a result of dif- ferentiation arrest, uncontrolled proliferation and diminished apoptosis of myeloid progenitor cells. It is the most common acute leukemia in adults, accounting for about 80% of all cases.1 Despite re- markable progress in uncovering the molecular- genetic changes underlying the pathogenesis of AML, a little has changed in the initial treatment of patients which is still based on the classification of patients into risk groups according to pretreat- ment karyotype analysis.2 The largest karyotype based risk group is AML with normal karyotype (AML-NK), representing almost 50% of de novo Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia240 adult AML cases. AML-NK is a highly heteroge- neous group with respect to genetic abnormali- ties and clinical outcome of the patients, but the whole group is still stratified into intermediate risk group. Some of molecular markers such as mutations in fms-related tyrosine kinase-3 (FLT3), nucleophosmin (NPM1), CCAAT/enhancer bind- ing protein alpha (CEBPA) and runt-related tran- scription factor 1 (RUNX1) gene have made an impact on prognosis of AML-NK patients, and have already been included into the revised World Health Organization (WHO) classification of mye- loid neoplasms and acute leukemia, and European LeukemiaNet (ELN).2,3 However, there is a con- stant need for the introduction of new molecular markers that have significant impact on the prog- nosis of patients. Since diminished apoptosis is one of the hall- mark traits of leukemic cells, research focused on the analysis of the expression profile of the main participants in the apoptotic process can be of great importance in the detection of new prognos- tic-relevant molecular markers of AML. Also, since the mechanism of action of cytotoxic drugs used in the treatment of AML involves activation of apop- totic process, the expression pattern of apoptotic factors could have an impact on the occurrence of resistance. Namely, multi drug resistance (MDR) is a main clinical obstacle to successful cancer treat- ment. Resistance to chemotherapy treatment in AML is still a major cause for initial treatment fail- ure and relapse of the disease, and it is caused by multifactorial mechanisms involving genetic fac- tors.4,5 Two main mechanisms of MDR are: pump (transport) resistance, associated with increased expression of proteins involved in drug efflux, and non-pump (apoptotic) resistance, associated with increased activity of anti-apoptotic system.6 Gene expression of the ATP-binding cassette (ABC) su- perfamily of membrane transporters (ABCB1 for- merly known as MDR1 gene) is a marker of pump- resistance, while markers of non-pump resistance are levels of expression of BCL2 and BAX genes.7-9 These markers could be considered as pharma- cotranscriptomics markers and their analysis in AML patients could be a basis for the assessment of their role in tumor resistance to several groups of drugs. The inhibitors of these markers might ef- fectively reverse MDR in AML patients.10 The process of apoptosis is under the control of two distinct but interconnected pathways, intrin- sic and extrinsic. Activation of intrinsic pathway is under the control of the BCL family of proteins. B-cell lymphoma 2 (BCL2) family of proteins in- clude both pro-apoptotic (BAX, BAK) and anti- apoptotic members (BCL2, BCL-XL, MCL1).11 BCL2 is the best-known member of BCL2-familly, with an anti-apoptotic function. Its high expression has been reported throughout the evolution of AML, at presentation, relapse and also during treatment resistance. Moreover, increased BCL2 correlated with failure to achieve complete remission (CR) and with shorter overall survival (OS) of AML patients, making it an important therapeutic tar- get.12,13 Indeed, these findings led to the design of potent and selective BCL2-inhibitor, venetoclax. This modern BCL2-inhibitor is used in combina- tion with classical therapy improving outcome in patients that are ineligible for intensive chemo- therapy.14 BCL2-associated X (BAX) is a pro-apoptotic pro- tein, transcriptionally activated by the tumor sup- pressor p53. BAX is essential in the final stages of apoptotic process and its activation leads to release of cytochrome c from mitochondria and direct cell death.15 Some studies found that high expres- sion of BAX is good prognostic marker in AML, while others failed to prove its prognostic signifi- cance.16-20 Because of the existing inconsistencies in as- sessment of the individual impact of BCL2 and BAX expression level on AML prognosis, research- ers have resorted to BAX/BCL2 ratio analysis.9,19 Namely, BAX and BCL2 regulate apoptotic process by binding to each other and thus forming heter- odimers. The BAX/BCL2 ratio determines the cell fate after apoptotic stimuli has been received. The overexpression of ABCB1 gene is consid- ered to be independent factor for the occurrence of multi-drug resistance in AML. ABCB1 gene is located at chromosome 7q21.31, and it encodes 120 kb permeability glycoprotein (P-gp), a member of ATP-binding cassette (ABC) superfamily of trans- porter proteins, also called adenosine triphosphate binding cassette transporter B1 (ABCB1). P-gp is an efflux pump, transporting toxic substances out of the cell.21,22 By decreasing intracellular concentra- tions of drugs P-gp confers resistance to a large number of therapeutics used in clinical oncology. Also, P-gp has a drug-independent role in AML causing the inhibition of apoptosis in AML blast cells via modulation of a sphingomyelin-ceramide pathway.23 In order to show how the expression level of main apoptotic factors, such as BCL2 and BAX, can influence the occurrence of resistance, and wheth- er their influence is independent from the impact of multi drug resistance (ABCB1) gene expression Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia 241 level, in this study we investigated the expression pattern of these genes, and examined the possi- bility of their mutual influence on the prognosis in AML-NK patients. In doing so, the expression level of these genes was analyzed in the context of other already established prognostic molecular markers. In this way, we aimed to determine how the expression pattern of these genes can be used for a more precise stratification of AML-NK pa- tients into risk groups. Patients and methods Patients and therapy protocol Bone marrow (BM) samples from the 51 newly di- agnosed AML-NK patients (25 females, 26 males; median age 51 years, range 23‒62 years) were col- lected at Clinic of Hematology, Clinical Center of Serbia. Research was conducted in accordance with the ethical standards of the World Medical Association’s Declaration of Helsinki. The study was approved by the Ethics Commit-tee of the Clinical Center of Serbia (No. 110/11), and written informed consent was obtained for all patients. Diagnostic procedures comprised cytomorphol- ogy, cytogenetics, and immunophenotyping of BM. Morphologic diagnosis was made according to the French-American-British (FAB) classification.24 Conventional G-band karyotyping was employed for cytogenetic analysis.25 Immunophenotyping by flow cytometry (FACS Calibur, BD Biosciences, USA) was carried out systematically in the whole group of patients according to standard protocols based on European LeukemiaNet (ELN), Work Package 10 (WP10) criteria.26 All patients received induction and consolida- tion chemotherapy with daunorubicin and cyta- rabine according to the protocol 3 + 7, followed by three consolidation cycles of high/intermediate doses of cytarabine.2 Patients aged ≤ 55 years un- derwent allogeneic stem cell transplantation (SCT), in total 15 (25.42%) patients. Definitions of CR, over- all survival (OS), disease free survival (DFS) and resistance were established by proposed criteria.27 Gene expression and mutational analyses Bone marrow mononuclear cells (BMMCs) from AML-NK patients and from 14 healthy controls (BM donors, 8 males and 6 females, median age 31 years), were purified on Ficoll-PaqueTM Plus (GE Healthcare, Buckinghamshire, UK) density gradient, suspended in TRI Reagent (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and total RNA was extracted according to man- ufacturer’s instructions. In brief, mononuclear cells where first homogenized in TRI Reagent, and then separation phase was initiated by add- ing chloroform and subsequent certification. Total RNA was precipitated from the aqueous phase using isopropyl alcohol, pelleted and washed in 70%‒75% ethanol. One microgram of total RNA was used for the cDNA synthesis using RevertAid Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Real time-PCR was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems). For expression anal- ysis of ABCB1 SYBR®Green chemistry was used and PCR reaction (10 µl) consisted of 1µl of cDNA (50 ng RNA equivalent) SYBR™ Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), primers in final concentration 400 nM (ABCB1 for- ward 5`-GTC TAC AGT TCG TAA TGC TGA CGT and ABCB1 reverse 5`-TGT GAT CCA CGG ACA CTC CTA C). In the case of BCL2 and BAX, expres- sion analysis was done as previously described.28 For all target genes GAPDH gene was used as endogenous control, and all reactions were run in duplicate. Relative quantification analysis was performed using comparative ddCt method, using healthy controls as calibrator.29,30 Detection of FLT3-ITD and NPM1 mutations were analyzed as previously described.31,32 Statistical analysis Data are presented as medians with range, means ± SD, or as absolute numbers with percentages. Differences in continuous variables were ana- lyzed using Mann-Whitney U test for distribu- tion between 2 groups. Analyses of frequencies were performed using Fisher exact test. Survival probabilities were estimated by the Kaplan-Meier method, and differences in survival distributions were evaluated using the LogRank test. The statistical analyses were performed using the SPSS computer software 21.0 (IBM). For all analyses, the P values were 2-tailed, and P < 0.05 was considered statistically significant. Results In the study, we analyzed the expression of BCL2, BAX and ABCB1 gene, in a cohort of 51 newly di- agnosed patients with AML-NK. Clinical and bio- Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia242 TABLE 1. Clinical characteristics for de novo acute myeloid leukemia with normal karyotype (AML-NK) patients stratified by the level of BCL2, BAX gene expression and BAX/BCL2 ratio Parameter BCL2 BAX BAX/BCL2 Total n=51 BCL2+ n=25 BCL2- n=26 P BAX+ n=25 BAX- n=26 P BAX/BCL2high n=25 BAX/BCL2low n=26 P Sex Male (%) 26 (51) 10 (38) 16 (62) 0.165 16 (62) 10 (38) 0.095 16 (62) 10 (38) 0.095 Female (%) 25 (49) 15 (60) 10 (40) 9 (36) 16 (64) 9 (36) 16 (64) Age* (years) 51(23-62) 53 (27-62) 49 (23-62) 0.137 49 (27-62) 52.5 (23-62) 0.597 48 (23-62) 54 (27-62) 0.111 WBC count* (x109/L) 22 (1-349) 7 (1-184) 22.5 (2-349) 0.159 22 (1-184) 22 (0.2-349) 0.808 29 (1-349) 6 (1-184) 0.041 HB* (g/L) 99(66-131) 103 (82-131) 98 (66-128) 97 (66-131) 105 (78-128) 98 (66-128) 103 (82-131) > 80 (g/L) 45 (88) 25 (56) 20 (44) 0.023 22 (49) 23 (51) 1.000 19 (53) 26 (47) < 80 (g/L) 6 (12) 0 6 (100) 3 (50) 3 (50) 0 6 (100) 0.010 Plts* (x109/L) 55(8-422) 60 (8-422) 53.5 (8-169) 0.528 55 (17-169) 54.5 (8-422) 0.497 52 (8-169) 60 (8-422) 0.685 LDH *(U/L) 321(1-2904) 175 (153-1992) 590.5 (1-2904) 0.010 386 (153-1992) 306.5 (1-2904) 0.816 605 (1-2904) 75 (153-1992) 0.002 PB blast* (%) 14(0-98) 15 (0-98) 11 (0-97) 0.737 11 (0-92) 16 (0-98) 0.623 11 (0-97) 15 (0-98) 0.865 BM blasts* (%) 62(30-97) 57 (30-90) 66.5 (32-90) 0.531 63 (30-97) 61 (31-90) 0.756 70 (30-90) 57 (31-97) 0.341 CD34 (%) 0.095 0.404 0.050 present 24 (47) 15 (63) 9 (38) 10 (42) 14 (58) 8 (33) 16 (67) absent 27 (53) 10 (37) 17 (363) 15 (56) 12 (44) 17 (63) 10 (37) FAB (%) 0.006 0.239 0.002 M0 4 (8) 4 (100) 0 1 (25) 3 (75) 0 4 (100) M1 5 (10) 4 (80) 1 (20) 3 (60) 2 (40) 1 (20) 4 (80) M2 18 (35) 10 (56) 8 (44) 7 (39) 11 (62) 6 (33) 12 (67) M4 17 (33) 3 (18) 14 (82) 8 (47) 9 (53) 14 (82) 3 (18) M5 7 (14) 4 (57) 3 (43) 6 (86) 1 (14) 4 (57) 3 (43) CR (%) 0.404 0.264 0.577 success 28(55) 12 (43) 16 (57) 16 (57) 12 (43) 15 (54) 13 (46) failure 23(45) 13 (57) 10 (43) 9 (39) 14 (61) 10 (43) 13 (57) Resistance (%) 0.024 0.703 0.044 yes 8 (16) 7 (88) 1 (12) 3 (38) 5 (62) 1 (13) 7 (87) no 43 (84) 18 (42) 25 ( 58) 22 (51) 21 (49) 24 (56) 19 (44) Relapse (%) 1.000 0.047 0.137 yes 17 (61) 7 (41) 10 (59) 7 (41) 10 (59) 7 (41) 10 (59) no 11 (39) 5 (45) 6 (55) 9 (82) 2 (18) 8 (73) 3 (27) FLT3- ITDmutations (%) 0.324 1.000 0.199 present 12 (24) 4 (33) 8 (67) 6 (50) 6(50) 8 (67) 4 (33) absent 39 (76) 21 (54) 18 (46) 19 (49) 20 (51) 17 (44) 22 (56) NPM1 mutations (%) 0.237 0.144 0.144 present 17 (33) 6 (35) 11 (65) 11 (65) 6 (35) 11 (65) 6 (35) absent 34 (67) 19 (56) 15 (44) 14 (41) 20 (59) 14 (41) 20 (59) BM = bone marrow; CR = complete remission; HB = hemoglobin; FAB = French-American-British classification; PB = peripheral blood; Plts = platelets; WBC = white blood cell count *median (range) Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia 243 logical characteristics of the patients are shown in Table 1. BCL2 expression Median expression level of BCL2 in cohort of 51 AML-NK patients at diagnosis was 1.22 (range 0.13–8.97), which was not significantly different compared to healthy controls (median 1.00, range 0.21–1.59) (P = 0.148). When BCL2 median expres- sion level detected among AML-NK patients (1.22) was applied as a cut-off value, 49% of patients ex- hibited high BCL2 expression, and were marked as BCL2+ (Table 1). Examining the association of BCL2 expression level with clinical characteristics of the patients, we have found that BCL2+ patients had lower LDH levels (P = 0.010) and higher hemoglobin level (P = 0.023) (Table 1.). Also, BCL2+ patients primarily belonged to the M0/M1 FAB group of patients (P = 0.006). The presence of BCL2+ status was not as- sociated with mutations in FLT3-ITD and NPM1 gene (P = 0.324 and P = 0.237, respectively). When we analyzed the prognostic impact of high BCL2 expression in our cohort of AML-NK patients we have found that BCL2+ status was asso- ciated with the presence of resistant disease, since 88% of resistant patient had elevated BCL2 expres- sion (P = 0.024). The CR rate among our group of patients was 55%. Among BCL2+-positive patients CR rate was lower (48%), but this was not signifi- cantly different compared to BCL2- group (62%) (P = 0.404). Survival analysis indicated that BCL2+ patients had longer duration of CR compared to BCL2- patients (11 months vs. 9.3 months), but this difference showed no statistical significance after survival analysis was performed (LogRank = 0.46, P = 0.831). A similar result was obtained when ana- lyzing the impact of BLC2 status on OS (BCL2+, 6 months vs..BCL2- , 8 months; LogRank = 2.030, P = 0.154). BAX expression Median expression level of pro-apoptotic BAX gene in our cohort of AML-NK patients was 0.92 (range 0.27‒2.64), which was not significantly dif- ferent compared to healthy controls (median 1.09, range 0.41‒1.55) (P = 0.704). Based on the BAX me- dian expression level the patients were divided into BAX+ and BAX- group. There were no signifi- cant associations between BAX expression level and clinical and molecular characteristics of the patients. Analysis of the potential prognostic impact of BAX status showed that BAX- patients had lower CR rate compared to BAX+ group, but without sta- tistical significance (P = 0.264) (Table 1). The nega- tive impact of low BAX expression was also reflect- ed in the fact that BAX- patients were more prone to relapse (P = 0.047). Also, BAX- patients had lower DFS (BAX-, 8 months vs.. BAX+, 11 months; LogRank = 0.020, P = 0.889), and lower OS (BAX-, 5 months vs.. BAX+, 7 months; LogRank = 0.020, P = 0.888) but it was not statistically significant. Combined BCL2 and BAX expression (BAX/BCL2 ratio) The possible cumulative effect of both BCL2 and BAX gene expression level was also analyzed us- ing BAX/BCL2 ratio. In AML-NK group median BAX/BCL2 ratio was 0.62 (range 0.11‒7.77), while in healthy samples it was 0.91 (range 0.59‒3.69). We haven’t found significant difference between BAX/ BCL2 values among patient and healthy control group (P = 0.185). When median BAX/BCL2 value detected in AML-NK patients (0.62) was applied as a cut-off value for discriminating BAX/BCL2 high and BAX/BCL2 low group, 49% of patients had BAX/ BCL2high status. Regarding the clinical characteristics of the pa- tients, BAX/BCL2 high status was associated with higher number of WBC (P = 0.041), hemoglobin levels lower than 80 g/L (P = 0.010), higher LDH level (P = 0.002), with M4 FAB group (P = 0.002), and with absence of CD34 (P = 0.050). BAX/BCL2 high status was not significantly associated with mutations in FLT3-ITD and NPM1 gene (P = 0.199 and P = 0.144) (Table 1). The prognostic significance of BAX/BCL2 ra- tio was evident only in terms of the presence of primary resistance, where BAX/BCL2low status patients were in 87% resistant to therapy (P = 0.044). Survival analysis didn’t show any signifi- cant difference in DFS and OS duration between BAX/BCL2high and BAX/BCL2low groups of patients (LogRank = 0.139, P = 0.710 and LogRank = -0.004, P = 0.951, respectively). ABCB1 expression In our cohort of AML-NK patients median ex- pression level of ABCB1 gene was significantly lower compared to healthy controls (0.16, range 0.00‒13.74 vs. 1.02, range 0.29‒5.27, respectively) (P = 0.025). According to median ABCB1 expression level, we have divided patients into ABCB1+ (25 Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia244 patients) and ABCB1- group (26 patients). We have found that ABCB1+ patients had lower LDH levels (P = 0.028), and were predominantly found in M0/ M1 FAB group (P<0.001). Furthermore, ABCB1+ sta- tus was associated with the absence of FLT3-ITD (P = 0.019), as well as, with the presence of CD34 antigen (P = 0.050) (Table 2). Interestingly, ABCB1 status was not associated with the occurrence of resistance (P = 1.000), nor did it affect the CR rate (P = 0.781). Also, expression level of ABCB1 did not affect DFS and OS duration (LogRank = 0.037, P = 0.848 and LogRank = 0.951, P = 0.329, respectively). In addition, patients with high expression of ABCB1 predominantly had high expression of BCL2, and therefore were frequently found in BAX/BCL2 low group (P < 0.001). When we performed substratification of AML- NK patients based on the presence of FLT3-ITD and NPM1 mutations into 3 risk groups (favorable NPM1+-11 patients, poor FLT3-ITD+-12 patients, and intermediate FLT3-ITD-/NPM1- -28 patients), we have found that ABCB1+ status was predomi- nant in the FLT3-ITD-/NPM1- group, because 71% of FLT3-ITD-/NPM1-patients had high ABCB1 expression (P = 0.001). Analyzing the potential prognostic significance of ABCB1 expression in this group of 28 patients, prominent impact was observed only in survival analysis for OS where ABCB1+ patients had shorter survival of 5 months, compared to ABCB1- patients with 10 months (LogRank = 3.447, P = 0.063). Discussion Loss of control in the process of programmed cell death is one of the basic events in the malignant transformation and the development of various types of tumors, including AML. For this reason, many of the participants in apoptosis are recog- nized as targets for the design and application of therapeutics. Aberrant expression of genes that control apoptosis, like BCL2-familly members, represent a recurrent feature of leukemic cells that can lead to increased cell survival and chemother- apy resistance.11,33,34 In this study we analyzed the expression pattern of two BCL2-family member genes, BCL2 and BAX, as well as BAX/BCL2 ratio in order to elucidate their influence on prognosis of AML-NK patients. We have found that the expression level of BCL2 among de novo AML patients was not dif- ferent compared to healthy controls, and showed TABLE 2. Clinical characteristics for de novo acute myeloid leukemia with normal karyotype (AML-NK) patients stratified by the level of MDR1 gene expression Parameter Total n=51 MDR1+ n=26 MDR1- n=25 P Sex 0.051 Male (%) 26 (51) 17 (65) 9 (35) Female (%) 25 (49) 9 (36) 16 (64) Age* (years) 51(23-62) 53 (23-62) 49 (23-62) 0.396 WBC count* (x109/L) 22(1-349) 7 (1-184) 26 (0-349) 0.071 Hemoglobin* (g/L) 99(66-131) 106 (78-124) 96 (66-131) 0.191 > 80 (g/L) 45 (88) 24 (53) 21 (47) < 80 (g/L) 6 (12) 1 (17) 5 (83) Platelets* (x109/L) 55(8-422) 42 (8-422) 69.5 (16-169) 0.129 LDH *(U/L) 321(1-2904) 175 (1-2904) 553.5 (175-1992) 0.028 PB blast* (%) 14(0-98) 14 (0-98) 13.5 (0-87) 0.900 BM blasts* (%) 62(30-97) 57 (30-90) 65 (33-97) 0.565 CD34 (%) 0.025 present 24 (47) 16 (67) 8 (33) absent 27 (53) 9 (33) 18 (67) FAB (%) <0.001 M0 4 (8) 4 (100) 0 M1 5 (10) 5 (100) 0 M2 18 (35) 11 (61) 7 (39) M4 17 (33) 3 (18) 14 (82) M5 7 (14) 2 (29) 5 (71) Complete remission (%) 0.781 success 28(55) 13 (46) 15 (54) failure 23(45) 12 (52) 11 (48) Resistance (%) 1.000 yes 8 (16) 4 (50) 4 (50) no 43 (84) 21 (49) 22 (51) Relapse (%) 0.460 yes 17 (61) 9 (53) 8 (47) no 11 (39) 4 (36) 7 (64) FLT3-ITD mutations (%) 0.019 present 12 (24) 2 (17) 10 (83) absent 39 (76) 23 (59) 16 (41) NPM1 mutations (%) 0.075 present 17 (33) 5 (29) 12 (71) absent 34 (67) 20 (59) 14 (41) BCL2+ 25 (49) 20 (80) 5 (20) <0.001 BCL2- 26 (51) 5 (24) 21 (76) BAX/BCL2high 25(49) 5 (20) 20 (80) BAX/BCL2low 26 (51) 20 (77) 6 (23) <0.001 BM = bone marrow; FAB = French-American-British classification; PB = peripheral blood; WBC = white blood cell count *median (range) Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia 245 extremely heterogeneous pattern, with wide range of detected values. Similar finding was reported by others.11,35 Also, consistent with some other previ- ously published findings, in our study BCL2+ status was not a predictor for reduced CR rate, and did not influence DFS and OS.9,36-38 However, in our cohort of patients a statistically significant association be- tween BCL2+ status and the existence of resistance was shown. We believe that this finding might be important because BCL2+ patients may benefit from specific anti-BCL2 therapy. Bilbao-Sieyro et al.38 came to the same conclusion after they reported that increased BCL2 expression found in CR and relapsed samples (but not in diagnosis samples) was associated with poor DFS and OS. Furthermore, we have found that high expres- sion of BCL2 was detected among patients with FAB M0/M1 subtypes. This is not surprising, given the fact that the expression of BCL2 is differentia- tion stage specific, being at its highest in immature myeloid progenitors, and decreasing at the final stages of differentiation.35 This finding is very important when anti-BCL2 therapy (venetoclax) is used in the treatment of AML. Namely, it was shown that AML patients belonging to FAB M4/ M5 subtype can exhibit resistance to this specific therapy. It is assumed that the cause of this resist- ance lies in the lack of therapeutic target i.e. BCL2, since leukemic cells belonging to these AML sub- types originate from more differentiated hemat- opoietic cells, having lower, or non-existent BCL2 expression.39,40 Our study showed significant association be- tween decreased BAX expression level and higher relapse rate. This finding is similar with already published data, but it has to be said that the influ- ence of BAX expression level on prognosis in AML was predominantly studied through its associa- tion with other apoptotic genes, like BCL2 (BAX/ BCL2 ratio).9,17,19,20,41,42 In our study, BAX/BCL2low ratio was significantly associated with the pres- ence of the resistance. Other studies showed that increased BAX/BCL2 ratio was associated with in- creased CR rate,18,41 while patients with low BAX/ BCL2 had shorter OS.18 Following the example of Del Poeta et al.41 we also tried to prove the associa- tion of BAX/BCL2high ratio with NPM1-/FLT3-ITD+ mutational status, but without any success since the presence of these mutations were not associ- ated with BCL2 and BAX expression level when analyzed individually. Of note is that the methodology used in the ref- erenced studies was different ranging from flow- cytometry, western-blot, to RT-PCR and RNA-seq, therefore their results cannot be entirely compara- ble. Also, the analysis focusing on only two apop- totic factors, BCL2 and BAX, represents only a sim- plification of the real situation, where other pro- and anti-apoptotic members of the BCL2-familly interact with each other and determine the final fate of the leukemic cell. When we analyzed expression pattern of ABCB1 gene known to be involved in the chemore- sistance, we observed some similarity with the re- sults obtained through BCL2 and BAX expression analysis. Thus, similar to BCL2 expression, ABCB1 expression analysis showed that ABCB1+ status was preferably found in M0/M1 FAB subgroup of patients. This was not surprising considering the fact that ABCB1 expression is dependent on the differentiation stage, i.e. that the highest ABCB expression was observed among cells with im- mature immunophenotype.43 In line with this was the finding that ABCB1+ status was associated with CD34+ status. This is also due to the fact that CD34 expression is present in pluripotent hematopoietic cells, and it’s down-regulated during differentia- tion process, as it is the case with ABCB1 expres- sion.44-46 In our study, we observed mutual exclusion be- tween FLT3-ITD+ and NPM1+ status, and high ex- pression of ABCB1. Similar finding was reported by others, and in the case of FLT3-ITD+ patients it is assumed to be a consequence of a loss of ABCB1 expression under increased proliferative activity caused by the presence of a FLT3-ITD mutation.47-50 In our study overexpression of ABCB1 was not associated with occurrence of chemoresistance, CR rate, and the duration of DFS and OS. This can be explained by the presence of age-dependent as- sociation between high expression of ABCB1 and adverse prognosis. Namely, the clinical relevance of ABCB1 expression is diminished or completely lost among young adult AML patients, and par- ticularly in pediatric AML patients.45,51-53 In our study median age of AM-NK patients was 51 years, and 61% of older patients (> 51 years) were ABCB1high. Based on this, we can say that our co- hort of patients is represented by younger adults, in whom the association between ABCB1 expres- sion and chemoresistance/adverse outcome, is not so evident. However, it may be that the analysis of a larger number of patients could provide a more accurate statistical power for significance. Also, in addition to the expression of ABCB1, the resistance in AML can be influenced by other members of the ABC-transporter family, or even by the expression pattern of some other prognostically Radiol Oncol 2023; 57(2): 239-248. Pravdic Z et al. / Expression of BCL2, BAX and ABCB1 gene in acute myeloid leukemia246 significant genes.55-57 Some studies have shown that contribution to the resistance, and to the overall prognosis is defined by co-expression patterns of many different ABC-transporters, and not by their individual influence.54,55,58 It is assumed that some of the ABC transporters have overlapping speci- ficity to a range of substrates, and that their co- expression is responsible for chemoresistance. This is particularly evident in the study by Marzac C et al.54 where resistant disease among AML patients increased from 21% to a 100% depending of number of overexpressed ABC transporter genes (0 to 3). In conclusion, our study showed that occur- rence of resistance was associated with increased expression of BCL2, while patients with low BAX expression were more prone to relapse. Combined impact of these two genes analyzed through BAX/ BCL2 ratio showed that AML-NK patients with BAX/BCL2low status were resistant to chemothera- py. Also, in our cohort of patients ABCB1 expres- sion level was not a predictor of resistant disease, but we have found association between ABCB1+ status and the absence of NPM1 and FLT3-ITD mutations, molecular markers with an already established prognostic significance in AML-NK. However, we were unable to demonstrate that ABCB1 expression could contribute to a more ac- curate risk stratification in these patients. This is the first study in which the expression of BCL2, BAX, BAX/BCL2 ratio and ABCB1 was examined solely in AML-NK group of patients in which the prognostic influence of cytogenetic ab- erration, either unfavorable or favorable, could be excluded. This cytogenetically homogenous group of patients are extremely heterogeneous regarding their outcome, and that is why it would be of great importance if expression pattern of some of these genes should prove to be significant for progno- sis and response to therapy. Since the research on pharmacotranscriptomics markers in AML is de- ficient, this study which includes the gene expres- sion analysis involved in both mechanisms of mul- tidrug resistance (apoptotic-dependent and efflux pump-dependent) has provided new data on these potential components of algorithm for individual- ized, personalized treatment of AML patients. Acknowledgments This work has been funded by grant from the Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. 451- 03-68/2022-14/200042). References 1. Shallis RM, Wang R, Davidoff A, Ma X, Zeidan AM. Epidemiology of acute myeloid leukemia: recent progress and enduring challenges. 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Veronika Kloboves-Prevodnik, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: vkloboves@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. The expression of CD56 in DLBCL is highly unusual. Little is known about its incidence and clinical importance. So far, no genetic profiling was performed in CD56 positive DLBCL. Patients and methods. Tissue microarrays have been constructed, sectioned, and stained by H&E and immuno- histochemistry for 229 patients with DLBCL diagnosed 2008–2017. For CD56 positive cases, clinical data was collected including age at diagnosis, stage of the disease, International Prognostic Index (IPI) score, treatment scheme and number of chemotherapy cycles, radiation therapy, treatment outcome, and possible relapse of the disease. Overall survival (OS) and progression-free survival (PFS) were calculated. For four patients, RNA was extracted and targeted RNA (cDNA) sequencing of 125 genes was performed with the Archer FusionPlex Lymphoma kit. Results. CD56 expression was found in 7 cases (3%). The intensity of expression varied from weak to moderate focal, to very intensive and diffuse. All patients had de novo DLBCL. The median age at the time of diagnosis was 54.5 years. Five of them were women and 2 males. According to the Hans algorithm, 6 patients had the germinal centre B cells (GBC) type and one non-GBC (activated B-cell [ABC]) type, double expressor. Genetic profiling of four patients ac- cording to Schmitz’s classification showed that 1 case was of the BN2 subtype, 1 of EZB subtype, 2 were unclassified. The six treated patients reached a complete response and did not experience progression of the disease during the median follow-up period of 80.5 months. Conclusions. We report on one of the largest series of CD56+DLBCL with detailed clinicopathological data and for the first time described genetical findings in a limited number of patients. Our results show that CD56 expression is rare, but seems to be present in prognostic favourable subtypes of DLBCL not otherwise specified (NOS) as tested by immunohistochemical or genetic profiling. Key words: diffuse large B-cell lymphoma not otherwise specified; CD56; immunohistochemistry; genetic profiling; prognosis Introduction CD56, also known as the neural cell adhesion mol- ecule (NCAM), is a member of the immunoglobu- lin superfamily that plays important functional roles during nervous system development, differ- entiation, and immune surveillance. In addition to neurons and glial cells, CD56 is normally also Radiol Oncol 2023; 57(2): 249-256. Gasljevic G et al. / CD56-positive diffuse large B-cell lymphoma250 expressed in neuroendocrine tissues and some cells of the hematopoietic system like NK cells and activated T lymphocytes.1 In the hematopathology service, it is mainly used as a marker of NK cells and their neoplastic counterparts. Its aberrant ex- pression is useful as a proof of clonal plasma cell proliferation, while it can also be used as prognos- tic marker in plasmacytoma, as well as in acute myeloid leukemia (AML) and acute lymphoblastic leukaemia (ALL).2-5 Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, representing approxi- mately one third of all non-Hodgkin lymphomas.2 Cases of DLBCL that do not fit the distinctive clinical presentation, tissue morphology, neoplas- tic cell phenotype, and/or pathogen-associated criteria of other subtypes of DLBCL are termed “DLBCL not otherwise specified (DLBCL NOS)’ and represent 80–85% of all DLBCL cases.2 The WHO 2016 classification of hematopoietic neo- plasms2 requires that the neoplastic cells in DLBCL NOS be further defined based on whether they are derived from germinal centre B cells or acti- vated B-cells as identified by gene expression pro- filing (GEP) or are germinal centre B cells (GBC) or non-GBC as identified by immunohistochemi- cal (IHC) analyses. In general, DLBCL NOS is an aggressive disease with an overall long-term sur- vival rate in patients treated with standard chem- otherapy regimens of ~60%.7,8 Patients with acti- vated B-cell (ABC) DLBCL and non-GBC variants have significantly worse prognoses than patients with the GBC variant.6 Expression of markers in DLBCL NOS neoplastic cells that have clinical significance as prognostic or predictive factors in- clude CD5, MYC, BCL2, BCL6, CD20, CD19, CD22, CD30, PD-L1, and PD-L2.2,6 For example, 5–10% of DLBCL NOS cases express CD5 and have a very poor prognosis that is not improved by even ag- gressive treatment regimens, while the expression of CD30 represents a favourable prognostic indi- cator.2 Very little is known about the incidence and clinical importance of CD56 expression in DLBCL. In the last 30 years, the literature has only a few case reports or small series of CD56+ DLBCL with conflicting results on its importance.10-18 It could have a prognostic value; however, since new tar- get drugs are becoming available and among them is also anti-CD56 antibody, CD56 could serve as a potential target for the treatment of patients who do not respond to standard therapeutic schemes. The purpose of this study was to evaluate CD56 expression in DLBCL in our series, to estimate its relationship to epidemiological factors, to roughly estimate its value as a prognostic marker, and to describe, for the first time the molecular findings in a subset of cases. Patients and methods Specimens Data bases of the Department of Pathology Institute of Oncology Ljubljana (IOL) have been searched for all cases of DLBCL diagnosed be- tween 2008 and 2017. Only the cases in which appropriate amount of material was present that could allow the construction of tissue microar- rays (229) have been chosen for the study. Tissue microarrays have been constructed, sectioned, and stained by H&E and immunohistochemistry for the Hans algorithm as previously described.19 Also, for the cases that were CD56 positive, flow cytometric and/or immunocytochemical stain- ing results and data were retrieved and re-an- alysed from the database of the Department of Cytopathology. Patients For selected patients, clinical data was collected including age at diagnosis, stage of the disease, IPI score, treatment scheme and number of cycles, potential radiation therapy, outcome and possible relapse of the disease were also noted. Overall sur- vival (OS) and progression-free survival (PFS) were calculated. Subjects were censored at their last visit to the IOL and for those who finished follow-up at IOL, a vital status from the Cancer Registry of the Republic of Slovenia. All procedures followed in this evaluation were in accordance with the ethical standards of the responsible committee on human experimentation (Ethical Committee of Institute of Oncology Ljubljana, approval number: ERID- KESOPKR-23 and the Ethical Committee of the Republic of Slovenia, approval number: 58/02/15) and the Helsinki Declaration of 1975, as revised in 2000. Immunohistochemistry 3–4 μm thick, formalin-fixed paraffin-embedded sections of constructed TMAs were used for im- munohistochemical staining with the monoclonal antibody CD56. Staining was performed on the Ventana Benchmark platform using the MRQ 42 clone (Cell Marque) in dilution 1:200. Radiol Oncol 2023; 57(2): 249-256. Gasljevic G et al. / CD56-positive diffuse large B-cell lymphoma 251 Flow cytometric analysis and immunocytochemistry The preparation of FNAB (fine needle aspiration biopsy) lymph node sample, cell counting, sample preparations for flow cytometric immunopheno- typing, acquisition of cells with flow cytometer and measurement result analysis were performed as previously described.20 Monoclonal antibodies against CD45, CD19, CD20, CD3, CD10, CD5, CD23, FMC7, κ and λ LCs (BD Biosciences, New Jersey, U.S.) were used. The samples were acquired us- ing a four-colour flow cytometer FACSCalibur (BD Biosciences, New Jersey, U.S.), a six-colour flow cy- tometer FACSCanto II (BD Biosciences, New Jersey, U.S.) or a ten-colour FACSCanto X (BD Biosciences, New Jersey, U.S.). The measurement results were analysed using CellQuest (BD Biosciences, New Jersey, U.S.) or BD FACSDiva software (BD Biosciences, New Jersey, U.S.). For immunocyto- chemical staining, methanol and Delaunay-fixed cytospines were prepared. Stainings were carried out on the Ventana Benchmark Ultra platform us- ing antibodies against CD56, CK AE1/AE3, CK18 (DAKO), CD20 (Cell Marque, Rocklin, California, U.S.), synaptophysin (Termo Scientific, Waltham, Massachusetts, U.S.), CD3 and TTF-1 (Leica Biosystems, Nussloch, Germany). Molecular analysis – NGS sequencing RNA was extracted from 4 paraffin-embedded tis- sue samples was extracted using the MagMAXTM FFPE DNA/RNA Ultra Kit (ThermoFisher, Waltham, MA, USA). Samples were treated with DNase, dur- ing the extraction process. Targeted RNA (cDNA) sequencing of 125 genes was performed with the Archer FusionPlex Lymphoma kit (Invitae- ArcherDX, San Francisco, CA, USA). The final NGS library was quantified using the KAPA Library Quantification Kit (KAPA Biosystems, Merck, Ljubljana, Slovenia) and pair-end sequenced on a MiSeqDx system (Illumina, San Diego, CA, USA). The trimmed FASTQ file was uploaded to Archer Analysis software Version 6.0.3.2, which performed variant and fusion calls along with the determina- tion of cell of origin (ABC or GCB). Variants were considered true positive if the frequency of the var- iant allele was above 10%, with minimum coverage of 100x.20 All variants reported in GnomAD were excluded. Fusions were considered true positive if the fusion event was covered with a minimum of 5 unique reads and the percentage of reads support- ing the event was above 10%.21,22 Statistical analysis For numeric and demographic variables descrip- tive statistics were used (median, range, standard deviation, percentage). Overall survival and pro- gression-free survival were calculated using the Kaplan-Meier method. Statistical analyses were performed using IBM SPSS Statistics, version 26. Results Among 229 DLBCL, NOS cases included in the study, CD56 expression was found in 7 cases (3%). The intensity of CD56 expression varied from moderate focal to very intensive and diffuse posi- tive reaction (Figure 1). Reanalysis of the five cases in which fine needle aspiration biopsy (FNAB) of the lymph node was performed prior to surgical biopsy and histological examination showed that CD56 was not included in routine flow-cytometry FIGURE 1. (A) Morphology of diffuse large B-cell lymphoma (DLBCL), CD56+; H&E, 20x; (B) Strong expression of CD56 in DLBCL not otherwise specified (NOS) (tissue microarray), 4x; (C) weak to moderate CD56 expression in DLBCL NOS (tissue microarray), 4x. A B C Radiol Oncol 2023; 57(2): 249-256. Gasljevic G et al. / CD56-positive diffuse large B-cell lymphoma252 work-out. There was only one case23 (case 1 in Table 1) in which immunocytochemistry for CD56 was stained since tumour cells showed co-expres- sion of cytokeratin and the diagnosis of metastatic neuroendocrine carcinoma has been made. All patients had de novo DLBCL. The median age at the time of diagnosis was 54.5 years (range 30–57). Five of them were women and 2 males. Five patients were diagnosed with DLBCL, GC type, 2 with DLBCL non-GC (ABC) type, one being a dou- TABLE 1. Clinicopathological characteristics of patients with CD56 positive diffuse large B-cell lymphoma (DLBCL), review of the literature with our series Publication Coun No of pat Case No Sex Age GC type** Non-GC type LN Extranodal disease and site Clinical stage IPI LDH Surg CT and No. of cycles RT Response FU Kern 199323 USA 1 1 NA NA NA NA NA NA NA NA NA NA NA NA NA Muroi 199817 Jap 2 1 2 M,49 F,62 Yes Yes Yes Yes Liver, Spleen, Pericard. Ef. Liver NA NA NA NA NA NA No No CHOP, NAx CHOP, NAx No No PR? PR? NA NA Sekita 199918 Jap 1 1 M,16 Yes Yes I NA NA No CHOP, 6x No CR 10 m Hammer 199815 USA 4 1 2 3 4 M,51 M,69 M,76 M,54 NA NA NA NA NA NA NA NA Yes No Yes Yes Stomach NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Otsuka 200414 Jap 2 1 2 NA NA Yes Yes NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Weisberger *200611 USA 10 1 2 3 4 5 6 7 8 9 10 M,41 M,52 M,54 M,83 M,49 F,57 F,69 M,77 M,84 M,77 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes No Yes No Yes Yes Yes No Ileocecal valve SpineAbdomen Brain NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Isobe 200713 Jap 3 1 2 3 M,80 F,87 M,73 Yes Yes Yes Yes No Yes Ascites Ileum Ileum NA NA NA NA NA NA NA NA NA No Yes Yes THP-COP, 3x No R-CHOP, 6x No No No NR CR CR DOD 22 m 22 m Chen 201016 Ch 1 1 NA NA NA NA NA NA NA NA NA NA NA NA Gomyo 20109 Jap 7 1 2 3 4 5 6 7 M,29 F,60 F,22 M,64 M,63 M,50 F,45 Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No No No Spleen WR WR Pl. Ef, Adr. gl, Submand. gl Nasal cavity Intra-extradural mass Subcutis IIIB IIA IA IIIA IA IA IVA HI L L H L L HI ↑ N N ↑ N N ↑ No No No No No No No R-CHOP, aPBSCT R-CHOP 3x CHOP 3x CHOP 5x RCHOP 3x Res+CHOP 4x R-CHOP 8x No Yes Yes No Yes Ye sNo CR CR CR CR CR RCR A, 24 m A, 50 m A, 57 m D, 4 m (pneumonia) A, 43 m A, 70 mA, 5 m Stacchini 201212 It 5 1 2 3 4 5 M,72 M,15 M,71 M,60 M,21 Yes Yes Yes Yes Yes Yes Yes No No Spleen, Stomach,Pancr. Stomach, Liver Nasopharynx NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA AWD 12m Gu 201310 SK 1 1 F,5 Yes WR I N Yes COPAD, 6x No CR NA Liu 20208 Ch 1 1 M,14 Yes, DH Yes Nasopharynx IV NA ↑ No CTX+CP R-Hyper-CVAD AB R-DA-EPOCH, 6x IT DM+CTB,4x No CR NA Gasljevic 2022 Slo 7 1 2 3 4 5 6 7 F,56 F,51 M,57 M,56 F,53 F,30 F,79 Yes Yes Yes Yes NA Yes, DE Yes NA Yes Yes Yes Yes Yes Skeletal muscle Small bowel Spleen, Liver, Adrenal gland IA IA IIA IVB IIA IVB IVB 0 0 0 0 3 3 5 N N ↑ ↑ N ↑ ↑ Yes No No No Yes No No R-CHOP, 3x R-CHOP, 3x R-CHOP, 6x R-EPOCH,6x +IT,2x CHOP, 3x R-CHOP,8x No No No No No Yes Yes No CR CR CR CR CR CR NA A ,63 m A, 73 m A, 55 m A, 40 m A, 62 m A, 182 m DOD A = alive; aPBSCT = autologous peripheral blood stem cell transplantation; AWD = alive with disease; Ch = China; CHOP = cyclophosphamide, doxorubicin hydrocloride, vincristine sulfat, prednisone; Coun = country; CP = prednisone; CR = complete response; CT = chemotherapy; CTX = cyclophosphamide; D = dead; DA-EPOCH = etoposide, doxorubicin, vindesine, dexamethasone, cyclophosphamide; DE = double expressor; DOD = dead of disease; F = female; FU = follow-up; Gl = gland; Hyper- CVAD AB = A: cyclophosphamide, vindesine, liposomal doxorubicin, dexamethason, B: methotrexate, cytarabine; IPI = International Prognostic Index; It = Italy; IT = intratechal; Jap = Japan; LN = lymph nodes; M = male; m = months; N = normal; NA = not available; NR = no response; Pancr = pancreas; Pl. E = pleural effusion; PR = partial response; R = rituximab; res = resection; RT = radiotherapy; SK = South Korea; Slo = Slovenia; Submand = submandibular; THP-COP = pirarubicin, cyclophosphamide, vincristine sulfat, prednisone; WR = Waldeyers ring * only histologically proven cases are considered ** on the basis of the CD10 positivity Radiol Oncol 2023; 57(2): 249-256. Gasljevic G et al. / CD56-positive diffuse large B-cell lymphoma 253 ble expressor (DE). One patient refused staging and treatment and died shortly after being diag- nosed and was therefore excluded from survival analysis. Among the six patients who received treatment, three patients were in clinical stage 1, one in stage 2 while two were in clinical stage 4. Only patients in clinical stage 4 had constitutional symptoms. Four patients had disease localised in the lymph nodes while two of them also had extranodal infil- trates – one in the pectoral muscles and the other in the renal fascia and small bowel. Three patients had elevated LDH levels, in fact, both patients in clinical stage 4B and one in stage 2A. Those pa- tients in stage 4 had the IPI score 3 and others had the IPI score 0. Three patients underwent surgical procedure and were later treated with adjuvant 3 cycles of CHOP (cyclophosphamide, doxorubicin, vincris- tine, prednisone) and R-CHOP (rituximab, cy- clophosphamide, doxorubicin, vincristine, pred- nisone). Other 3 patients were treated with 6 or 8 cycles of R-CHOP. Two patients were also treated with adjuvant radiotherapy after completion of sys- temic treatment. The patient with non-GC type DE of DLBCL was treated with 6 cycles of R-EPOCH (rituximab, etoposide, cyclophosphamide, doxo- rubicin, vincristine, prednisone) together with 2 doses of intrathecally administered methotrexate and cytosine arabinoside for central nervous sys- tem prophylaxis. The 6 treated patients reached a complete re- sponse and did not experience progression of the disease during the follow-up period, meaning that 5-year PFS and OS are 100%. Median follow-up was 80.5 months (range 42–197). The clinicopathological characteristics of our cohort together with all cases reported in the lit- erature are shown in Table 1. Genetic profiling of 4 patients was performed as described in Patients and methods, and the results are presented in Table 2. Discussion CD56 expression in DLBCL NOS is very rare. Its incidence is reported to be 0.5 to 7% of DLBCLs, but is actually unknown since CD56 is generally not included in the immunohistochemical or flow cytometric panel for the diagnosis of DLBCL.10-18 In our series of patients with DLBCL NOS expression of CD56 was present in 3% of patients and varied in intensity from weak to very strong and diffuse. In one of those cases, that phenomenon resulted in an incorrect diagnosis of lymph node metasta- sis of the neuroendocrine tumour. In fact, in the general pathology service the main use of CD56 is to prove neuroblastoma and neuroendocrine dif- ferentiation in tumours of different origin while in hematopathology service it is used as a marker of NK cells, as a proof of clonal plasma cell prolifera- tion, and as a prognostic marker in plasmacytoma, acute myeloid leukemia (AML), and acute lympho- blastic leukemia (ALL).2-5 Since neuroendocrine carcinomas could be unevenly and weakly posi- tive or even negative for cytokeratins24, it is of the greatest importance for the pathologist to be aware that strong expression of CD56 could be present al- TABLE 2. Genetic profile of CD56 positive diffuse large B-cell lymphoma (DLBCL) samples Case number in Table 2 COO IHC COO AFPL fusion variants VAF (%) variant classiification Schmitz et al., 201832 classification gene nucleotide change amino acid change 1 GCB GCB ND RANBP1 NM_002882.3:c.23A>G NP_002873.1:p.(His8Arg) 13,7 Uncertain significance unclassified 2 GCB GCB ND ND ND ND ND ND unclassified 3 GCB GCB ND CD79B NM_000626.2:c.587A>T NP_000617.1:p.(Tyr196Phe) 49,0 Pathogenic EZB CD79B NM_000626.2:c.568A>G NP_000617.1:p.(Met190Val) 50,1 Uncertain significance EZH2 NM_001203247.1:c.1922A>G NP_001190176.1:p.(Tyr641Cys) 53,7 Pathogenic MYD88 NM_001172567.1:c.656C>G NP_001166038.1:p.(Ser219Cys) 37,7 Uncertain significance SH3BP5 NM_004844.4:c.460G>A NP_004835.2:p.(Ala154Thr) 19,3 Uncertain significance 4 ABC ABC IGH-BCL6 CD79B NM_000626.2:c.587A>C NP_000617.1:p.(Tyr196Ser) 25,9 Pathogenic BN2 SH3BP5 NM_004844.4:c.460G>A NP_004835.2:p.(Ala154Thr) 12,6 Uncertain significance AFPL = Archer SusionPlex lymphoma; COO= cell of origin; IHC = immunohistochemical analyses; ND = not detected; VAF = variant allele freqency; Radiol Oncol 2023; 57(2): 249-256. Gasljevic G et al. / CD56-positive diffuse large B-cell lymphoma254 so in some entities that are by definition not CD56 positive. Throughout the papers published so far, there has been much speculation about this phenom- enon with regard to its expression in special clin- icopathological settings and its possible prognos- tic value. From an epidemiological point of view, some authors9 suggested that it could be related to racial and/or geographical factors since, at the time of the publication of the paper, almost 50% of all reported cases were reported from Japan. Thorough analysis of all the cases with available information shows that 18 out of 45 cases (40%) have arisen in the population of far east (Japan, Korea, China; Table 1), while 27 (60%) were report- ed in the western population, Caucasians mainly (USA, Italy, Slovenia; Table 1). These results sug- gest that CD56+DLBCL is not related to racial / ethnic factors opposite to some other CD56 posi- tive lymphoproliferative diseases such as NK/T cell lymphoma, nasal type.2 The age distribution is very wide with cases described in paediatric/ adolescent population as well as in the older pa- tient most of the patients being in 6–7th decade of life. In our series, the vast majority of patients were middle aged, in the beginning of the sixth decade. The distribution of gender showed that among the far east patients, somewhat higher number of men are reported (6 female vs. 9 males; for 3 cases there is no information about gender) while in the western world there is a predominance of males (7 females vs. 19 males; 26% vs. 74%). However, our series shows contradictory results in which most patients (70%) are women, so it can be assumed that the higher incidence reported in males so far could be only a mere coincidence. There are two main biologically distinct mo- lecular subtypes of DLBCL: GCB and ABC. ABC DLBCL is associated with substantially worse outcomes when treated with standard chemoim- munotherapy. Based on gene expression studies, Hans et al.25 developed an algorithm to discrimi- nate GBC from non-GBC types in regard to im- munohistochemical expression of CD10, bcl6 and MUM1 with cutoff of 30%. In addition to GCB and ABC subtypes, double-hit lymphomas and dou- ble-expressor lymphomas, which overexpress myc and bcl2 protein, are aggressive DLBCLs and are also associated with a poor prognosis. On the basis of immunohistochemical results, a few authors9,11-13 found a relation of CD56 expression to DLBCL of GBC origin. Of the 45 summarized cases, for 8 cases there was no information about immunophe- notype. Twenty-eight out of 36 (76%) were of GBC type and the remaining 24% were of non-GBC (ABC) type. One reported case8 was double hit lymphoma with translocations of MYC and bcl-6, while in our series one DLBCL of non-GC (ABC) type DLBCL showed so-called double expressor profile with expression of bcl2 and myc protein expression being > 30%. Somewhat lower percent- age of GBC types are reported in Eastern patients compared to the Western (10/15 and 17/21 or 75% vs. 81%). This finding could be related to the previ- ously recognized and reported lower frequency of the DLBCL GBC subtype in Asian countries.26 In addition, it has been suggested that CD56 expression in DLBCL could be related to a more frequent extranodal presentation associated to the adhesive properties of CD56.9,11 In neural cells, it mediates cell-to-cell adhesion by CD56 molecules of adjacent cells binding together.27 It may be in- volved in homophilic adhesion for NK and T cells due to the C2-set Ig regions and fibronectin re- gions within its extracellular domain.28 However, its function with respect to B-cell ontogeny is un- clear. The expression of CD56 has been detected in a human pluripotent stem cell.28 A subset of very early precursor B cells has the innate capac- ity for CD56 expression that is down-regulated and extinguished later in differentiation. It has been shown that lymphomagenesis is a stepwise process progression of which is enabled by accu- mulation of genetic events.8 In follicular or mantle cell lymphoma, for example30, first events such as t(14,18) and t(11,14) namely, do occur in progenitor B cells. Drawing parallels to this, we could assume that CD56+ DLBCL could arise from the precursor B-cell that, for whatever reason, did not down- regulate CD56 expression and then collected addi- tional mutations that resulted in lymphoma devel- opment. Some authors9,11-13 underlined frequent ex- tranodal infiltrates in CD56+DLBCL with spleen, stomach, ileum, and nasal cavity being most fre- quently involved. Of 40 cases with available infor- mation, 16 (40%) presented with isolated lymphad- enopathy while 24 (60%) had extranodal infiltrates with or without lymphadenopathy (14 vs. 10). Four of our patients presented with isolated lymphad- enopathy while two had extranodal disease, which is concordant with majority of our patients having limited stage disease and were therefore treated adjuvantly after surgery. The expression of CD 56 can be used as a prog- nostic marker in certain hematopathological enti- ties; it can predict the occurrence of brain infiltra- tion in ALL5, the aggressiveness of multiple my- eloma3, and relapsed AML.4 So far, its prognostic Radiol Oncol 2023; 57(2): 249-256. Gasljevic G et al. / CD56-positive diffuse large B-cell lymphoma 255 importance in DLBCL has not been confirmed. All of our patients achieved complete remission, and remained in remission which can be at least par- tially attributed to low IPI scores and low clinical stages; however, two patients with clinical stage 4 also achieved and maintained complete remission. None of our patients had a high IPI score of 4 or 5 which are known to have the lowest survival.31 In most of them, DLBCL was of GCB subtype, which also carry a better prognosis.24 Schmitz et al.32 classified DLBCL cases accord- ing to genetic findings into 4 categories, namely MCD (based on the cooccurrence of MYD88L265P and CD79B mutations), BN2 (based on bcl6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations) and the EZB group (based on EZH2 mutations and bcl2 translocations). These subtypes differed phenotypically and in response to immu- nochemotherapy, with favourable survival in the BN2 and EZB groups. Genetic profiling of four pa- tients from our series according to Schmitz classi- fication32, showed that 1 case was of BN2 subtype, one belongs to the EZB group, while two were un- classified. Although data are limited and demand testing in larger cohorts of patients, so far it can be concluded that CD56 expression is more often pre- sent in cases of DLBCL NOS with prognostically favourable genetical findings. CD56 is expressed in some aggressive tumour types such as small lung cell carcinoma and neu- roblastoma. To date, it has been used as a target molecule for antibody-based immunotherapy in phase I and II clinical trials for small cell lung car- cinoma33; a favourable safety profile has been dem- onstrated. That led to the development of CAR-T therapy directed against CD56 in neuroblastoma. In the xenograft neuroblastoma model, anti-CD56 therapy led to the tumour burden control but had only modest effect on survival.34 More studies are needed in regard to neuroblastoma therapy and other CD56 positive tumours but CD56 could even- tually serve as a potential target for the treatment of CD56+ DLBCL patients who do not respond to the standard therapeutic schemes. In conclusion, here we report one of the larg- est series of CD56+DLBCL with detailed clinico- pathological data and for the first time described genetic findings in a limited number of patients. Our results show that CD56 expression is rare but seems to be present in prognostic favourable sub- types of DLBCL NOS as tested by immunohisto- chemical or genetic profiling. Acknowledgement The publication of this article was financed by the Slovenian Research Agency (ARRS), grant No. P3-0289. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors’ contributions: GG and VKP conceived the idea, designed the study, collected the data, and wrote the initial draught of the article. LB and BJN collected and analysed the data and revised the manuscript. SN and VŠD performed the mo- lecular testing, interpreted the data, and revised the manuscript. References 1. Van Acker H, Capsomidis A, Smits EL, Van Tendeloo VF. CD56 in the immune system: more than a marker for cytotoxicity? Front Immunol 2017; 8: 892- 90. doi: 10.3389/fimmu.2017.00892 2. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumours of hematopoietic and lymphoid tissues. 4th edition. Lyon: IARC; 2017. 3. Chang H, Samiee S, Yi QL. Prognostic relevance of CD56 expression in multiple myeloma: a study including 107 cases treated with high-dose melphalan-based hemotherapy and autologous stem cell transplant. Leuk Lymphoma 2006; 47: 43-7. doi: 10.1080/10428190500272549 4. Chaudhri NA, Almhareb F, Walter CU, Nounou R, Khalil S, Bakshi N, et al. Expression of CD56 in acute myeloid leukemia (AML) is associated with poor outcome when patients treated with stem cell transplant in second remis- sion but not in the first remission. Blood 2011; 118: 4880-86. doi: 10.1182/ blood.V118.21.4880.4880 5. Ravandi F, Cortes J, Estrov Z, Thomas D. 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Oncogene 2018; 37: 3686-97. doi:10.1038/s41388-018- 0187-2 Radiol Oncol 2023; 57(2): 257-269. doi: 10.2478/raon-2023-0023 257 research article Quantitative dynamic contrast-enhanced parameters and intravoxel incoherent motion facilitate the prediction of TP53 status and risk stratification of early-stage endometrial carcinoma Hongxia Wang1, Ruifang Yan1, Zhong Li1, Beiran Wang1, Xingxing Jin1, Zhenfang Guo2, Wangyi Liu1, Meng Zhang1, Kaiyu Wang3, Jinxia Guo3, Dongming Han1 1 Department of MR, the First Affiliated Hospital of Xinxiang Medical University, Weihui, China 2 Department of Neurology, the First Affiliated Hospital of Xinxiang Medical University, Weihui, China 3 MR Research China, GE Healthcare, Beijing, China Radiol Oncol 2023; 57(2): 257-269. Received 9 February 2023 Accepted 6 April 2023 Correspondence to: Dr. Dongming Han, 88 Jiankang Road, Weihui 453100, PR China. E-mail: 625492590@qq.com Disclosure: Kaiyu Wang and Jinxia Guo, who are employees of GE Healthcare, gave guidance to this paper in terms of technical parameters and language embellishment. The remaining authors declare no relationships with any companies whose products or services may be related to the subject matter of the article. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. The aim of the study was to investigate the value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and intravoxel incoherent motion (IVIM) in differentiating TP53-mutant from wild type, low-risk from non-low-risk early-stage endometrial carcinoma (EC). Patients and methods. A total of 74 EC patients underwent pelvic MRI. Parameters volume transfer constant (Ktrans), rate transfer constant (Kep), the volume of extravascular extracellular space per unit volume of tissue (Ve), true diffusion coefficient (D), pseudo-diffusion coefficient (D*), and microvascular volume fraction (f) were compared. The com- bination of parameters was investigated by logistic regression and evaluated by bootstrap (1000 samples), receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). Results. In the TP53-mutant group, Ktrans and Kep were higher and D was lower than in the TP53-wild group; Ktrans, Ve, f, and D were lower in the non-low-risk group than in the low-risk group (all P < 0.05). In the identification of TP53-mutant and TP53-wild early-stage EC, Ktrans and D were independent predictors, and the combination of them had an optimal diagnostic efficacy (AUC, 0.867; sensitivity, 92.00%; specificity, 80.95%), which was significantly better than D (Z = 2.169, P = 0.030) and Ktrans (Z = 2.572, P = 0.010). In the identification of low-risk and non-low-risk early-stage EC, Ktrans, Ve, and f were independent predictors, and the combination of them had an optimal diagnostic efficacy (AUC, 0.947; sensitiv- ity, 83.33%; specificity, 93.18%), which was significantly better than D (Z = 3.113, P = 0.002), f (Z = 4.317, P < 0.001), Ktrans (Z = 2.713, P = 0.007), and Ve (Z = 3.175, P = 0.002). The calibration curves showed that the above two combinations of independent predictors, both have good consistency, and DCA showed that these combinations were reliable clinical prediction tools. Conclusions. Both DCE-MRI and IVIM facilitate the prediction of TP53 status and risk stratification in early-stage EC. Compare with each single parameter, the combination of independent predictors provided better predictive power and may serve as a superior imaging marker. Key words: early-stage endometrial carcinoma; dynamic contrast-enhanced MRI; intravoxel incoherent motion; p53 status; risk stratification Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma258 Introduction Endometrial carcinoma (EC) is a common ma- lignant tumor of the female reproductive sys- tem worldwide, and approximately 80% of new- ly diagnosed EC patients are in the early stage (International Federation of Gynecology and Obstetrics (FIGO) stage IA, IB).1 The TP53 is an important suppressor gene that is deeply involved in tumorigenesis and can control cell growth, ap- optosis and regulate angiogenesis. Several studies have shown that high expression of TP53 is closely associated with poor prognosis in EC patients.2,3 Risk stratification based on the histologic subtype, grade, FIGO stage, and lymphovascular space in- vasion (LVSI) is the primary basis for determining treatment strategies for early-stage EC.4 For non- low-risk (intermediate-, high-intermediate-, and high-risk) patients, lymphadenectomy (LND) is required in addition to the standard treatment of total hysterectomy with bilateral salpingo-oopho- rectomy, since it can significantly improve patient benefit. But for low-risk patients, LND is not rec- ommended as it is likely to lead to complications and increased care costs.5 Currently, preoperative biopsy and routine magnetic resonance imaging (MRI) are the primary means of obtaining the TP53 status and risk stratification information of EC, respectively.6 However, biopsy may not be suf- ficient for a reliable diagnosis due to shortcomings such as unstable sampling depending on operator experience, inadequate sampling, and invasive- ness.7,8 At the same time, conventional T1-weighted imaging (T1WI) and T2-weighted imaging (T2WI) not only fail to reflect the TP53 status, histologi- cal subtype, and grade information of the lesion but also likely to have a poor to moderate pooled sensitivity in detecting high-risk factors, including deep myometrial invasion and cervical stromal in- filtration, due to the presence of adenomyosis and leiomyomas and the loss of the junctional zone.9,10 Therefore, finding a noninvasive and effective means to assess the TP53 status and risk stratifica- tion in early-stage EC is of great benefit to patients. Dynamic contrast-enhanced MRI (DCE-MRI) is a promising quantitative MRI sequence that can detect blood supply in biological tissues by ana- lyzing the dynamic distribution of contrast agents through pharmacokinetic models.11,12 Intravoxel incoherent motion (IVIM) can also be used to re- flect blood perfusion, and compared to DCE-MRI, it not only eliminates the need for contrast agents but also provides additional information on the diffusion of water molecules within the lesion.13-16 Recently, some authors have used IVIM and DCE- MRI for EC-related studies. For example, Satta et al. and Fu et al. applied IVIM and DCE-MRI to assess the grade, stage, and other histopathological fea- TABLE 1. Imaging protocol parameters Parameters T1WI T2WI DWI IVIM DCE-MRI Sequence 2D-FSE 2D-FSE 2D-SS-EPI 2D-SS-EPI 3D-LAVA Orientation Oblique Axial Oblique Axial Oblique Axial Oblique Axial Oblique Axial TR/TE (ms) 659/12.3 6000/95 3708/74.3 2000/80.7 3.5/1.7 FOV (cm2) 40 × 40 40 × 40 40 × 40 40 × 40 36 × 36 Matrix 288 × 192 320 × 320 96 × 128 128 × 192 288 × 192 Flip angle (°) 160 160 90 90 15 Slice thickness (mm) 6 6 6 6 6 No. of sections 20 20 20 Based on lesion’s size 26 NEX 1 1 1, 4 1, 1, 1, 1, 1, 1, 2, 4, 4, 6 0.73 Fat suppression / STIR STIR STIR FLEX b-values (s/mm2) / / 0, 800 0, 20, 40, 80, 160, 200, 400, 600, 800, 1000 / Respiratory compensation Free Free Free Free Free Scan time 1 min 56 s 48 s 1 min 04 s 3~6min 6 min 08 s (40 phases) DCE-MRI = dynamic contrast-enhanced magnetic resonance imaging; DWI = diffusion-weighted imaging; FOV = field of view; FLEX = FLEXible; FSE = fast spin echo; IVIM = intravoxel incoherent motion; LAVA = liver acquistion with volume assessmeNT; NEX = number of excitations; SS-EPI = single shot echo planar imaging; STIR = short-inversion time(TI) recovery; TR/TE = repetition time/echo time; T1WI = T1-weighted imaging; T2WI = T2-weighted imaging Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma 259 tures of EC and showed that some of the derived parameters helped to identify the histopathologi- cal features of EC.17,18 Zhang et al. and Meng et al. used IVIM19,20, while Ye et al.12 used DCE-MRI for the preoperative risk assessment of EC, and their results showed that some parameters of DCE-MRI or IVIM could play a positive role in the risk strati- fication prediction of EC. However, not only did none of these studies address TP53 status but also risk stratification was assessed either by applying only one of the IVIM or DCE-MRI techniques or the subjects were not early-stage EC. The purpose of this study was to investigate the contributory value of quantitative parameters de- rived from DCE-MRI and IVIM in differentiating TP53-mutant from TP53-wild, low-risk from non- low-risk early-stage EC, offering a potential refer- ence for the clinical management of early-stage EC. Patients and methods Study patients This prospective study was complied with ethi- cal committee standards and approved by the ethics committee of the First Affiliated Hospital of Xinxiang Medical University (NO. EC-022-002) and informed consent was taken from all individ- ual participants. From January 2021 to April 2022, 114 female patients underwent pelvic MRI due to suspected EC by clinical examination, ultrasound (US), or computed tomography (CT). Forty par- ticipants were excluded during this study: 1) 7 pa- tients were diagnosed with an endometrial polyp, atypical hyperplasia, or other non-EC diseases; 2) 16 patients had FIGO stage ≥ II; 3) 4 patients re- ceived radiotherapy or neoadjuvant chemothera- py; 4) 3 patients had claustrophobia or other dis- eases that prevented them from completing all the sequences; 5) 6 patients had inadequate DCE-MRI or IVIM imaging quality for analysis due to severe artifacts, and 6) 4 patients decided to perform his- tological analysis and treatment in other institutes. Ultimately, 74 patients were enrolled in the study (Figure 1). MRI protocols A 1.5 T MR system (Optima MR360, Waukesha, WI, USA) with a 12-channel phased-array body coil was used in this study. The imaging protocol included oblique axial (perpendicular to the long axis of the uterus) T1WI, T2WI, DWI, IVIM, and DCE-MRI. For DWI and DCE-MRI sequences, the scans covered the anterior superior iliac spine to the symphysis pubis. For IVIM (b = 0, 20, 40, 80, 160, 200, 400, 600, 800, and 1000 s/mm2), to minimize scan time, the scan was limited to the lesion area (determined by an experienced radiologist from the DWI images), and its location, layer thickness, and layer spacing were consistent with the corre- sponding layer of DWI.18 DCE-MRI was performed by a three-dimensional liver acquisition with vol- ume acceleration (3D-LAVA) sequence with 40 phases (time resolution, 9s), and gadopentetate dimeglumine (Gd-DTPA, Bayer Pharmaceutical, Berlin, Germany) was injected intravenously with an automatic injector (0.2 mL/kg, 3.0 mL/s). The protocol details are provided in Table 1. Image postprocessing All images were transferred to the Advantage Workstation (version 4.7), and the IVIM and DCE- MRI images were analyzed within the workstation FIGURE 1. Flowchart of the present study. EC = endometrial carcinoma Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma260 using vendor-provided software named MADC and GenIQ, respectively. The IVIM parameters were calculated by the following formula: Sb/S0 = (1 - f) × exp(-b×D) + f × exp (-b×D*) [1] where S0 was the signal intensity at the b value of 0; Sb was the signal intensity at the b value de- noted by the subscript; D was the true diffusion coefficient of a water molecule; D* was the pseudo- diffusion coefficient due to microcirculation; and f was the microvascular volume fraction, indicating the fraction of diffusion related to microcircula- tion.11 The DCE-MRI perfusion parameters were quantitatively calculated based on the Tofts mod- el. The arterial input function (AIF) was obtained from the internal iliac artery. The imaging param- eter Ktrans, known as the volume transfer constant, represents the diffusion of contrast medium from the vessel to the extravascular extracellular space (EES); Kep, known as the rate transfer constant, rep- resents the diffusion of contrast medium from the EES to the vessel; and Ve represents the volume of EES per unit volume of tissue 13; thus, Kep = Ktrans/Ve. For regions of interest (ROI), first, images of DCE-MRI and IVIM were co-registered, and then on the DCE-MRI images of the phase with the clearest lesion display21, ROIs were delineated layer by layer for all slices containing the tumor, and these ROIs were manually drawn along the inside margin of the primary tumor, avoiding ar- eas with cystic degeneration, necrosis, apparent signs and hemorrhage artifacts, and blood vessels. Subsequently, all completed ROIs were automati- cally copied to the pseudo-color maps of the DCE- MRI and IVIM-derived parameters to calculate the mean values based on the volume of interest (VOI). All of these procedures were completed indepen- dently by two radiologists with 7 and 15 years of experience who were blinded to each other’s re- sults and the patient’s clinicopathological data. Histopathologic analysis All lesion specimens were obtained surgically, and the median interval from pelvic MRI examination to surgery was 12 days (1-25 days). The specimens were processed by an experienced pathologist. The histological subtype, grade, and LVSI were confirmed by hematoxylin/eosin (HE) staining. The stage was estimated with the FIGO staging system.22 According to the European Society for Medical Oncology (ESMO) clinical practice guide- lines, low-risk patients were classified into the low- risk group, while intermediate-risk, high-interme- diate-risk, and high-risk patients were classified into the non-low-risk group.4 The TP53 status was evaluated by immunohistochemical (IHC) stain- ing, where non-staining was viewed as the wild group, and faint, moderate, and strong staining was viewed as the mutant group. Ultimately, risk stratification was evaluated in all 74 patients, and TP53 status was evaluated in 46 patients (28 pa- tients declined IHC for financial or other reasons). TABLE 2. Clinicopathologic features of the patients Variable Data Age (mean ± SD) (years) 54.00 ± 7.91 Maximum diameter (mean ± SD) (mm) 25.10 (13.76, 42.58) FIGO stage n (%) IA 44 (59.46) IB 30 (40.54) Histologic subtype n (%) Adenocarcinoma 67 (90.54) Non-adenocarcinoma 7 (9.46) Clear-cell 3 (4.06) Undifferentiated carcinoma 2 (2.70) Carcinosarcoma 2 (2.70) Lymphovascular space invasion n (%) Positive 10 (6.76) Negative 64 (93.24) Histologic grade n (%) Grade 1 54 (72.98) Grade 2 10 (13.51) Grade 3 10 (13.51) Risk stratification n (%) Low 44 (59.46) Intermediate 20 (27.03) High-intermediate 0 (0.00) High 10 (13.51) TP53 expression Mutant 21 (28.38) Wild 25 (33.78) No result 28 (37.84) FIGO = International Federation of Gynecology and Obstetrics; SD = standard deviation Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma 261 Statistical analysis All data were analyzed with Stata version 16.0 (Stata Corp) and MedCalc version 15.0 (MedCalc Software). P < 0.05 was considered statistically significant. The interobserver consistency of two radiologists was classified using the intraclass correlation coefficient (ICC) as poor (ICC < 0.40), fair (0.40 ≤ ICC < 0.60), good (0.60 ≤ r < 0.75), or ex- cellent (ICC ≥ 0.75).23 The Shapiro–Wilk test was employed to check the normality of the data. The Mann–Whitney U test and the independent sam- ples t-test were used for nonnormally distributed data (median and interquartile range) and normal- ly distributed data (mean ± standard deviation), respectively. The area under the receiver operating characteristic (ROC) curve (AUC) was employed to quantify the diagnostic efficacy of different pa- rameters, and the differences were assessed using DeLong analysis. The combination of parameters was investigated by logistic regression, evaluated by bootstrap (random number set 123, repeated sampling 1000 times, backward strategy, bounded by a value of 0.1), calibration curves, and decision curve analysis (DCA).24 FIGURE 2. (A–I) A 53-year-old woman with low-risk endometrial carcinoma (EC) (arrowheads, endometrioid type, grade 2, stage IA, lymphovascular space invasion (LVSI) negative, and TP53-wild). (J–R) A 56-year-old woman with non-low-risk (intermediate) EC (arrowheads, endometrioid type, grade 1, stage IB, LVSI negative, and TP53-mutant). (A, J) Sagittal T2-weighted imaging maps; (B, K) Oblique axial pseudo colored maps of volume transfer constant (Ktrans); (C, L) Oblique axial pseudo colored maps of rate transfer constant (Kep); (D, M) Oblique axial pseudo colored maps of the volume of extravascular extracellular space per unit volume of tissue (Ve); (E, N) Oblique axial colored maps of true diffusion coefficient (D); (F, O) Oblique axial colored maps of pseudo-diffusion coefficient (D*); (G, P) Oblique axial colored maps of microvascular volume fraction (f), and (H, Q) Histopathological images (magnification = 100), and (I, R) Immunohistochemical image (magnification = 200). A E B F C G D H I J LK M RP QON Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma262 Results Basic information The clinicopathological and imaging characteris- tics are shown in Table 2 and Figure 2, respectively. Interobserver consistency The D, D*, f, Ktrans, Ve, and Kep measured by 2 radiol- ogists had excellent consistency, and the ICCs were 0.864 (95% CI: 0.788 - 0.913), 0.799 (95% CI: 0.696 - 0.867), 0.855 (95% CI: 0.729 - 0.918), 0.868 (95% CI: 0.799 - 0.915), 0.834 (95% CI: 0.748 - 0.892), and 0.828 (95% CI: 0.739 - 0.888), respectively. The average re- sults were used for the ultimate analysis. Differences in parameters The Ktrans and Kep were higher and D was lower in the TP53-mutant group than in the TP53-wild group (P = 0.038, 0.002, and 0.037, respectively), f, D*, and Ve were not significantly different between the two groups (P = 0.750, 0.604, and 0.434, respectively). The Ktrans, Ve, f, and D values were lower in the non- low-risk group than in the low-risk group (P < 0.001, < 0.001, 0.002, and < 0.001, respectively), Kep and D* were not significantly different between the two groups (P = 0.218 and 0.601) (Table 3, Figure 3). Regression analyses In the identification of TP53-mutant and TP53-wild early-stage EC, the potential related factors such as age, tumor size, risk stratification, FIGO stage, sub- type, grade, LVSI, D, D*, f, Ktrans, Ve, and Kep were all enrolled in regression analysis. Univariate analy- sis demonstrated that grade, D, Ktrans, and Kep were all risk predictors (P all < 0.1), while multivariate analysis showed that only D and Ktrans were inde- pendent predictors (P = 0.003, 0.016). TABLE 3. Comparison of different parameters Parameters D (×10−3mm2/s) D* (×10−3mm2/s) f (%) K trans (min-1) Ve Kep (min-1) Risk stratification High-risk (n = 10) 0.63 (0.40, 0.73) 58.40 (40.10, 88.73) 1.64 ± 0.60 0.35 (0.15, 0.43) 0.30 ± 0.07 1.23 (0.48, 1.54) High-intermediate-risk (n = 0) / / / / / / Intermediate-risk (n = 20) 0.55 (0.40, 0.81) 52.00 (26.88, 74.33) 1.74 ± 0.96 0.37 (0.29, 0.47) 0.33 ± 0.14 1.24 (0.82, 1.98) Low-risk (n = 44) 0.86 (0.64, 1.16) 44.35 (21.93, 95.33) 2.43 ± 1.08 0.61 (0.43, 1.14) 0.58 ± 0.25 1.53 (0.79, 2.21) P-value 0.033 a 0.464 a 0.012 a < 0.001 a < 0.001 a 0.191 a P-value (High vs Intermediate) 0.880 b 0.248 b 0.735 c 0.397 b 0.532 c 0.307 b P-value (High vs Low) 0.009 b 0.238 b 0.004 c < 0.001 b 0.001 c 0.099 b P-value (Intermediate vs Low) 0.001 b 0.937 b 0.014 c < 0.001 b < 0.001 c 0.582 Low-risk (n = 44) 0.86 (0.64, 1.16) 44.35 (21.93, 95.33) 2.43 ± 1.08 0.61 (0.43, 1.14) 0.58 ± 0.25 1.53 (0.79, 2.21) Non-low-risk (High + Intermediate, n = 30) 0.58 (0.40, 0.77) 55.25 (34.63, 72.78) 1.71 ± 0.84 0.37 (0.28, 0.45) 0.32 ± 0.12 1.23 (0.82, 1.87) z/t value - 3.793 -0.523 3.234 -5.109 5.304 -1.233 P-value < 0.001 b 0.601 b 0.002 c < 0.001 b < 0.001 c 0.218 b TP53 expression Mutant (n = 21) 0.72 ± 0.31 43.70 (16.30, 90.75) 2.30 ± 1.09 0.67 (0.41, 1.14) 0.32 (0.25, 0.91) 1.67 (1.17, 2.09) Wild (n = 25) 0.91 ± 0.29 50.60 (26.90, 82.75) 2.20 ± 1.02 0.43 (0.37, 0.49) 0.49 (0.36, 0.76) 0.90 (0.58, 1.55) Z/t value -2.155 -0.518 0.321 -2.073 -0.783 -3.165 P-value 0.037 c 0.604 b 0.750 c 0.038 b 0.434 b 0.002 b The bold typeface in the table indicates the comparison with statistical significance. a Comparisons were performed by Analysis of variance (ANOVA) test; b comparisons were performed by Mann–Whitney U test; c comparisons were performed by independent t test. Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma 263 In the identification of non-low-risk and low- risk early-stage EC, potential risk-related factors such as age, tumor size, TP53 status, D, D*, f, Ktrans, Ve, and Kep were all enrolled in regression analysis. Univariate analysis demonstrated that tumor size, D, f, Ktrans, and Ve were all risk predictors (P all < 0.1), while multivariate analysis showed that only f, Ktrans, and Ve were independent predictors (P = 0.036, 0.003, and 0.024, respectively) (Table 4). Diagnostic performance of different parameters In the differentiation of TP53-mutant and TP53- wild early-stage EC, the combination of inde- pendent predictors (Ktrans and D) showed the op- timal diagnostic efficacy (AUC = 0.867; sensitivity, 92.00%; specificity, 80.95%; P < 0.001), which was significantly better than D (AUC = 0.694, Z = 2.169, FIGURE 3. Plots show individual data points, averages, and standard deviations of true diffusion coefficient (D) (A, G), pseudo-diffusion coefficient (D*) (B, H), microvascular volume fraction (f) (C, I), volume transfer constant (Ktrans) (D, J), the volume of extravascular extracellular space per unit volume of tissue (Ve) (E, K), and rate transfer constant (Kep) (F, L) in low-risk and non-low-risk groups (A–F), TP53-mutant and TP53-wild groups (G–L). Individual points are averages of values calculated by 2 readers. *P < 0.05, **P < 0.01, ***P < 0.001, and ● P > 0.005. A E B F C G D H I J LK Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma264 P = 0.030), and Ktrans (AUC = 0.679, Z = 2.572, P = 0.010). However, the difference between the com- bination of independent predictors and Kep (AUC = 0.773) was not significant (AUC = 0.773, Z = 1.272, P = 0.203) (Figure 4A, Table 5). In the differentiation of low-risk and non-low- risk early-stage EC, the combination of independ- ent predictors (f, Ktrans, and Ve) showed the opti- mal diagnostic efficacy (AUC = 0.947; sensitivity, 83.33%; specificity, 93.18%; P < 0.001), which was significantly better than D (AUC = 0.761, Z = 3.113, P = 0.002), f (AUC = 0.688, Z = 4.317, P < 0.001), Ktrans (AUC = 0.852, Z = 2.713, P = 0.007), and Ve (AUC = 0.808, Z = 3.175, P = 0.002) (Figure 4B, Table 5). Validation Bootstrapped samples were used to validate the combination of independent predictors. The ROC and the calibration curve indicated that the valida- tion models not only had high accuracy in iden- tifying TP53-mutant and TP53-wild early-stage EC (AUC, 0.815; 95% CI, 0.782 - 0.846, Figure 5A), and low-risk and risk early-stage EC (AUC, 0.922; 95% CI, 0.895 - 0.940, Figure 6A), but also highly had good consistency (Figure 5B, 6B). Also, DCA showed that the above combinations of independ- ent predictors were both reliable clinical decision tools (Figure 5C, Figure 6C). Discussion Prediction of TP53 status and risk stratification of early-stage EC by IVIM The parameter D of IVIM can reflect the diffusion movement of water molecules in the tissue, and usually, the more obvious the restriction of water molecule diffusion, the smaller the D value.13 In this study, the D value of the TP53-mutant group was significantly lower than that of the TP53-wild group, which was similar to the results of Wang et al. in the field of epithelial ovarian cancer25, sug- gesting that D values can be used to predict TP53 status of early-stage EC. Presumably, the reason was that TP53-mutant has a faster rate of cell pro- liferation than TP53-wild, which easily impedes the diffusion of water molecules, resulting in a lower D value.26 In addition, D could also be used to assess the risk stratification of early-stage EC in the present study, which was consistent with previous studies.17,19 The reason may be that there were differences in histological grade, FIGO stage, and lymph node metastasis between the low-risk FIGURE 4. Receiver operating characteristic (ROC) curves, (A) shows each parameter and the combination of independent predictors for differentiation of TP53-mutant and TP53-wild early-stage endometrial carcinoma (EC); (B) shows each parameter and the combination of independent predictors for differentiation of low-risk and non-low-risk early-stage EC. FIGURE 5. In the prediction of TP53 status, receiver operating characteristic curves (A), calibration curves (B), and decision curve analysis (C) of the validation model. A B A B C Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma 265 and non-low-risk early-stage EC, resulting in dif- ferent degrees of influence on the diffusion of water molecules and ultimately leading to sig- nificant differences in D values between the two groups.18,20 D* was a perfusion parameter of IVIM that is mainly correlated with the velocity of blood flow within the microcirculation.13 Previous publica- tions have demonstrated that D* values with poor stability and repeatability could not effectively evaluate histopathological information of early- stage EC due to the influence of the scanning parameters, the ROI determination method, the signal-to-noise ratio (SNR), and other factors.17-20 In this study, there was no statistically significant dif- ference in D* between the TP53-mutant and TP53- wild groups, and the low-risk and the non-low-risk groups, which was consistent with the above re- search, further proving that the D* value was un- able to play a role in the assessment of TP53 status and risk stratification in early-stage EC. As another perfusion parameter derived from IVIM, f was mainly related to the microvascular density of the tissue.13,27 A study by Zhang et al. involving 53 participants showed that although high-risk early-stage EC is metabolically active and rich in neovascularization, due to the dense tissue structure and more necrotic tissue, its over- all internal microvascular density is instead re- duced compared to that of low-risk early-stage EC, so the f value decreases.20 This trial was conduct- ed on a larger sample size of patients (n = 74) and obtained results consistent with those of Zhang et al.19 Further analysis also identified the f value as an independent predictor for discriminating be- tween low-risk and non-low-risk early-stage EC. However, there were also studies that have shown conflicting results of f values in the assessment of lesions. For example, the study by Meng et al. showed that high-risk early-stage EC had higher f values than low-risk early-stage EC.20 Similarly, in the assessment of gliomas, the study of Bai et al. showed that low-grade gliomas had higher f values than high-grade gliomas14, while Shen et al. concluded that high-grade gliomas have greater f values.16 We speculate that the above phenom- enon may be caused by the variations in scanning equipment and b-value settings28, as well as the shortcoming that the f value itself is susceptible to T2 contribution and relaxation effects.29 In ad- dition, the results of this study also showed that the f was similar to D* and could not differenti- ate TP53-mutant from TP53-wild early-stage EC, which to some extent suggests that the use of IVIM perfusion parameters to assess the TP53 status of early-stage EC may still need further exploration. Prediction of TP53 status and risk stratification of early-stage EC by DCE- MRI Ktrans is the most significant perfusion-related pa- rameter in DCE-MRI, mainly reflecting the trans- fer rate of the contrast agent from the vessel to the EES.30 Previous studies have shown that the more neovascularization in the tissue and the greater the permeability, the greater the Ktrans value.31 In terms of TP53 status assessment, the present study found a significantly higher Ktrans value in the TP53-mutant group compared with the TP53- wild group, which we suggest may be related to the ability of TP53 gene overexpression to pro- mote angiogenesis.2,3 In terms of risk stratification assessment, several studies have shown that EC with aggressive characteristics, such as grade 3, FIGURE 6. In the prediction of risk stratification, receiver operating characteristic curves (A), calibration curves (B), and decision curve analysis (C) of the validation model. A B C Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma266 advanced FIGO stage, and non-endometrioid sub- type, grows quickly without sufficient neoangio- genesis (i.e., blood support), resulting in tissue hy- poxia. Hypoxia will lead to tissue necrosis and the formation of hypoperfused areas, thus eventually causing a decrease in overall tumor perfusion and a decrease in Ktrans values.12,17,32,33 In this work, the Ktrans value was significantly lower in the non-low- risk group than in the low-risk group, which was consistent with the above findings and further demonstrates that the Ktrans value can play a role in the risk stratification of early-stage EC. Kep was designed to reflect the transfer rate of the contrast agent from the EES into vessels, so similar to Ktrans, its size was closely related to the number of new vessels and vascular permeability.29 In this study, since TP53 overexpression can promote angi- ogenesis2,3, the Kep value of the TP53-mutant group was significantly higher than those of the TP53- wild group, and the diagnostic efficacy was 0.773. However, the Kep value did not show significant value in the identification of different risk stratifi- cations, which was not consistent with the study of Ye et al.12 We speculated that this may be because the study by Ye et al. included both early-stage (stage I) and advanced-stage (stage II, III, and IV) EC, whereas the present study population included only early-stage EC, which reduced the differences TABLE 4. Logistic regression analyses Parameters Univariate Analyses P-value Multivariate Analyses P-value OR for 1 SD (95% CI) OR for 1 SD (95% CI) Low vs non-low risk Age (year) 1.462 (0.894–2.388) 0.130 / / Tumor size (mm) 1.055 (1.003–1.110) 0.038 1.083 (0.979–1.197) 0.123 TP53 mutant 1.506 (0.407–5.578) 0.540 / / D (×10−3mm2/s) 0.089 (0.021–0.373) 0.001 0.144 (0.015–1.334) 0.088 D* (×10−3mm2/s) 0.867 (0.533–1.412) 0.567 / / f (%) 0.419 (0.226–0.776) 0.006 0.292 (0.093–0.921) 0.036 Ktrans (min-1) 0.009 (0.001–0.153) 0.001 0.001 (0.000–0.089) 0.003 Ve 0.173 (0.069–0.432) < 0.001 0.130 (0.022–0.766) 0.024 Kep (min-1) 0.642 (0.367–1.126) 0.122 / / TP53 mutant vs wild Age (year) 0.855 (0.465–1.548) 0.605 / / Tumor size (mm) 1.175 (0.649–2.127) 0.594 / / Subtype 77.708 (0.001–100.5) 0.999 / / Grade 2.099 (0.957–4.602) 0.064 1.961 (0.816–4.717) 0.132 Risk stratification 1.506 (0.407–5.578) 0.540 / / FIGO stage 1.360 (0.739–2.505) 0.323 / / LVSI 802.578 (0.001–1150.5) 0.999 / / D (×10−3mm2/s) 2.063 (1.016–4.191) 0.045 8.274 (2.066–33.136) 0.003 D* (×10−3mm2/s) 1.020 (0.567–1.835) 0.948 / / f (%) 0.906 (0.504–1.629) 0.742 / / Ktrans (min-1) 0.487 (0.236–1.003) 0.051 0.155 (0.034–0.710) 0.016 Ve 1.008 (0.560–1.812) 0.979 / / Kep (min-1) 0.501 (0.244–1.032) 0.061 1.172 (0.425–3.234) 0.759 D = true diffusion coefficient; D* = pseudo-diffusion coefficient; f = microvascular volume fraction; FIGO = international federation of gynecology and obstetrics; CI = confidence interval; Kep = rate transfer constant; Ktrans = volume transfer constant; LVSI = lymphovascular space invasion; OR = odds ratio;. SD = standard deviation; Ve = volume of extravascular extracellular space per unit volume of tissue The bold typeface in the table indicates the logistic regression analyses with statistical significance. In the analysis of the high- and low-risk group, the TP53 mutant data were analysed only for these patients who had the p53 gene test. The remaining parameters, such as diameter, were analysed for all 74 patients. Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma 267 in patients between the different groups and ulti- mately resulted in nonfunctional Kep values. Ve is a parameter in DCE-MRI that can reflect the volume of EES. In the present study, there was no significant difference in Ve between the TP53- mutant and TP53-wild groups, which may be relat- ed to the fact that TP53 overexpression promotes both cell proliferation and angiogenesis, resulting in difficulty in significant changes in EES.2,3 In terms of risk stratification assessment, Ve values in the non-low-risk group were significantly smaller than those in the low-risk group, which was simi- lar to the results of previous studies17,34, and we speculated that the reason for this result may lie in the fact that the non-low-risk group had greater in- vasiveness and therefore greater cell density, tight- er tissue structure, and smaller EEC compared with the low-risk group. However, some studies have also concluded that Ve was difficult to use in the evaluation of diseases such as EC and breast cancer.12,35 This may be related to the fact that Ve is less stable and susceptible to factors such as lesion edema and microcystic changes.36 In a follow-up study, we will expand the sample size and further explore the role of Ve in EC assessment to obtain more convincing results. Diagnostic performance comparison The diagnostic efficacy of the combination of in- dependent predictors and each individual param- eter was compared in this study, and the results showed that the diagnostic efficacy of the former was significantly higher than that of the latter, which may be because the combination of inde- pendent predictors concentrates the advantages of different parameters and therefore can reflect the lesion characteristics more comprehensively and accurately. Therefore, we suggest that the com- bined application of IVIM and DCE-MRI in clini- cal routine may provide a more reliable basis for the TP53 status and risk stratification prediction of early-stage EC when conditions permit. Correlation of risk stratification with TP53 mutation In this study, TP53 mutation and risk stratification in early-stage EC were included in each other’s regression analysis, and the results showed that neither was a predictor of the other. Although the small sample size may have affected the reliability of the above results to a certain extent, it indicates TABLE 5. Predictive performance of different parameters Parameters AUC (95% CI) P-value Cutoff Sensitivity Specificity Comparison with combined diagnosis Low vs non-low risk D (×10−3mm2/s) 0.761 (0.648–0.853) < 0.001 0.691 73.33% 72.73% Z = 3.113, P = 0.002 D* (×10−3mm2/s) 0.536 (0.416–0.653) 0.598 / / / / f (%) 0.688 (0.569–0.790) 0.003 1.240 36.67% 93.18% Z = 4.317, P < 0.001 Ktrans (min-1) 0.852 (0.750–0.924) < 0.001 0.487 90.00% 68.18% Z = 2.713, P = 0.007 Ve 0.808 (0.700–0.890) < 0.001 0.401 83.33% 70.45% Z = 3.175, P = 0.002 Kep (min-1) 0.585 (0.652–0.849) 0.204 / / / / Combined diagnosis 1 0.947 (0.869–0.986) < 0.001 / 83.33% 93.18% / TP53 mutant vs wild D (×10−3mm2/s) 0.694 (0.541–0.821) 0.019 0.605 92.00% 47.62% Z = 2.169, P = 0.030 D* (×10−3mm2/s) 0.545 (0.391–0.692) 0.498 / / / / f (%) 0.535 (0.382–0.648) 0.388 / / / / Ktrans (min-1) 0.679 (0.525–0.809) 0.036 0.499 80.00% 61.90% Z = 2.572, P = 0.010 Ve 0.568 (0.413–0.713) 0.675 / / / / Kep (min-1) 0.773 (0.626–0.884) < 0.001 1.557 80.00% 66.67% Z = 1.272, P = 0.203 Combined diagnosis 2 0.867 (0.734–0.949) < 0.001 / 92.00% 80.95% / AUC = area under the receiver operating characteristic (ROC) curve; D = true diffusion coefficient; D* = pseudo-diffusion coefficient; f = microvascular volume fraction; Kep = rate transfer constant; Ktrans = volume transfer constant; Ve = volume of extravascular extracellular space per unit volume of tissue The combined diagnosis 1 represents f + Ktrans + Ve ; the combined diagnosis 2 represents D + Ve Radiol Oncol 2023; 57(2): 257-269. Wang H et al. / DCE-MRI in risk stratification of endometrial carcinoma268 to some extent that the TP53 status in early-stage EC is not significantly correlated with risk stratifi- cation. In the future, as the sample size increases, we will conduct more in-depth studies on the rela- tionship between the two, with a view to obtain- ing more accurate results. This study has several limitations. First, our study was designed at a single institution with a relatively small number of patients, especially since some patients forgo immunohistochemical testing for financial reasons, which may have led to selection bias. Second, due to the small sample size, this study did not set up a separate validation set but used the bootstrap (1000 samples) method to validate the combination of independent predic- tors, which may have reduced the reliability of the experimental results. Third, areas of cystic degen- eration, necrosis, apparent signs and hemorrhage artifacts, or vessels were avoided in the delineation of the ROI, which may influence the determination of some parameters. Finally, the machine used in this study was a 1.5 T MRI, and its imaging quality and parameter reliability may be inferior to those of a 3.0 T MRI. Conclusions Both DCE-MRI and IVIM facilitate the prediction of TP53 status and risk stratification in early-stage EC. Comparison with each single parameter, the combination of independent predictors provided better predictive power and may serve as a supe- rior imaging marker. Acknowledgement This work was supported by the Roentgen Imaging Research Project (HN-20201017-002), the Key Project of Henan Province Medical Science and Technology Project (LHGJ20200519). The datasets and analyses during the current study are avail- able from the corresponding author on reasonable request. References 1. 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Dynamic contrast-enhanced magnetic reso- nance imaging of prostate cancer: a review of current methods and applica- tions. World J Radiol 2017; 9: 416-25. doi: 10.4329/wjr.v9.i12.416 32. Bredholt G, Mannelqvist M, Stefansson IM, Birkeland E, Bø TH, Øyan AM, et al. Tumor necrosis is an important hallmark of aggressive endometrial can- cer and associates with hypoxia, angiogenesis and inflammation responses. Oncotarget 2015; 6: 39676-91. doi: 10. 18632/oncotarget.5344 33. Vaupel P, Mayer A. Hypoxia in tumors: pathogenesis-related classification, characterization of hypoxia subtypes, and associated biological and clinical implications. Adv Exp Med Biol 2014; 812: 19-24. doi: 10.1007/978-1-4939- 0620-8_3 34. Haris M, Gupta RK, Singh A, Husain N, Husain M, Pandey CM, et al. Differentiation of infective from neoplastic brain lesions by dynamic contrast-enhanced MRI. Neuroradiology 2008; 50: 531-40. doi: 10.1007/ s00234-008-0378-6 35. Liu C, Wang K, Chan Q, Liu Z, Zhang J, He H, et al. Intravoxel incoherent motion MR imaging for breast lesions: comparison and correlation with pharmacokinetic evaluation from dynamic contrast-enhanced MR imaging. Eur Radiol 2016; 26: 3888-98. doi: 10.1007/s 00330-016-4241-6 36. Cho N, Im SA, Park IA, Lee KH, Li M, Han W, et al. Breast cancer: early prediction of response to neoadjuvant chemotherapy using parametric response maps for MR imaging. Radiology 2014; 272: 385-96. doi: 10.1148/ radiol.14131332 Radiol Oncol 2023; 57(2): 270-278. doi: 10.2478/raon-2023-0026 270 research article Two-stage hepatectomy in resection of colorectal liver metastases − a single- institution experience with case-control matching and review of the literature Spela Turk1,2, Irena Plahuta1,2, Tomislav Magdalenic1, Tajda Spanring1,2, Kevin Laufer1,2, Zan Mavc1, Stojan Potrc1,2, Arpad Ivanecz1,2 1 Clinical Department of Abdominal and General Surgery, University Medical Centre Maribor, Maribor, Slovenia 2 Department of Surgery, Faculty of Medicine, University of Maribor, Maribor, Slovenia Radiol Oncol 2023; 57(2): 270-278. Received 18 March 2023 Accepted 15 May 2023 Correspondence to: Assist. Prof. Arpad Ivanecz, M.D., Ph.D., Clinical Department of Abdominal and General Surgery, University Medical Centre Maribor, Ljubljanska ulica 5, SI-2000 Maribor, Slovenia. E-mail: arpad.ivanecz@ukc-mb.si Spela Turk and Irena Plahuta contributed equally to this work. Disclosure: No potential conflicts of interest were disclosed. The abstract was accepted for presentation at the 15th Biennial Congress of the European Hepato-Pancreato-Biliary Association in June 2023 in Lyon, France. This is an open-access article distributed under the terms of the CC-BY licence (https://creativecommons.org/licenses/by/4.0/). Background. Two-stage hepatectomy (TSH) has been proposed for patients with bilateral liver tumours who have a high risk of posthepatectomy liver failure after one-stage hepatectomy (OSH). This study aimed to determine the outcomes of TSH for extensive bilateral colorectal liver metastases. Patients and methods. A retrospective review of a prospectively maintained database of liver resections for colorectal liver metastases was conducted. The TSH group was compared to the OSH group in terms of perioperative outcomes and survival. Case-control matching was performed. Results. A total of 632 consecutive liver resections for colorectal liver metastases were performed between 2000 and 2020. The study group (TSH group) consisted of 15 patients who completed TSH. The control group included 151 patients who underwent OSH. The case-control matching-OSH group consisted of 14 patients. The major morbidity and 90-day mortality rates were 40% and 13.3% in the TSH group, 20.5% and 4.6% in the OSH group and 28.6% and 7.1% in the case-control matching-OSH group, respectively. The recurrence-free survival, median overall survival, and 3- and 5-year survival rates were 5 months, 21 months, 33% and 13% in the TSH group; 11 months, 35 months, 49% and 27% in the OSH group; and 8 months, 23 months, 36% and 21%, respectively, in the case-control matching-OSH group, respectively. Conclusions. TSH used to be a favourable therapeutic choice in a select population of patients. Now, OSH should be preferred whenever feasible because it has lower morbidity and equivalent oncological outcomes to those of completed TSH. Key words: colorectal cancer; liver metastases; hepatectomy; future liver remnant; posthepatectomy liver failure; survival analysis Introduction Colorectal cancer is the third most diagnosed cancer worldwide.1 At diagnosis, the disease has spread to the liver in 15% to 25% of patients, and another 25% develop colorectal liver metastases metachronously.2 Liver resection remains the on- ly potentially curative treatment option for these patients.2 Despite the ability of the liver to regen- erate after significant tissue loss, a future liver Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases 271 remnant, which contributes 25–30% of the total liver volume, has been the minimal requirement in patients with a noncirrhotic liver.2 Therefore, major hepatectomies are associated with a high risk of posthepatectomy liver failure.3 Innovative approaches have been developed to improve colo- rectal liver metastases resectability, i.e., two-stage hepatectomy (TSH).2,4 Their initial phase can be portal vein embolization or intraoperative selec- tive portal vein ligation. The novelist approach is the associating liver partition and portal vein liga- tion for staged hepatectomy (ALPPS) procedure.2 This study aimed to determine the feasibility and safety of TSH for patients with extensive bi- lateral colorectal liver metastases by comparing perioperative and long-term outcomes between TSH and one-stage hepatectomy (OSH) groups. Patients and methods Study population A retrospective review of a prospectively ob- tained database of 632 consecutive liver proce- dures for colorectal liver metastases at the Clinical Department of Abdominal and General Surgery of the University Medical Centre Maribor in Slovenia was performed. This department is a specialised referral centre for hepato-pancreato-biliary sur- gery. The study period was from 1 January 2000 until 31 December 2020. Before the surgery, patients consented to their anonymous data being used for research. Therefore, their records were anonymised and dei- dentified before analysis. Ethical approval for this study was obtained from the National Medical Ethics Committee of the Republic of Slovenia (0120-455/2020/3). All methods were performed following the relevant guidelines and regulations. Inclusion and exclusion criteria The inclusion criteria were patients with bilateral colorectal liver metastases who: • completed TSH or • underwent their first OSH for colorectal liver metastases, • the TSH group was formed from patients who underwent portal vein embolization or portal vein ligation, as proposed by Regimbeau.5 The exclusion criteria were as follows: • explorative laparotomies without liver re- sections, • repeated liver resections, • patients with unilateral colorectal liver me- tastases, • radiofrequency ablation (RFA) or its combi- nations with liver resections. Definitions Routinely available clinical characteristics were analysed, including patient demographics, perfor- mance status defined according to the American Society of Anaesthesiologists Classification (ASA classification)6, application of neoadjuvant chemo- therapy, preoperative carcinoembryonic antigen (CEA) level, and presence of extrahepatic disease. Primary colorectal tumour variables included the tumour location and nodal invasion. Liver metasta- sis variables included synchronous/metachronous metastases and the number and size of metastases. Patients were presented at the multidisciplinary team meeting.2 Bilateral colorectal liver metastases were resected in a single procedure when both the volume and function of the future liver remnant were considered sufficient. The parenchyma-spar- ing principle of liver surgery for colorectal liver me- tastases was applied.7 The types of liver resections were classified according to the Brisbane terminol- ogy.8 Major liver resections involved three or more adjacent liver segments, including conventional major resections (left/extended left hepatectomies, right/extended right hepatectomies, central hepa- tectomies).8 The analysis of future liver remnant consisted of computed tomography (CT) volume- try, laboratory liver tests (prothrombin time and al- bumins), and the indocyanine green clearance test.2 Specimens were analysed by a gastrointestinal histopathologist who assessed the resection mar- gin. The histological surgical margins for malig- nant lesions were defined as microscopically nega- tive (R0) or positive (<1 mm, R1).7 In addition, the Clinical Risk Score devised by Fong et al. was ap- plied.9 Two-stage hepatectomy Portal vein embolization, intraoperative selective portal vein ligation, or the ALPPS procedure were performed when the analysis suggested an insuf- ficient future liver remnant.2 Portal vein emboli- zation was followed by atrophy of the embolized hemiliver and hypertrophy of the other hemiliver.2 TSH with portal vein ligation was performed when the intraoperative findings were unfavourable.4 In the first stage, the metastasectomy of one hemiliver Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases272 was performed along with portal vein ligation for the other hemiliver.4 The effect was similar to that of preoperative portal vein embolization.10 The second stage followed a few weeks later and consisted of a major hepatectomy.2,4 ALPPS was performed with the same rationale; the difference was the addition of liver parenchyma transection in the first stage.11 RFA has been applied where radical liver re- section has been infeasible due to the proximity of large vessels.2 Therefore, RFA has been applied intraoperatively as an independent procedure or adjunct to liver resection.2 RFA has also been used as a percutaneous procedure. However, these pa- tients were excluded from the analyses. Follow-up Patients were followed-up at the outpatient clinic at periodic intervals. The follow-up protocol con- sisted of a CEA level, a chest radiograph or CT, an abdominal ultrasound, CT, or magnetic resonance imaging every three months for the first two years and every six months afterwards.2 Study endpoints Primary outcomes ‒ overall survival and recurrence-free survival The primary outcome was overall survival (OS). It was defined as the interval between the date of liver resection (the second stage in the TSH group) of colorectal liver metastases and the date of death or the last follow-up in surviving patients. The second primary outcome was recurrence- free survival (RFS). It was calculated from the date of liver resection (the second stage in the TSH group) to the date of any detected recurrence or the last follow-up in patients without recurrence. Secondary outcomes ‒ morbidity and mortality Morbidity was reported according to the Clavien- Dindo (CD) classification.12 Major morbidity was defined as CD ≥ 3a. Mortality rates were reported as the number of patients who died within 90 post- operative days. Posthepatectomy haemorrhage, bile leak- age, and liver failure were graded according to the International Study Group of Liver Surgery (ISGLS).13-15 Statistical analysis IBM SPSS for Windows Version 28.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. Percentages are reported to one decimal place. A P value ≤ 0.05 was considered statistically significant. Categorical variables are displayed as numbers with percentages. The differences between cat- egorical variables were tested using the chi-square or Fisher-Freeman Halton test when more than two categories were present. Continuous vari- ables were expressed as medians (minimum-max- imum, interquartile range) and analysed with the Mann-Whitney test since the distribution analysis showed the non-normal distribution of data. Survival data for median OS and RFS are pre- sented as Kaplan-Meier curves, and groups were compared by a log-rank test. The results are ex- pressed in months as the median (95% confidence interval (95% CI)). Survival tables were used for 3- and 5-year OS and RFS, given in percentages. Case–control matching CCM-OSH group n=14 Liver procedures for CLMs n=632 Liver resection n=469 Liver resection and RFA n=29 Open RFA n=42 Percutaneous RFA n=22 Exploration only n=30 One-stage procedures n=592 OSH group Bilateral liver metastases n=151 Two-stage hepatectomy Only the first stage performed due to disease progression n=6 TSH group n=15 Both stages completed First stage n=23 Right PVE: n=6 Right PVL: n=13 ALPPS: n=4 Excluded: Unilateral CLMs or repeated liver resection n=318 Excluded: Unilateral CLMs n=2 FIGURE 1. The study flowchart. The study period covers 1 January 2000 to 31 December 2020. ALPPS = associating liver partition and portal vein ligation for staged hepatectomy; CCM-OSH = case-control matching one-stage hepatectomy; CLMs = colorectal liver metastases; OSH = one-stage hepatectomy; PVE = portal vein embolization; PVL = portal vein ligation; RFA = radiofrequency ablation; TSH = two-stage hepatectomy Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases 273 TABLE 1. Clinical characteristics and perioperative outcomes of the 166 patients Clinical characteristics OSH (n=151) TSH (n=15) P value Male sex a 109 (72.2%) 13 (86.7%) 0.365 Age (years) b 62 (34–84; 14) 64 (45–75; 12) 0.819 ASA score ≥ 3 a 32 (21.2%) 2 (13.3%) 0.701 Primary tumour location c Right colon 27 (17.9%) 1 (6.7%) 0.166 Left colon 61 (40.4%) 6 (40.0%) Rectum 61 (40.4%) 7 (46.7%) > 1 primary tumour 2 (1.3%) 1 (6.7%) Primary tumour nodal invasion a 99 (66.0%) 10 (66.7%) 1.000 Synchronous liver metastases a 81 (53.6%) 11 (73.3%) 0.179 Number of liver metastases b 3 (1–19; 3) 5 (2–12; 6) 0.001 Size of liver metastases (cm) b 4 (0.6–20; 3) 5 (1.5–11; 5.5) 0.183 Neoadjuvant chemotherapy a 79 (52.3%) 13 (86.7%) 0.013 Preoperative CEA level (ng/mL) b 14 (1–1359; 47.5) 12 (2–1312; 60) 0.464 Extrahepatic disease a 27 (17.9%) 3 (20.0%) 1.000 Clinical risk score 3–5 a 92 (60.9%) 10 (66.7%) 0.875 Major liver resection a 58 (38.4%) 15 (100%) <0.001 Atypical resection 29 (19.2%) 0 (0.0%) / Segmentectomy/segmentectomy & atypical resection 3 (2.0%)/14 (9.3%) 0 (0.0%) / Bisegmentectomy/bisegmentectomy & atypical resection 10 (6.6%)/37 (24.5%) 0 (0.0%) / Right/extended right hepatectomy 28 (18.5%)/6 (4.0%) 13 (86.7%)/2 (13.3%) / Left/extended left hepatectomy 6 (4.0%)/3 (2.0%) 0 (0.0%) / Trisegmentectomy 6 (4.0%) 0 (0.0%) / Trisegmentectomy & atypical resection 5 (3.3%) 0 (0.0%) / Central resection 4 (2.6%) 0 (0.0%) / R0 resection a 118 (78.1%) 10 (66.7%) 0.492 CD ≥ 3a a 31 (20.5%) 6 (40.0%) 0.161 90-day mortality a 7 (4.6%) 2 (13.3%) 0.189 ISGLS haemorrhage grade C a 2 (1.3%) 0 (0.0%) 1.000 ISGLS bile leakage grade C a 5 (3.3%) 1 (6.7%) 1.000 ISGLS liver failure – any grade a 40 (26.5%) 12 (80.0%) <0.001 Grade A a 16 (10.6%) 5 (33.3%) 0.034 Grade B a 19 (12.6%) 6 (40.0%) 0.014 Grade C a 5 (3.3%) 1 (6.7%) 1.000 Hospital stay (days) b 10 (5–63; 7) 14 (8–158; 11) 0.028 a Categorical variable reported as n (%), chi-square test; b continuous variable, non-normal distribution, reported as median (minimum-maximum, interquartile range), Mann-Whitney test; c categorical variable with more than two groups, reported as n (%), Fisher-Freeman-Halton test; ASA = American Society of Anaesthesiologists; CEA = carcinoembryonic antigen; CD = Clavien-Dindo classification; ISGLS = International Study Group of Liver Surgery; OSH = one-stage hepatectomy; TSH = two-stage hepatectomy Case-control matching was performed for 15 patients from the TSH group.16 Patients from the OSH group (controls) were selected based on the variables that were statistically significant in a bivariate analysis. The sampling was performed without replacement and with maximising execu- Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases274 TABLE 2. Clinical characteristics of the matched groups Clinical characteristics CCM-OSH (n = 14) TSH (n = 15) P value Male sex a 11(78.6%) 13(86.7%) 1.000 Age (years) b 60 (53–78; 13) 64 (45–75; 12) 0.463 ASA score ≥ 3 a 2 (14.3%) 2 (13.3%) 1.000 Primary tumour in right colon a 2 (14.3%) 1 (6.7%) 1.000 Primary tumour in left colon a 10 (71.4%) 6 (40%) 0.125 Primary tumour in rectum a 2 (14.3%) 7 (46.7%) 0.063 > 1 primary tumour a 0 (0.0%) 1 (6.7%) 1.000 Primary tumour nodal invasion a 10 (71.4%) 10 (66.7%) 1.000 Synchronous liver metastases a 11(78.6%) 11(73.3%) 1.000 Number of liver metastases b 5 (2–12; 6) 5 (2–12; 6) 0.317 Size of liver metastases (cm) b 4.6 (1–20; 7) 5 (1.5–11; 5.5) 0.463 Neoadjuvant chemotherapy a 13 (92.9%) 13 (86.7%) 1.000 Preoperative CEA level (ng/mL) b 9 (1–261; 76) 12 (2–1312; 60) 0.975 Extrahepatic disease a 1 (7.1%) 3 (20.0%) 1.000 Clinical risk score 3–5 a 13 (92.9%) 10 (66.7%) 0.250 Major hepatectomy a 14(100%) 15 (100%) 1.000 Right/extended right hepatectomy 8 (57.1%)/2 (14.3%) 13 (86.7%)/2 (13%) / Left hemihepatectomy 1 (7.1%) 0 (0.0%) / Trisegmentectomy & atypical resection 3 (21.4%) 0 (0.0%) / R0 resection a 8 (57.1%) 10 (66.7%) 1.000 CD ≥ 3a a 4 (28.6%) 6 (40.0%) 0.688 90-day mortality a 1 (7.1%) 2 (13.3%) 1.000 ISGLS haemorrhage grade C a 0 (0.0%) 0 (0.0%) 1.000 ISGLS bile leakage grade C a 1 (7.1%) 1 (6.7%) 1.000 ISGLS liver failure – any grade a 10 (71.4%) 12 (80%) 1.000 Grade A a 4 (28.6%) 5 (33.3%) 1.000 Grade B a 5 (35,7%) 6 (40.0%) 1.000 Grade C a 1 (7.1%) 1 (6.7%) 1.000 Hospital stay (days) b 16 (6–63; 13) 14 (8–158; 11) 0.406 a Categorical variable reported as n (%), McNemar test; b continuous variable, nonnormal distribution, reported as the median (minimum- maximum, interquartile range), Wilcoxon signed ranks test ASA = American Society of Anaesthesiologists; CCM-OSH = case-control matching one-stage hepatectomy; CEA = carcinoembryonic antigen; CD = Clavien-Dindo classification; ISGLS = International Study Group of Liver Surgery; TSH = two-stage hepatectomy tion performance modality. Matched patients were assigned to the case-control matching-OSH group. The statistical analysis of continuous variables was performed with the Wilcoxon signed ranks test. The analysis of categorical variables was per- formed with the McNemar test.16 Survival was es- timated as described previously. Results The study population was stratified into two groups. The study group TSH consisted of 15 pa- tients who completed TSH. The control group OSH included 151 patients. The study flowchart is shown in Figure 1. Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases 275 Clinical characteristics of patients This study included 166 patients: 151 in the OSH group and 15 in the TSH group. Their clinical char- acteristics and perioperative outcomes are sum- marised in Table 1. Case-control matching To reduce the bias and equilibrate the number of group members, case-control matching was conducted. Patients from the OSH group (con- trols) were selected based on the predictors that were statistically significant in bivariate analysis (Table 1): number of liver metastases, neoadjuvant chemotherapy, and extent of liver resection. Case-control matching returned 14 controls among the OSH group, annotated as case-control matching-OSH. All three variables were statisti- cally significant in the case-control matching mod- el (P < 0.001). The Wilcoxon signed ranks test for the median of differences between before and after matching was insignificant (P = 0.317). Analyses after case-control matching After case-control matching, the TSH and OSH groups were compared (Table 2). Morbidity and mortality Perioperative morbidity and 90-day mortality rates are provided in Table 1 and Table 2. In the OSH group, seven (4.6%) patients died postoperatively. The causes of death were sepsis TABLE 3. Survival analysis Overall OSH (n = 151) TSH (n = 15) P value Median OS (months) [95% CI] 35 [30–40] 35 [31–39] 21 [17–25] 0.063 3-year OS 48% 49% 33% 0.107 5-year OS 26% 27% 13% 0.107 RFS (months) [95% CI] 11 [8–14] 11 [9–13] 5 [2–8] 0.138 3-year RFS 14% 15% 13% 0.070 5-year RFS 10% 10% 7% 0.070 After case-control matching Overall CCM-OSH (N=14) TSH (N=15) P value Median OS (months) [95% CI] 23 [19–27] 23 [5–41] 21 [17.0–25.0] 0.575 3-year OS 34% 36% 33% 0.743 5-year OS 17% 21% 13% 0.743 RFS (months) [95% CI] 7 [4–10] 8 [1–15] 5 [2–8] 0.888 3-year RFS 14% 14% 13% 0.498 5-year RFS 3% 0% 7% 0.498 CI = confidence interval; CCM = case-control matching; OS = overall survival; OSH = one-stage hepatectomy; RFS = recurrence-free survival; TSH = two-stage hepatectomy FIGURE 2. Overall survival after case-control matching (TSH vs. case-control matching-OSH groups), P = 0.575. CCM-OSH = case-control matching one-stage hepatectomy; TSH = two-stage hepatectomy Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases276 (n = 1), cardiorespiratory failure (n = 1), multiorgan failure (n = 3), and posthepatectomy liver failure (n = 2). In the TSH group, two (13.3%) patients died postoperatively. One patient suffered from colonic perforation, was reoperated on several times, and died of multiorgan failure. The second patient died of cardiorespiratory failure after acute myocardi- al infarction. In the case-control matching-OSH group, the patient (7.1%) died of posthepatectomy liver failure. Survival analysis Patients were followed until their death or until 31 December 2022. The median follow-up was 174 (95% CI 113–235) months. A summary of the sur- vival analysis is provided in Table 3 and Figure 2. The RFS, median OS, and 3- and 5-year survival rates were 5 months, 21 months, 33% and 13% in the TSH group; 11 months, 35 months, 49% and 27% in the OSH group; and 8 months, 23 months, 36% and 21% in the case-control matching-OSH group, respectively. Discussion The main finding of our research is that the TSH group had a similar survival to that of the case- control matching-OSH group (median OS: 21 vs. 23 months), while the major morbidity rate was lower in the case-control matching-OSH group (40% vs. 28.6%). The first TSH was performed on the proposition that a liver resection where some tumour tissue remains in place could be justified if it could be removed by second liver resection.4 The time dur- ing surgeries was intended for liver hypertrophy, which was enhanced by portal vein embolization or portal vein ligation.4,17 Our first TSH was performed in 2005, and 23 pa- tients with the most difficult patterns of colorectal liver metastases were allocated for this demand- ing treatment (Figure 1).18-20 Only 15 patients who finished both stages were eligible for this study. This figure is among the lowest, especially com- pared to the most recent multicentre studies, but close to those in earlier studies.2 Regimbeau et al. TABLE 4. Literature review of surgical outcomes and survival after two-stage hepatectomy for colorectal liver metastases Authors Year Study period N of patients Major liver resection (%) R0 resection (%) Morbidity (%) Mortality (%) Median follow-up (months) Median RFS (months) 3-y RFS (%) 5-y RFS (%) Median OS (months) 3-y OS (%) 5-y OS (%) Adam et al., France4 2000 1992–1999 13 62 NR 45 15 22 NR 31 31 44 35 NR Tanaka et al., Japan17 2007 1992–2004 22 67 87 23 0 NR NR 6 NR NR 33 NR Wicherts et al., France24 2008 1992–2007 41 76 NR 59 7 24 NR 26 13 39 60 42 Narita et al., France25 2011 1996–2009 61 95 NR 54 0 30 NR 15 8 40 59 32 Turrini et al., France26 2012 2000–2010 34 91 100 20 6 41 NR 24 14 44 59 35 Omichi et al., Japan27 2022 2013–2019 32 NR 78 22 0 17 6 NR NR 41 61 NR The present study, Slovenia 2023 2000–2020 15 100 67 40 13 174 7 13 7 21 33 13 Multicentre studies Tsai et al., USA and Portugal28 2010 1994–2008 35 80 NR 26 5 NR NR NR NR 16 58 NR Regimbeau et al. LiverMetSurvey registry5 2017 2000–2014 625 NR 58 25 9 84a 41a 43 23 40a 45 23 Petrowsky et al. ALPPS registry29 2020 2009–2019 510 100 73 21 5 38 11 19 12 37 52 27 Chavez et al. Five centres in the USA22 2021 2000–2016 196 76 92 23 5 28 NR 19 18 50 64 44 a = mean ALPPS = associating liver partition and portal vein ligation for staged hepatectomy; N = number; NR = not reported; OS = overall survival; RFS = recurrence-free survival; USA = United States of America Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases 277 and Chavez et al. published large multicentre stud- ies.5,22 Their study periods overlap with previous analyses from included centres, as shown in some reviews (Table 4).21,23 The diversity of first-step procedures challeng- es further analysis.30 Nevertheless, this research adheres to the criteria by Regimbeau et al.5 except for one case of completed ALPPS. The clinical characteristics of patients in the OSH and TSH groups differed only in the medi- an number of colorectal liver metastases (3 vs. 5) and the rate of chemotherapy treatment (52.3% vs. 86.7%). Both characteristics denote an extensive tu- mour burden in the TSH group.27 The concept of parenchyma-sparing liver sur- gery for colorectal liver metastases was estab- lished approximately 30 years ago.7 This explains the large group of one-stage hepatectomies (n = 151), even in the case of bilateral colorectal liver metastases, and the lower rate of major hepatecto- mies in this group (38.4%) (Tables 1 and 2). In the TSH group, the rate of major hepatectomies was 100%, and the difference from the OSH group was statistically significant. The reported rates of major liver resections are given in Table 4. There was a significant difference in posth- epatectomy liver failure when comparing the TSH (80%) and OSH groups (26.5%) (P < 0.001) (Table 1). However, this difference disappeared after case- control matching because the matched group was selected based on neoadjuvant chemotherapy, the extent of liver resection and the number of liver metastases. In the case-control matching-OSH group, 71% of patients had any grade of posthepa- tectomy liver failure (Table 2), and one patient died of it. However, the reported rates of posthepatec- tomy liver failure are from 2.6 to 16%.5,25,27-28 This wide range may also be due to several definitions of it.31 The most commonly used definitions were the following: peak bilirubin > 7 mg/dL32, the “50- 50” criteria (50% of normal for the prothrombin in- dex and 50 µmol/L for bilirubin on postoperative day 5)33, and the definition by the ISGLS used in this study.14 The high rate of major morbidity (40% in the TSH group) reflects the burden of demanding two- stage procedures. On the other hand, 4 (28.6%) pa- tients suffered from major morbidity in the case- control matching-OSH group, and the difference between these two groups was statistically insig- nificant (P = 0.688). The 90-day mortality in our TSH group (13%) did not exceed the reported 15% (Table 4). TSH aims to improve the survival of patients by resecting all tumoral tissue and enabling sufficient future liver remnant. Our last TSH was performed in 2016, a year before Torzilli et al. published re- sults of enhanced OSH as a safe alternative to TSH for multiple bilateral deep-located colorectal liver metastases.34 The prognosis was thought to depend on the size and number of colorectal liver metastases.2 Whether the resection of colorectal liver metasta- ses could achieve R0, survival was the same re- gardless of the number of lesions.2 However, Fong et al. showed that the prognosis depends on the combination of survival factors, i.e., Clinical Risk Score.9 There was no significant difference (P = 0.063) in the median OS between the OSH (35 months) and TSH groups (21 months). The rate of R0 re- sections and the high Clinical Risk Score did not differ. After case-control matching, the OS in the case-control matching-OSH group was 23 months. However, the median OS in recent reports is long- er (37–50 months) (Table 4). Our study showed an insignificant difference in the 3-year RFS between the TSH group (13%) and the case-control matching-OSH group (14%). The 3-year RFS in our study was similar to that re- ported by Narita et al. and Chavez et al., but much shorter than the 43% reported by Regimbeau et al. (Table 4).5,22,25 Limitations of our study could explain these dif- ferences in survival. First, this was a single-institu- tion, retrospective study covering a wide period. We had a small group of patients who underwent TSH. Furthermore, the operative technique and use of portal vein embolization have changed over time; thus, it is difficult to apply this study to other modern scenarios. In addition, the interpretation of data and their comparison to other reports was difficult because the TSH group consisted of vari- ous first-stage procedures. To conclude, parenchyma-sparing surgery is a principle of liver surgery for colorectal liver me- tastases. TSH used to be a safe and favourable therapeutic choice in a select population of pa- tients because it could prevent posthepatectomy liver failure and enable good oncological results. Now, OSH should be preferred whenever feasible because it has lower morbidity and equivalent on- cological outcomes as completed TSH. Radiol Oncol 2023; 57(2): 270-278. Turk S et al. / Two-stage hepatectomy in resection of colorectal liver metastases278 Acknowledgements The University Medical Centre Maribor funded this study but had no role in its design, practice, or analysis. References 1. Morgan E, Arnold M, Gini A, Lorenzoni V, Cabasag CJ, Laversanne M, et al. Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 2023; 72: 338-44. doi: 10.1136/ gutjnl-2022-327736 2. Kow AWC. Hepatic metastasis from colorectal cancer. J Gastrointest Oncol 2019; 10: 1274-98. doi: 10.21037/jgo.2019.08.06 3. Müller PC, Linecker M, Kirimker EO, Oberkofler CE, Clavien PA, Balci D, et al. Induction of liver hypertrophy for extended liver surgery and partial liver transplantation: state of the art of parenchyma augmentation-assisted liver surgery. Langenbecks Arch Surg 2021; 406: 2201-15. doi: 10.1007/ s00423-021-02148-2 4. Adam R, Laurent A, Azoulay D, Castaing D, Bismuth H. Two-stage hepatec- tomy: a planned strategy to treat irresectable liver tumors. Ann Surg 2000; 232: 777-85. doi: 10.1097/00000658-200012000-00006 5. Regimbeau JM, Cosse C, Kaiser G, Hubert C, Laurent C, Lapointe R, et al. Feasibility, safety and efficacy of two-stage hepatectomy for bilobar liver metastases of colorectal cancer: a LiverMetSurvey analysis. HPB 2017; 19: 396-405. doi: 10.1016/j.hpb.2017.01.008 6. Doyle DJ, Hendrix JM, Garmon EH. American Society of Anesthesiologists Classification. 2022. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing LLC. [cited 2923 Feb 15]. PMID: 28722969. Available at: https:// www.ncbi.nlm.nih.gov/books/NBK441940/ 7. Torzilli G, Vigano L, Gatti A, Costa G, Cimino M, Procopio F, et al. Twelve-year experience of “radical but conservative” liver surgery for colorectal metasta- ses: impact on surgical practice and oncologic efficacy. HPB (Oxford) 2017; 19: 775-84. doi: 10.1016/j.hpb.2017.05.006 8. Strasberg SM, Belghiti J, Clavien PA, Gadzijev E, Garden JO, Lau WY, et al. The Brisbane 2000 terminology of liver anatomy and resections. HPB (Oxford) 2000; 2: 333-39. doi: 10.1016/s1365-182x(17)30755-4 9. Fong Y, Fortner J, Sun RL, Brennan MF, Blumgart LH. Clinical score for pre- dicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases. Ann Surg 1999; 230: 309-18; discussion 18-21. doi: 10.1097/00000658-199909000-00004 10. Broering DC, Hillert C, Krupski G, Fischer L, Mueller L, Achilles EG, et al. 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HPB (Oxford) 2011; 13: 528-35. doi: 10.1111/j.1477-2574.2011.00319.x 14. Rahbari NN, Garden OJ, Padbury R, Brooke-Smith M, Crawford M, Adam R, et al. Posthepatectomy liver failure: a definition and grading by the International Study Group of Liver Surgery (ISGLS). Surgery 2011; 149: 713- 24. doi: 10.1016/j.surg.2010.10.001 15. Koch M, Garden OJ, Padbury R, Rahbari NN, Adam R, Capussotti L, et al. Bile leakage after hepatobiliary and pancreatic surgery: a definition and grading of severity by the International Study Group of Liver Surgery. Surgery 2011; 149: 680-8. doi: 10.1016/j.surg.2010.12.002 16. Pearce N. Analysis of matched case-control studies. BMJ 2016; 352: i969. doi: 10.1136/bmj.i969 17. Tanaka K, Shimada H, Matsuo K, Ueda M, Endo I, Togo S. Remnant liver re- generation after two-stage hepatectomy for multiple bilobar colorectal me- tastases. Eur J Surg Oncol 2007; 33: 329-35. doi: 10.1016/j.ejso.2006.10.038 18. Plahuta I, Magdalenić T, Turk Š, Potrč S, Ivanecz A. Achievements in surgical treatment for colorectal liver metastases from 2000 until 2020. AMB Acta Medico-Biotechnica 2022; 15: 41-53. doi: 10.18690/actabiomed.231 19. Turk Š, Plahuta I, Magdalenić T, Mavc Ž, Ivanecz A. [How can we prevent liver failure after extensive resections of colorectal liver metastases]? [Slovenian]. [Internet]. Gastroenterolog 2022; 26: 37-47. [cited 2023 Feb 16]. Available at: https://dk.um.si/IzpisGradiva.php?lang=slv&id=83727 20. Ivanecz A, Krebs B, Stozer A, Jagric T, Plahuta I, Potrc S. Simultaneous pure laparoscopic resection of primary colorectal cancer and synchronous liver metastases: a single institution experience with propensity score matching analysis. Radiol Oncol 2018; 52: 42-53. doi: 10.1515/raon-2017-0047 21. Lam VW, Laurence JM, Johnston E, Hollands MJ, Pleass HC, Richardson AJ. A systematic review of two-stage hepatectomy in patients with initially un- resectable colorectal liver metastases. HPB (Oxford) 2013; 15: 483-91. doi: 10.1111/j.1477-2574.2012.00607.x 22. Chavez MI, Gholami S, Kim BJ, Margonis GA, Ethun CG, Tsai S, et al. Two-stage hepatectomy for bilateral colorectal liver metastases: a multi- institutional analysis. Ann Surg Oncol 2021; 28: 1457-65. doi: 10.1245/ s10434-020-09459-6 23. Vauthey JN, Kawaguchi Y, Adam R. Colorectal liver metastasis. 1st edition. Cham (CH): Springer; 2022. 24. Wicherts DA, Miller R, de Haas RJ, Bitsakou G, Vibert E, Veilhan LA, et al. Long-term results of two-stage hepatectomy for irresectable colorectal cancer liver metastases. Ann Surg 2008; 248: 994-1005. doi: 10.1097/ SLA.0b013e3181907fd9 25. Narita M, Oussoultzoglou E, Jaeck D, Fuchschuber P, Rosso E, Pessaux P, et al. Two-stage hepatectomy for multiple bilobar colorectal liver metastases. Br J Surg 2011; 98: 1463-75. doi: 10.1002/bjs.7580 26. Turrini O, Ewald J, Viret F, Sarran A, Goncalves A, Delpero JR. Two-stage hepatectomy: who will not jump over the second hurdle? Eur J Surg Oncol 2012; 38: 266-73. doi: 10.1016/j.ejso.2011.12.009 27. Omichi K, Inoue Y, Mise Y, Oba A, Ono Y, Sato T, et al. Hepatectomy with perioperative chemotherapy for multiple colorectal liver metastases is the available option for prolonged survival. Ann Surg Oncol 2022; 29: 3567-76. doi: 10.1245/s10434-022-11345-2. 28. Tsai S, Marques HP, de Jong MC, Mira P, Ribeiro V, Choti MA, et al. Two-stage strategy for patients with extensive bilateral colorectal liver metastases. HPB (Oxford) 2010; 12: 262-9. doi: 10.1111/j.1477-2574.2010.00161.x 29. Petrowsky H, Linecker M, Raptis DA, Kuemmerli C, Fritsch R, Kirimker OE, et al. First long-term oncologic results of the ALPPS procedure in a large cohort of patients with colorectal liver metastases. Ann Surg 2020; 272: 793-800. doi: 10.1097/sla.0000000000004330 30. Baumgart J, Jungmann F, Bartsch F, Kloth M, Mittler J, Heinrich S, et al. Two-stage hepatectomy and ALPPS for advanced bilateral liver metastases: a tailored approach balancing risk and outcome. J Gastrointest Surg 2019; 23: 2391-400. doi: 10.1007/s11605-019-04145-9 31. Joechle K, Goumard C, Vega EA, Okuno M, Chun YS, Tzeng CD, et al. Long-term survival after post-hepatectomy liver failure for colorectal liver metastases. HPB (Oxford) 2019; 21: 361-69. doi: 10.1016/j.hpb.2018.07.019 32. Mullen JT, Ribero D, Reddy SK, Donadon M, Zorzi D, Gautam S, et al. Hepatic insufficiency and mortality in 1,059 noncirrhotic patients undergoing ma- jor hepatectomy. J Am Coll Surg 2007; 204: 854-62; discussion 62-4. doi: 10.1016/j.jamcollsurg.2006.12.032 33. Balzan S, Belghiti J, Farges O, Ogata S, Sauvanet A, Delefosse D, et al. The “50-50 criteria” on postoperative day 5: an accurate predictor of liver failure and death after hepatectomy. Ann Surg 2005; 242: 824-8, discussion 28-9. doi: 10.1097/01.sla.0000189131.90876.9e 34. Torzilli G, Viganò L, Cimino M, Imai K, Vibert E, Donadon M, et al. Is Enhanced one-stage hepatectomy a safe and feasible alternative to the two-stage hepatectomy in the setting of multiple bilobar colorectal liver metastases? A comparative analysis between two pioneering centers. Dig Surg 2018; 35: 323-32. doi: 10.1159/000486210 Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. I Radiol Oncol 2023; 57(2): 141-149. doi: 10.2478/raon-2023-0029 Elektroskleroterapija z bleomicinom (BEST) za zdravljenje žilnih malformacij. Poročilo študijske skupine Mednarodne mreže za izmenjavo praks o elektrokemoterapiji (InspECT) Muir T, Bertino G, Groselj A, Ratnam L, Kis E, Odili J, McCafferty I, Wohlgemuth WA, Cemazar M, Krt A, Bosnjak M, Zanasi A, Battista M, de Terlizzi F, Campana LG, Sersa G Izhodišča. Biomedicinske aplikacije elektroporacije širimo s področja onkologije na področja cepljenja, zdravljenja aritmij in tudi na zdravljenja žilnih malformacij. Bleomicin široko uporabljamo kot sklerotizira- joče sredstvo pri zdravljenju različnih žilnih malformacij. Uporaba električnih pulzov ob hkratni uporabi bleomicina poveča učinkovitost zdravila, kar dokazuje elektrokemoterapija z bleomicinom pri zdravljenju tumorjev. Enak princip upoštevamo pri elektroskleroterapiji z bleomicinom (angl. bleomycin electroscle- rotherapy, BEST). Zdi se, da ta način omogoča učinkovito zdravljenje žilnih malformacij z nizkim pretokom (venskim in limfnim) in morda celo malformacij z visokim pretokom (arteriovenskim). Čeprav je bilo do zdaj objavljenih le nekaj poročil, se kirurška stroka zanj zanima in vse več centrov uporablja metodo BEST pri zdravljenju žilnih malformacij. V okviru konzorcija Mednarodne mreže za izmenjavo praks o elektro- kemoterapiji (angl. International Network for Sharing Practices on Electrochemotherapy, InspECT) je bila ustanovljena posebna delovna skupina za razvoj standardnih operativnih postopkov za BEST in spodbu- janje kliničnih preizkušanj. Zaključki. S standardizacijo zdravljenja in uspešnim zaključkom kliničnih preskušanj, ki dokazujejo učin- kovitost in varnost pristopa, je mogoče doseči kakovostnejše rezultate in klinični izhod bolezni. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. II Radiol Oncol 2023; 57(2): 150-157. doi: 10.2478/raon-2023-0021 Korelacija srednjega difuzijskega koeficienta (ADC) in vrednost maksimalnega standardiziranega privzema (SUVmax) pri difuzijsko obteženem magnetno resonančnem slikanju (MRI) in 18F-FDG-PET/CT pri otrocih z Hodgkinovim limfomom. Raziskava izvedljivosti Rosbach N, Fischer S, Koch V, Vogl TJ, Bochennek K, Lehrnbecher T, Mahmoudi S, Grünewald L, Grünwald F, Bernatz S Izhodišča. Želeli smo ugotoviti, ali lahko slikanje z magnetno resonanco (MRI) deluje kot nadomestna diagnostična metoda, pri kateri bolnik ni izpostavljen sevanju. glede na (18)F-fluorodeoksiglukozo (FDG) pozitronsko emisijsko tomografijo/računalniško tomografijo (PET/CT) pri otrocih s histološko potrjenim Hodgkinovim limfomom (HL) pred zdravljenjem. Analizirali smo potencialno korelacijo med difuzijskim koeficientom (angl. apparent diffusion coefficient, ADC) pri MRI in največjo standardizirano vrednostjo privzema (SUVmax) pri FDG-PET/CT. Bolniki in metode. Retrospektivno smo analizirali 17 bolnikov s histološko potrjenim HL (6 žensk, 11 mo- ških; srednja starost 16 let, razpon: 12–20 let). Bolnikom smo pred začetkom zdravljenja naredili preiskavi s MRI in PET/CT z (18)F-FDG. Zbrali smo podatke slikanja z F-FDG PET/CT in korelacijske zemljevide ADC pri MRI. Za vsako HL-lezijo sta dva neodvisna ocenjevalca določila vrednost SUVmax in korelacijsko povpre- čje ADC. Rezultati. Pri 17 bolnikih smo ugotovili skupno 72 lezij HL in med moškimi in ženskami ni bilo pomembne razlike v številu lezij (srednja vrednost pri moških 15, razpon 12–19 let; pri ženskah 17, razpon: 12–18 let; p = 0,021). Povprečen razmik med MRI in PET/CT preiskavo je bil 5,9 ± 5,3 dni. Ujemanje med ocenjevalci, ocenjeno s korelacijskim koeficientom znotraj razreda (angl. intraclass correlation coefficient, ICC), je bilo odlično (ICC = 0,98; 95 % interval zaupanja [CI]: 0,97–0,99). Korelirani SUVmax in srednji ADC vseh 17 bolnikov (ROI n = 72) sta pokazala močno negativno korelacijo -0,75 (95 % CI: -0,84, -0,63; p = 0,001). Analiza je pokazala razliko v korelaciji ocenjevanih področij. Korelirani SUVmax in srednji ADC sta poka- zala močno korelacijo pri pregledih vratu in prsnega koša (vrat: -0,83, 95 % CI: -0,93, – -0,63; p < 0,0001; prsni koš: -0,82, 95 % CI: -0,91, – -0,64 , p < 0,0001) in dobro korelacijo pri abdominalnih pregledih -0,62 (95 % CI: -0,83, -0,28; p = 0,001). Zaključki. SUVmax in srednji ADC sta pokazala močno negativno korelacijo pri pediatričnih lezijah HL. Ocena lezij se je zdela trdna. Rezultati kažejo, da lahko zemljevidi ADC in srednji ADC nadomestijo PET/ CT pri analizi aktivnosti bolezni pri pediatričnih bolnikih s HL. To bi lahko zmanjšalo število preiskav PET/CT in zmanjšalo izpostavljenost otrok sevanju. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. III Radiol Oncol 2023; 57(2): 158-167. doi: 10.2478/raon-2023-0024 Biopsija neopredeljenih sumljivih intrahepatičnih lezij z računalniškotomografskim vodenjem. Označevanje z lipiodolom pred posegom izboljša uspešnost biopsije Langenbach MC, Vogl TJ, Buchinger A, Eichler K, Scholtz JE, Hammerstingl R, Gruber-Rouh T Izhodišča. Jetrne biopsije, vodene z računalniško tomografijo (CT), običajno izvajamo brez uporabe kontrastnega sredstva (KS). Uporaba KS pa je koristna predvsem pri zahtevnih vbodnih poteh in zahtevnih lokacijah lezij. Namen raziskave je bil oceniti natančnost CT vodenih biopsij jetrnih lezij brez uporabe KS, z uporabo intravenskega KS ali pa z označenjem lezij z intraarterijsko aplikacijo lipiodola. Bolniki in metode. Retrospektivno sm o ovrednotili 607 bolnikov (358 moških [59,0 %]; povprečna starost 61 let; standardna deviacija [SD] ± 12,04) s suspektnimi jetrnimi lezijami, pri katerih smo izvedli CT vodeno biopsijo. Uspešne biopsije so bile tiste, pri katerih histološko nismo videli tipičnega jetrnega tkiva ali pa ne- specifičnih jetrnih sprememb. Glede na način izvedbe CT vodene biopsije, smo bolnike razdelili v tri skupi- ne in jih med seboj primerjali: skupina brez KS, skupina z lipiodolom in skupina z intravenskim KS. Ugotavljali smo tehnično uspešnost in tudi dejavnike, ki bi lahko vplivali na uspešnost biopsije ter zaplete. Rezultate smo analizirali z uporabo Wilcoxon-Man-Whitneyjevega t-testa, hi-kvadrat testa in testa Spearman-Rho. Rezultati. Celokupni delež uspešnih biopsij je bil 73,1 %, Uspešnost pa je bila značilno pri lezijah, ozna- čenih z lipiodolom (79,3 %), v primerjavi s skupino brez KS (73,8 %) in skupino z intravenskem KS (65,2 %) (p = 0,037). Pri manjših lezijah (premer < 20 mm) je označevanje z lipiodolom še v večji meri izboljšalo uspešnost biopsij (71,2%) v primerjavi s skupino brez KS (65,5 %) in skupino z intravenskim KS (47,7 %) (p = 0,021). Jetrna ciroza (p = 0,94) in etiologija lezij (p = 0,78) nista vplivala na stopnjo uspešnosti biopsij med skupinami. Med posegi ni prišlo do pomembnejših zapletov. Zaključki. Označevanje sumljivih jetrnih lezij z lipiodolom pred biopsijo znatno poveča uspešnost biopsij in je še posebej koristno za biopsije manjših lezij s premerom manj kot 20 mm. Dodatno, pri lezijah, ki jih ne vidimo s CT-jem brez uporabe kontrasta, je označevanje z lipiodolom boljše kot uporaba intravenskega kontrasta. Entiteta tarčne lezije ne vpliva na stopnjo uspešnih biopsij. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. IV Radiol Oncol 2023; 57(2): 168-177. doi: 10.2478/raon-2023-0025 Radiološka ocena indeksa skeletne mišičnine in miosteatoze ter njun vpliv na pooperativne zaplete po presaditvi jeter Petrič M, Jordan T, Karteek P, Ličen S, Trotovšek B, Tomažič A Izhodišča. Presaditev jeter pri bolnikih z akutno ali kronično jetrno odpovedjo predstavlja možnost oz- dravitve in izboljša kvaliteto življenja. Vpliv prehranskega status na pooperativni potek po presaditvi jeter še ni jasno določen. Z raziskavo smo skušali ugotoviti napovedno vrednost indeksov skeletne mišičnine in miosteatoze na rezultate zdravljenja s presaditvijo jeter. Bolniki in metode. Retrospektivno smo zbrali podatke 138 polnoletnih bolnikov, ki so imeli prvo ortoto- pno presaditev jeter. Z analizo slik računalniške tomografije na nivoju tretjega ledvenega vretenca smo izračunali indeks skeletne mišičnine in miosteatoze. Analizirali smo njun vpliv na pooperativni potek po presaditvi jeter. Rezultati. Nizke vrednosti indeksa skeletne mišičnine so bile prisotne pri 63 % moških in 28,9 % ženskih prejemnikov jeter. Miosteatoza je bila prisotna pri 45 (32,6 %) bolnikih. Moški prejemniki z visokim indeksom skeletne mišičnine so imeli daljšo ležalno dobo v enoti intenzivne terapije (P < 0.025) v primerjavi z nizkim indeksom. Indeks skeletne mišičnine pri ženskah pa ni imel vpliva na ležalno dobo v enoti intenzivne terapije (P = 0,544), prav tako ne prisotnost miosteatoze ne glede na spol (P = 0,161). Indeksa skeletne mišičnine in miosteatoza nista vplivala na čas hospitalizacije (moški, P > 0,05 ter ženske, P = 0,843), sto- pnjo pooperativnih zapletov (moški, P = 0,883 ter ženske, P = 0,113), okužb (moški, P = 0,293 ter ženske, P = 0,285; P = 0,173) in zavrnitve presadka (moški, P = 0,875 ter ženske, P = 0,135). Zaključki. V pričujoči raziskavi indeksa skeletne mišičnine in miosteatoza nista imela vpliva na poopera- tivne zaplete po presaditvi jeter. Uporaba CT analize sestave telesa ter določitev specifičnih parametrov so ključni za pridobitev verodostojnih podatkov v prihodnosti. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. V Radiol Oncol 2023; 57(2): 178-183. doi: 10.2478/raon-2023-0022 Vrednost koeficienta ADC kot biološkega označevalca zorenja možganovine ploda Kobal L, Šurlan Popović K, Avsenik J, Vipotnik Vesnaver T Izhodišča. Pri razvoju plodovih možganov ima pomembno vlogo mielinizacija, ki poteka v točno dolo- čenem zaporedju. Količina vode v možganih je sorazmerna z mielinizacijo – bolj kot so možgani mielinizi- rani, manjša je vsebnost vode v njih. Difuzijo vodnih molekul lahko kvantitativno ocenimo s pomočjo difu- zijskega koeficienta (angl. apparent diffusion coefficient, ADC). Zanimalo nas je, ali lahko z določanjem vrednosti ADC kvantitativno ocenimo razvoj možganovine ploda. Bolniki in metode. V retrospektivno raziskavo smo vključili 42 plodov gestacijske starosti med 25 in 35 tedni. Na sekvencah difuzijsko poudarjenega magnetnoresonančnega slikanja smo ročno izbrali 13 po- dročij, v katerih smo izmerili vrednosti ADC. Statistično značilne razlike med vrednostmi ADC smo preverili s pomočjo enosmerne analize variance in Tukeyevim post hoc testom. Povezavo med vrednostmi ADC možganskih področij in gestacijsko starostjo plodov smo nato ocenili s pomočjo linearne regresije. Rezultati. Povprečna gestacijska starost plodov je bila 29,8 ± 2,4 tednov. Vrednosti ADC v talamusih, ponsu in malih možganih so se statistično značilno razlikovale med seboj ter od vrednosti ADC v vseh preostalih možganskih področjih. V talamusih, ponsu in malih možganih je linearna regresija pokazala statistično značilen upad vrednosti ADC z naraščajočo gestacijsko starostjo. V raziskavi smo upoštevali stopnjo značilnosti p = 0,05. Zaključki. Vrednosti ADC se z naraščanjem gestacijske starosti ploda spreminjajo, razlikujejo se tudi med različnimi možganskimi področji. V ponsu, malih možganih in talamusih je koeficient ADC uporaben bio- loški označevalec zorenja možganovine ploda, saj se vrednosti ADC z naraščanjem gestacijske starosti linearno znižujejo. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. VI Radiol Oncol 2023; 57(2): 184-190. doi: 10.2478/raon-2023-0020 Longitudinalno spremljanje difuzijskega koeficienta (ADC) pri bolnikih z rakom prostate, ki jih zdravimo z magnetnoresonančno vodeno radioterapijo na napravi MR-Linac z 1,5 T. Prospektivna raziskava izvedljivosti Almansour H, Schick F, Nachbar M, Afat S, Fritz V, Thorwarth D, Zips D, Bertram F, Müller AC, Nikolaou K, Othman AE, Wegener D Izhodišča. Hibridni magnetnoresonančni (MR) linearni pospeševalniki (angl. linear accelerator, MR- Linak) bi lahko omogočili individualno neposredno prilagajanje radioterapije (RT) z uporabo kvantitativ- nih sekvenc MR, kot je difuzijsko tehtano slikanje. Namen raziskave je bil proučiti dinamiko difuzijskega koeficienta (angl. apparent diffusion coefficient, ADC) lezij pri bolnikih z rakom prostate, pri katerih smo z 1,5 T obteženim MR-Linakom izvajali RT z MR vodenjem. Kot referenčni standard smo uporabili vrednosti ADC na diagnostičnem MR s 3T. Bolniki in metode. V prospektivno raziskavo smo vključili bolnike z biopsijsko potrjenim rakom prosta- te, ki smo jim opravili preiskavo s 3 T obteženem MR, (MR3T), in preiskavo na 1,5 T obteženem MR-Linak (MRL) pred in med RT. Vrednosti ADC lezij sta izmerila radiolog in radioterapevt na rezini z največjo lezijo. Vrednosti ADC na obeh sistemih smo primerjali s pomočjo parnih t-testov. Izračunali smo Pearsonov ko- relacijski koeficient in soglasje med odčitovalcema. Rezultati. Vključili smo 9 bolnikov, starih 67 ± 6 let (razpon od 60 do 67 let). Pri 7 bolnikih je bila rakava sprememba v perifernem območju, pri 2 bolnikih pa v prehodnem območju. Zanesljivost med odčito- valcema glede merjenja ADC lezije je bila odlična, s koeficientom znotraj razreda (angl. intraclass cor- relation coefficient, ICC) > 0,90 pred in med RT, tako navajamo rezultati prvega odčitovalca. Pri obeh sistemih je prišlo do statistično značilnega zvišanja ADC lezij med RT (povprečno MRL-ADC pred RT 0,97 ± 0,18 x 10‒3 mm2/s v primerjavi s MRL-ADC med RT 1,38 ± 0,3 x 10‒3 mm2/s, kar pomeni povprečno zvišanje ADC lezij za 0,41 ± 0,20 x 10‒3 mm2/s; p < 0,001). Povprečna vrednost MR3T-ADC pred RT je bila 0,78 ± 0,165 x 10‒3 mm2/s v primerjavi s povprečno vrednostjo MR3T-ADC med RT 0,99 ± 0,175 x 10‒3 mm2/s, kar pomeni povprečno povišanje ADC lezije za 0,21 ± 0,96 x 10‒3 mm2/s,;p < 0,001). Absolutne vrednosti ADC iz MRL so bile pred in med RT stalno pomembno višje od vrednosti iz MR3T (p < = 0,001). Kljub temu je bila med MRL-ADC in MR3T-ADC pred (r = 0,798, p = 0,01) in med RT (r = 0,863, p = 0,003) močna pozitivna korelacija. Zaključki. ADC lezij, izmerjen na MRL, se je med RT znatno povečal, meritve ADC lezij na obeh siste- mih pa so pokazale podobno dinamiko. To kaže, da bi ADC lezij, izmerjen na MRL, lahko uporabljali kot biološki označevalec za oceno odziva na zdravljenje. Nasprotno pa so absolutne vrednosti ADC, izračunane z algoritmom proizvajalca MRL, pokazale sistematična odstopanja od vrednosti, pridobljenih na diagnostičnem sistemu MR3T. Pričujoče preliminarne ugotovitve so obetavne, vendar jih je potrebno potrditi z večjo raziskavo. Po potrditvi, bi lahko ADC lezij na MRL uporabljali za oceno tumorskega odziva v realnem času pri bolnikih z rakom prostate, ki jih zdravimo z MR vodenim obsevanjem. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. VII Radiol Oncol 2023; 57(2): 191-200. doi: 10.2478/raon-2022-0052 Kraniotomija pri budnem bolniku med operativnim zdravljenjem možganskih gliomov. Izkušnje iz Univerzitetnega kliničnega centra Ljubljana Žele T, Velnar T , Koritnik B, Bošnjak R, Markovič-Božič J Izhodišča. Kraniotomija pri budnem bolniku (ang. awake craniotomy) je nevrokirurška tehnika, ki omogoča intraoperativno nevrofiziološko testiranje s sodelovanjem bolnika med odstranjevanjem mo- žganskega tumorja v regionalni anesteziji. To omogoči prepoznavanje vitalno najpomembnejših (i.e. elokventnih) možganskih področij pri operaciji in zmanjša možnost njihove poškodbe. Namen raziskave je bil predstaviti izkušnje s kraniotomijo pri budnem bolniku med operativnim zdravljenjem gliomov v Univerzitetnem kliničnem centru Ljubljana od 2015 do 2019. Bolniki in metode. Kandidati za kraniotomijo v budnem stanju so bili bolniki z glialnim tumorjem znotraj ali v bližini govornih možganskih področji, tumorjem v področju inzule in tumorjem znotraj ali v bližini pri- marne motorične skorje. Bolnike smo klinično ocenili pred in po operaciji. Rezultati. V 5-letnem obdobju smo opravili 24 kraniotomij pri budnem bolniku (18 moških in 6 ženskih; povprečna starost 41 let). Njihovo sodelovanje med operacijo smo pri večini bolnikov ocenili kot »odlič- no«, stopnjo neugodja kot »blažje neugodje« in bolečino kot »zmerno«. Po operaciji smo opažali blago nevrološko poslabšanje pri 13 % bolnikov (3/24). Popolno resekcijo smo lahko naredili pri malignih gliomih v 60 % (6/10) pri nizkomalignih gliomih pa v 29 % (4/14). Primerjava predoperativnega in pooperativnega funkcionalnega stanja bolnika z lestvico po Karnovskem in kvalitete življenja z anketnim vprašalnikom kratke oblike 36 (angl. Short-Form 36 health survey, SF-36) je pokazala, da operacija ni pomembno ne- gativno vplivala na telesno zmogljivost ali kakovost življenja bolnikov (p > 0,05). Zaključki. Izkušnjah iz terciarnega centra kažejo, da je kraniotomija pri budnem pacientu izvedljiva in varna nevrokirurška tehnika. Izbira ustreznih bolnikov, natančno načrtovanje in predoperativna priprava ter timski pristop so ključni pri doseganju najboljšega poteka operacije in uspeha tovrstnega zdravljenja. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. VIII Radiol Oncol 2023; 57(2): 201-210. doi: 10.2478/raon-2023-0009 Kognitivno funkcioniranje v kohorti bolnikov z visokomalignimi gliomi Škufca Smrdel AC, Podlesek A, Skoblar Vidmar M, Markovič J, Jereb J, Kuzma Okorn M, Smrdel U Izhodišča. Visoko maligni gliomi so povezani s kognitivnimi motnjami. V raziskavi smo želeli preučiti kogni- tivno funkcioniranje v kohorti bolnikov z malignimi gliomi, glede na stanje izocitratdehidrogenaze (IDH) in metil gvanin metil transferaze (MGMT) ter druge klinične značilnosti. Bolniki in metode. V raziskavo smo vključili bolnike z visoko malignimi gliomi, zdravljenimi v opazo- vanem času. Pooperativno smo naredili nevropsihološko oceno in pri tem uporabili Slovenski test ver- balnega učenja, Slovenski test črkovne fluentnosti, Test psihomotorične hitrosti (angl. Trail Making Test, Part A and B) ter samoocenjevalni vprašalnik. Rezultate smo analizirali kot vrednost ‘z‘ (angl. z-score) in dihotomizirano glede na stanje IDH in MGMT. Razlike med skupinami smo proučevali z uporabo T-testa ter testov Mann-Whitney U, χ2 in Kendall‘s Tau. Rezultati. Od 275 bolnikov v kohorti smo jih lahko vključili 90. Med njimi 46 % bolnikov ni bilo sposobnih sodelovati zaradi slabega stanja zmogljivosti ali dejavnikov, povezanih z boleznijo. Bolniki, kjer je bila pri- sotna mutacija IDH, so bili mlajši in bolj zmogljivi, imeli so več tumorjev gradusa III in več metilacij MGMT. V tej skupini je bilo kognitivno funkcioniranje statistično značilno boljše na področjih neposrednega ver- balnega priklica, kratkoodloženega in dolgoodloženega priklica, kot tudi na področjih izvršilnih funkcij ter prepoznavanja. Glede na stanje MGMT pa razlik nismo našli. Tumorji gradusa III so bili povezani s po- gostejšo metilacijo. Samoocena se je pokazala kot šibko orodje, povezano le z neposrednim priklicom. Zaključki. Glede na stanje MGMT nismo našli razlik v kognitivnem funkcioniranju, je pa bilo boljše ob prisotnosti IDH mutacije. V naši kohorti bolnikov z visokomalignimi gliomi jih skoraj polovica ni bila sposob- na sodelovanja in jih nismo vključili v raziskavo, kar kaže na veliko zastopanost prognostično ugodnejših bolnikov v raziskavah. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. IX Radiol Oncol 2023; 57(2): 211-219. doi: 10.2478/raon-2023-0019 Spremembe v kakovosti življenja pri bolnicah z zgodnjim rakom dojk in primerjava z normativno slovensko populacijo Grašič Kuhar C, Gortnar Cepeda T, Kurzeder C, Vetter M Izhodišča. Želeli smo proučiti spremembe v kakovosti življenja bolnic, zdravljenih zaradi raka dojk, in primerjati njihovo kakovost življenja z normativnimi podatki slovenske populacije. Bolniki in metode. Izvedli smo prospektivno kohortno raziskavo. Vključili smo 102 bolnici z zgodnjim rakom dojk, ki smo jih zdravili na Onkološkem inštitutu Ljubljana s kemoterapijo. Eno leto po zaključeni ke- moterapiji je izpolnilo vprašalnike 71 % vseh bolnic. Uporabili smo slovensko različico vprašalnikov EORTC QLQ C30 in BR23. Primarni cilj je bila primerjava globalnega zdravstvenega stanja/kakovosti življenja in skupnega rezultata C30 (angl. C30-Summary Score, C30-SumSc) pred kemoterapijo in eno leto po zaključeni kemoterapiji z normativno slovensko populacijo. V eksplorativni analizi smo proučili razlike v simptomih in funkcionalnih lestvicah QLQ C-30 in QLQ BR-23 med izhodiščnim stanjem in stanjem eno leto po koncu kemoterapije. Rezultati. Izhodiščni C30-SumSc bolnic je bil za 2,6 točke (p = 0,04) nižji od predvidenega C30-SumSc populacije, po enem letu po zaključku kemoterapije je bil za 6,5 točke (p <0,001) nižji od predvidenega C30-SumSc populacije. Nasprotno pa se globalno zdravstveno stanje bolnic ni statistično razlikovalo od napovedi iz populacije niti izhodiščno niti po enem letu. Eksploratorna analiza je pokazala, da so imele bolnice eno leto po kemoterapiji v primerjavi z začetkom kemoterapije statistično značilne in klinično pomembne nižje ocene pri telesni podobi in kognitivnem funkcioniranju ter višje ocene simptomov za bolečino, utrujenost in simptome roke. Zaključki. C30-SumSc je po enem letu po kemoterapiji znižan. Zgodnje intervencije je potrebno usmeriti v preprečevanje kognitivnega upada in telesne podobe ter v zmanjšanje utrujenosti, bolečine in simp- tomov roke. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. X Radiol Oncol 2023; 57(2): 220-228. doi: 10.2478/raon-2023-0028 Pomen PIK3CA aktivirajočih mutacij za izhod bolezni in sistemsko zdravljenje operabilnega invazivnega lobularnega karcinoma dojke Ribnikar D, Jerič Horvat V, Ratoša I, Veitch ZW, Grčar Kuzmanov B, Novaković S, Langerholc E, Amir E, Šeruga B Izhodišča. Namen raziskave je bil ovrednotiti vpliv PIK3CA aktivirajočih mutacij za izhod bolezni in učin- kovitost doponilnega endokrinega zdravljenja pri bolnicah z operabilnim invazivnim lobularnim karcino- mom. Bolniki in metode. V raziskavo smo vključili bolnice z zgodnjim invazivnim lobularnim karcinomom dojke, ki smo jih na Onkološkem inštitutu Ljubljana zdravili med leti 2003 in 2008. S pomočjo metode Kaplan-Meier smo izračunali čas do prvega oddaljenega razsoja bolezni in celokupno preživetje v ce- lotni skupini bolnic z ozirom na mutacijski status PIK3CA v primarnem tumorju dojke. Povezanost med PIK3CA aktivirajočo mutacijo in učinkovitostjo dopolnilnega endokrinega zdravljenja pa smo analizirali s pomočjo Coxovih modelov v skupini bolnic, ki so imele v tumorju pozitivne estrogenske in progesteronske receptorje. Rezultati. Srednja starost vseh bolnic ob diagnozi je bila 62,8 let in srednji čas opazovanja 10,8 let. Med 365 bolnicami, ki smo jih vključili v raziskavo, smo odkrili PIK3CA aktivirajočo mutacijo pri 45 %. Ugotovili smo, da prisotnost PIK3CA mutacije v primarnem ILC dojke ne vpliva na čas do prvega oddaljenega razsoja bolezni (p = 0,36) in ne na celokupno preživetje (p = 0,42). Pri bolnicah s prisotno aktivirajočo PIK3CA mutacijo je vsako leto zdravljenja s tamoksifenom zmanjšalo tveganje za smrt za 27 %, pri bol- nicah zdravljenih z zaviralci aromataze pa za 21 % v primerjavi z bolnicami, ki niso prejele endokrinega zdravljenja. Vrsta in trajanje endokrinega zdravljenja nista imela vpliva na čas do prvega oddaljenega razsoja bolezni, je pa podaljšano endokrino zdravljenje imelo ugoden vpliv na celokupno preživetje. Zaključki. Prisotnost aktivirajoče mutacije PIK3CA v primarnem operabilnem ILC dojke ne vpliva na čas do prvega oddaljenega razsoja bolezni in tudi ne na celokupno preživetje. Bolnice s prisotno PIK3CA aktivirajočo mutacijo so imele statistično pomembno zmanjšanje tveganja smrti ne glede na to, ali so v dopolnilnem hormonskem zdravljenju prejele tamoksifen ali zaviralce aromataze. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. XI Radiol Oncol 2023; 57(2): 229-238. doi: 10.2478/raon-2023-0018 Subplevralne fibrozne intersticijske nepravilnosti v pljučih vplivajo na izid radioterapije nedrobnoceličnega pljučnega raka Ito M, Katano T, Okada H, Sakuragi A, Minami Y, Abe S, Adachi S, Oshima Y, Ohashi W, Kubo A, Fukui T, Ito S, Suzuki K Izhodišča. Povezava med intersticijskimi pljučnimi nepravilnostmi in rezultati zdravljenja pljučnega raka z radioterapijo je nejasna. Namen raziskave je bil ugotoviti, ali so specifični podtipi intersticijskih pljučnih nepravilnosti dejavniki tveganja za radiacijski pnevmonitis. Bolniki in metode. V retrospektivni raziskavi smo analizirali bolnike z nedrobnoceličnim pljučnim ra- kom, ki smo jih zdravili z radikalno ali rešilno radioterapijo. Bolnike smo razdelili v skupino normalnih (brez nepravilnosti), skupino z intersticijskimi pljučnimi nepravilnostmi in skupino z intersticijsko pljučno boleznijo. Skupino z intersticijskimi pljučnimi nepravilnostmi smo nadalje razdelili na nesubpleuralno, subpleuralno nefibrotično in subpleuralno fibrotično skupino. Za določitev radiacijskega pnevmonitisa in preživetja ter primerjavo teh rezultatov med skupinami smo uporabili Kaplan-Meierjevo in Coxovo regresijsko metodo. Rezultati. V raziskavo smo vključili 175 bolnikov: normalni, n = 105; intersticijske pljučne nepravilnosti- -nesubpleuralno, n = 5; intersticijske pljučne nepravilnosti-subpleuralno nefibrotično, n = 28; intersticijske pljučne nepravilnosti-subpleuralno fibrotično, n = 31; intersticijska pljučna bolezen, n = 6. Radiacijski pnev- monitis stopnje ≥ 2 smo opazili pri 71 bolnikih (41 %). Intersticijske pljučne nepravilnosti (razmerje tveganj [HR]: 2,33; p = 0,008), radioterapija z modulirano intenzivnostjo (HR: 0,38; p = 0,03) in prostornina pljuč, ki je prejela 20 Gy (HR: 54,8; p = 0,03), so prispevali h kumulativni pojavnosti radiacijskega pnevmonitisa. V skupini intersticijske pljučne nepravilnosti je bilo 8 bolnikov z radiacijskim pnevmonitisom 5. stopnje, od tega jih je imelo 7 subpleuralno fibrotično obliko. Med radikalno zdravljenimi bolniki je imela skupina intersticijske pljučne nepravilnosti slabše 2-letno celokupno preživetje kot normalna skupina (35,3 % proti 54,6 %; p = 0,005). Multivariatna analiza je pokazala, da je skupina z intersticijskimi pljučnimi nepravilnost- mi-subpleuralno fibrotičnimi prispevala k slabšemu celokupnemu preživetju (HR: 3,07; p = 0,02). Zaključki . Intersticijske pljučne nepravilnosti, zlasti subpleuralne fibrotične, so lahko pomembni dejavniki tveganja za radiacijski pnevmonitis, ki lahko poslabšajo napoved poteka bolezni. Te ugotovitve lahko pomagajo pri odločanju glede radioterapije. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. XII Radiol Oncol 2023; 57(2): 239-248. doi: 10.2478/raon-2023-0017 Vpliv izražanja genov BCL2, BAX in ABCB1 na napoved poteka bolezni pri odraslih bolnikih z de novo akutno mieloično levkemijo z normalnim kariotipom Pravdić Z, Suvajdžić Vuković N, Gasić V, Marjanović I, Karan-Djurašević T, Pavlović S, Tosić N Izhodišča. Deregulacija apoptotičnega procesa je v ozadju patogeneze številnih vrst raka, vključno z levkemijo, in je zelo pomembna tudi za uspeh kemoterapevtskega zdravljenja. Zato bi lahko profil izra- žanja genov glavnih apoptotičnih dejavnikov, kot sta antiapoptotični BCL2 (protein 2 limfoma celic B) in proapoptotični BAX (BCL2-associated X), ter genov, vključenih v odpornost na več zdravil (ABCB1), pomembno vplival na napoved poteka bolezni. Uporabili bi ga lahko tudi kot tarčo za specifično zdra- vljenje. Bolniki in metode. Z metodo verižne reakcije s polimerazo v realnem času smo analizirali izražanje BCL2, BAX in ABCB1 v vzorcih kostnega mozga, ki smo jih zbrali ob diagnozi pri 51 odraslih bolnikih z aku- tno mieloično levkemijo z normalnim kariotipom (AML-NK), in preučili njihovo napovedno moč. Rezultati. Povečano izražanje BCL2 (BCL2+) je bilo povezano s kemorezistenco (p = 0,024), medtem ko so bili bolniki z nizkim izražanjem BAX (BAXnizko) bolj nagnjeni k ponovitvi bolezni (p = 0,047). Analiza skupnega učinka izražanja BCL2 in BAX je pokazala, da je bilo 87 % bolnikov s statusom BAX/BCL2nizko odpornih na zdravljenje (p = 0,044). Visoko izražanje ABCB1 je bilo povezano s statusom BCL2+ (p < 0,001) in odsotnostjo mutacij FLT3-ITD (p = 0,019). Zaključki. Pričujoča analiza profilov izražanja genov BCL2, BAX in ABCB1 je prva raziskava, ki obravnava izključno bolnike z AML-NK. Preliminarni rezultati so pokazali, da so bolniki z visokim izražanjem BCL2 v večji meri odporni na kemoterapijo in bi jim lahko koristilo specifično zdravljenje proti BCL2. Z nadaljnjimi razi- skavami na večjem številu bolnikov bi lahko pojasnili dejanski napovedni pomen teh genov pri bolnikih z AML-NK. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. XIII Radiol Oncol 2023; 57(2): 249-256. doi: 10.2478/raon-2023-0016 CD56-pozitivni difuzni velikocelični limfom B. Celovita analiza kliničnih, patoloških in molekularnih značilnosti s pregledom literature Gašljević G, Boltežar L, Novaković S, Šetrajčič-Dragoš V, Jezeršek-Novaković B, Kloboves-Prevodnik V Izhodišča. Difuzni velikocelični limfom B (angl. diffuse large B-cell lymphoma, DLBCL) brez dodatnih oznak (angl. not otherwise specified, NOS) je najpogostejši ne-Hodgkinov limfom. Izražanje CD56 na ce- licah DLBCL-NOS, je zelo neobičajno. O pojavnosti izražanja in njegovem kliničnem pomenu pri DLBCL je malo znanega. Pri takšnih primerih še niso naredili genetskega profila. Bolniki in metode. V raziskavo smo vključili 229 bolnikov s histološko potrjenim DLBCL-NOS, ki so bili di- agnosticirani na Onkološkem inštitutu Ljubljana v obdobju 2008–2017. Iz tkivnih blokov vseh bolnikov smo izdelali tkivne mreže in naredili tkivne rezine za barvanje s hematoksilin eozinom in imunohistokemična barvanja. Za vse primere DLBCL-NOS, kjer smo na limfomskih celicah imunohistokemično dokazali izraža- nje CD56, smo pridobili klinične podatke, vključno s starostjo ob diagnozi, stadijem bolezni, mednarodnim napovednim indeksom (angl. International Prognostic Index, IPI), shemo zdravljenja in številom krogov kemoterapije, zdravljenjem z radioterapijo, izidom zdravljenja in morebitno ponovitvijo bolezni. Izračunali smo celokupno preživetje in preživetje brez napredovanja. Pri štirih bolnikih s CD56 pozitivnim DLBCL-NOS smo izolirali RNA in opravili ciljno sekvenciranje RNA (cDNA) 125 genov s kompletom Archer FusionPlex Lymphoma. Rezultati. Izražanje CD56 smo ugotovili v 7 od 229 primerov (3%). Stopnja izražanja je bila zmerna žarišč- na do zelo intenzivna in difuzna. Vsi bolniki so imeli na novo nastal DLBCL-NOS. Srednja starost bolnikov ob diagnozi je bila 54,5 let, med njimi je bilo pet žensk in 2 moška. Po Hansovem algoritmu je imelo 6 bolnikov podtip GBC in en podtip ne-GBC (ABC), dvojni ekspresor. Genetski profil štirih bolnikov, določen po Schmitzovi klasifikaciji, je pokazal, da je bil en primer podtipa BN2, en podtipa EZB, dva pa sta bila nerazvrščena. Pri vseh 6 zdravljenih bolnikih smo dosegli popoln odgovor na zdravljenje in nismo ugotovili napredovanja bolezni v srednjem obdobju spremljanja 80,5 mesecev. Zaključki. Naša serija CD56 pozitivnih DLBCL-NOS je ena izmed največjih do sedaj objavljenih, v kateri so podrobno opisane klinično-patološke značilnosti. Prav tako je to prva serija CD56 pozitivnih DLBCL- NOS, pri kateri smo določili genetski profil. Rezultati kažejo, da je izražanje CD56 v DLBCL-NOS redko, vendar prisotno v napovedno ugodnih imunohistokemičnih in genetskih podtipih DLBCL-NOS. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. XIV Radiol Oncol 2023; 57(2): 257-269. doi: 10.2478/raon-2023-0023 Kvantitativni dinamični kontrastni parametri in znotrajvokselno nekoherentno gibanje omogočajo napovedovanje statusa TP53 in opredelitev tveganja pri začetnem endometrijskem karcinomu Wang H, Yan R, Li Z, Wang B, Jin X, Guo Z, Liu W, Zhang M, Wang K, Guo J, Han D Izhodišča. Namen raziskave je bil raziskati vrednost dinamičnega kontrastnega magnetnoresonančne- ga slikanja (DCE-MRI) in znotrajvokselnega inkoherentnega gibanja (IVIM) za razlikovanje med mutiranim TP53 in nemutiranim, nizkim in visokim tveganjem pri začetnem endometrijskem karcinoma. Bolniki in metode. Pri 74 bolnikih z endometrijskim karcinomom smo naredili MRI medenice. Primerjali smo parametre: konstanta volumskega prenosa (Ktrans), konstanta hitrosti prenosa (Kep), volumen ekstravaskularnega zunajceličnega prostora na enoto volumna tkiva (Ve), pravi difuzijski koeficient (D), psevdodifuzijski koeficient (D*) in mikrovaskularni volumski delež (f). Kombinacijo parametrov smo analizi- rali z logistično regresijo in ovrednotili z metodo zagonskega traku (angl. bootstrap) (1000 vzorcev), ROC krivuljami, kalibracijskimi krivuljami in analizo krivulje odločanja (DCA). Rezultati. V skupini z mutiranim TP53 sta bila Ktrans in Kep višja, D pa nižji kot v skupini z divjim tipom TP53; Ktrans, Ve, f in D so bili nižji v skupini z visokim tveganjem kot v skupini z nizkim tveganjem (vsi P < 0,05). Pri prepozna- vanju začetnega endometrijskega karcinoma z mutiranim TP53 in nemutiranim TP53 sta bila Ktrans in D ne- odvisna napovedna dejavnika, njuna kombinacija pa je imela optimalno diagnostično učinkovitost (AUC 0,867; občutljivost 92 %; specifičnost 80,95 %), kar je bilo pomembno boljše od D (Z = 2,169; P = 0,030) in Ktrans (Z = 2,572, P = 0,010). Pri prepoznavanju začetnega endometrijskega karcinoma z nizkim in visokim tveganjem so bili neodvisni napovedni kazalci Ktrans, Ve in f, njihova kombinacija pa je imela optimalno diagnostično učinkovitost (AUC 0,947; občutljivost 83,33 %; specifičnost 93,18 %), kar je bilo bistveno boljše od D (Z = 3,113; P = 0,002), f (Z = 4,317; P < 0,001), Ktrans (Z = 2,713; P = 0,007) in Ve (Z = 3,175; P = 0,002). Umeritvene krivulje so pokazale, da imata obe zgornji kombinaciji neodvisnih napovednih dejavnikov do- bro skladnost, DCA pa je pokazala, da sta ti kombinaciji zanesljivi klinični napovedni orodji. Zaključki. Tako DCE-MRI kot IVIM olajšata napoved statusa TP53 in opredelitev tveganja pri začetnem endometrialnem karcinomu. V primerjavi s posameznimi parametri je imela kombinacija neodvisnih na- povednih dejavnikov boljšo napovedno moč in lahko služi kot boljši slikovni označevalec. Slovenian abstracts Radiol Oncol 2023; 57(2): I-XV. XV Radiol Oncol 2023; 57(2): 270-278. doi: 10.2478/raon-2023-0026 Dvostopenjska hepatektomija pri resekciji jetrnih metastaz raka debelega črevesa in danke. Izkušnje terciarne ustanove s primerjavo primerov in kontrol ter pregled literature Turk S, Plahuta I, Magdalenič T, Španring T, Laufer K, Mavc Ž, Potrč S, Ivanecz A Izhodišča. Dvostopenjsko hepatektomijo so uvedli pri bolnikih z bilateralnimi jetrnimi tumorji, ki imajo visoko tveganje za jetrno odpoved po enostopenjski hepatektomiji. Namen raziskave je bil analizirati dvo- stopenjske hepatektomije pri obsežnih obojestranskih jetrnih zasevkih raka debelega črevesa in danke. Bolniki in metode. Retrospektivno smo pregledali prospektivno vodene podatkovne baze jetrnih resekcij zaradi zasevkov raka debelega črevesa in danke. Skupino dvostopenjskih hepatektomij smo primerjali s skupino enostopenjskih hepatektomij glede pooperativnih izidov in preživetja. Izvedli smo uje- manje primerov in kontrol (angl. case-control matching). Rezultati. Med leti 2000 in 2020 smo naredili 632 jetrnih resekcij zaradi zasevkov raka debelega črevesa in danke. Skupina z dvostopenjsko hepatektomijo je obsegala 15 bolnikov, kontrolna skupina z enosto- penjskih hepatektomijo pa je vključeval 151 bolnikov. Skupina enostopenjskih hepatektomij, kjer smo izvedli ujemanje primerov je vsebovala 14 bolnikov. Huda obolevnost in 90-dnevna smrtnost sta bili v skupini z dvostopenjsko hepatektomijo 40 % in 13,3 %; v skupini z enostopenjsko hepatektomijo pa 20,5 % in 4,6 % ter v skupini z enostopenjsko hepatektomijo in hkratno ujemanje primerov 28,6 % oziroma 7,1 %. Preživetje brez bolezni, srednje celokupno preživetje ter 3- in 5-letni preživetji so v skupini dvostopenjskih hepatektomij bili 5 mesecev, 21 mesecev, 33 % in 13 %; v skupini enostopenjskih hepatektomij pa 11 mesecev, 35 mesecev, 49 % in 27 % ter v skupini z enostopenjsko hepatektomijo in hkratno ujemanje primerov 8 mesecev, 23 mesecev, 36 % in 21 %. Zaključki. Dvostopenjska hepatektomija je bila dokaj varen in učinkovit postopek zdravljenja. Prednost enostopenjske hepatektomije pa je, da z njo lahko dosegamo ob enakovrednih onkoloških rezultatih nižjo hudo obolevnost po operaciji. 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 Pomembno: Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Pred predpisovan- jem zdravila Verzenios si preberite zadnji veljavni Povzetek glavnih značilnosti zdravil. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila http://www.ema.europa.eu Reference: 1. Povzetek glavnih značilnosti zdravila Verzenios, zadnja odobrena verzija. 2. ZZZS, Spremembe liste zdravil_2023_03_07 Eli Lilly farmacevtska družba, d.o.o., Dunajska cesta 167, 1000 Ljub lja na, te le fon 01 / 580 00 10, faks 01 / 569 17 05 PP-AL-SI-0186, 9.3.2023, Samo za strokovno javnost. SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli do- mnevnem neželenem učinku zdravila. IME ZDRAVILA: Verzenios 50 mg/100 mg/150 mg filmsko obložene tablete KAKOVOSTNA IN KOLIČINSKA SESTAVA: Ena filmsko obložena tableta vsebuje 50 mg/100 mg/150 mg abema- cikliba. Ena filmsko obložena tableta vsebuje 14 mg/28 mg/42 mg laktoze (v obliki monohidrata). Terapevtske indikacije: Zgodnji rak dojk: Zdravilo Verzenios je v kombinaciji z endokrinim zdravljenjem indicirano za adjuvantno zdravljenje odraslih bolnikov z na hormonske receptorje (HR – Hormone Receptor) pozitivnim, na receptorje humanega epidermalnega rastnega fak- torja 2 (HER2 – Human Epidermal Growth Factor Receptor 2) negativnim zgodnjim rakom dojk s pozitivnimi bezgavkami, pri katerih obstaja veliko tveganje za ponovitev. Pri ženskah v pred- ali perimenopavzi je treba endokrino zdravljenje z zaviralcem aromataze kombinirati z agonistom gonadoliberina (LHRH – Luteinizing Hormone–Releasing Hormone). Napredovali ali me- tastatski rak dojk: Zdravilo Verzenios je indicirano za zdravljenje žensk z lokalno napredovalim ali metastatskim, na hormonske receptorje (HR – Hormone Receptor) pozitivnim in na receptorje humanega epidermalnega rastnega faktorja 2 (HER2 – Human Epidermal Growth Factor Receptor 2) negativnim rakom dojk v kombinaciji z zaviralcem aromataze ali s fulvestran- tom kot začetnim endokrinim zdravljenjem ali pri ženskah, ki so prejele predhodno endokrino zdravljenje. Pri ženskah v pred- ali perimenopavzi je treba endokrino zdravljenje kombinirati z agonistom LHRH. Odmerjanje in način uporabe: Zdravljenje z zdravilom Verzenios mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. Zdravilo Verzenios v kombinaciji z endokrinim zdravljenjem: Priporočeni odmerek abemacikliba je 150 mg dvakrat na dan, kadar se uporablja v kombinaciji z endokrinim zdravljenjem. Zgodnji rak dojk: Zdravilo Verzenios je treba jemati neprekinjeno dve leti, ali do ponovitve bolezni ali pojava nesprejemljive toksičnosti. Napredovali ali metastatski rak dojk: Zdravilo Verzenios je treba jemati, dokler ima bolnica od zdravljenja klinično korist ali do pojava nesprejemljive toksičnosti. Če bolnica bruha ali izpusti odmerek zdravila Verzenios, ji je treba naročiti, da naj nas- lednji odmerek vzame ob predvidenem času; dodatnega odmerka ne sme vzeti. Obvladovanje nekaterih neželenih učinkov lahko zahteva prekinitev in/ali zmanjšanje odmerka. Zdravljenje z abemaciklibom prekinite v primeru povišanja vrednosti AST in/ali ALT >3 x ZMN SKUPAJ s celokupnim bilirubinom > 2,0 x ZMN v odsotnosti holestaze ter pri bolnicah z intersticijsko pljučno boleznijo (ILD)/pnevmonitis stopnje 3 ali 4. Sočasni uporabi močnih zaviralcev CYP3A4 se je treba izogibati. Če se uporabi močnih zaviralcev CYP3A4 ni mogoče izogniti, je treba odmerek abemacikliba znižati na 100 mg dvakrat na dan. Pri bolnicah, pri katerih je bil odmerek znižan na 100 mg abemacikliba dvakrat na dan in pri katerih se sočasnemu dajanju močnega zaviralca CYP3A4 ni mogoče izogniti, je treba odmerek abemacikliba dodatno znižati na 50 mg dvakrat na dan. Pri bolnicah, pri katerih je bil odmerek znižan na 50 mg abemacikliba dvakrat na dan in pri katerih se sočasnemu dajanju močnega zaviralca CYP3A4 ni mogoče izogniti, je mogoče z odmerkom abemacikliba nadaljevati ob natančnem spremljanju znakov toksičnosti. Alterna- tivno je mogoče odmerek abemacikliba znižati na 50 mg enkrat na dan ali prekiniti dajanje abemacikliba. Če je uporaba zaviralca CYP3A4 prekinjena, je treba odmerek abemacikliba pove- čati na odmerek, kakršen je bil pred uvedbo zaviralca CYP3A4 (po 3–5 razpolovnih časih zaviralca CYP3A4). Prilagajanje odmerka glede na starost in pri bolnicah z blago ali zmerno ledvično okvaro ter z blago (Child Pugh A) ali zmerno (Child Pugh B) jetrno okvaro ni potrebno. Pri dajanju abemacikliba bolnicam s hudo ledvično okvaro sta potrebna previdnost in skrbno spremlja- nje glede znakov toksičnosti. Način uporabe: Zdravilo Verzenios je namenjeno za peroralno uporabo. Odmerek se lahko vzame s hrano ali brez nje. Zdravilo se ne sme jemati z grenivko ali grenivkinim sokom. Bolnice naj odmerke vzamejo vsak dan ob približno istem času. Tableto je treba zaužiti celo (bolnice je pred zaužitjem ne smejo gristi, drobiti ali deliti). Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Pri bolnicah, ki so prejemale abemaciklib, so poročali o nevtropeniji, o večji pogostnosti okužb kot pri bolnicah, zdravljenih s placebom in endokrinim zdravljenjem, o povečanih vrednostih ALT in AST. Pri bolnicah, pri katerih se pojavi nevtropenija stopnje 3 ali 4, je priporočljivo prilagoditi odmerek. Do primerov nevtropenične sepse s smrtnim izidom je prišlo pri < 1 % bolnic z metastatskim rakom dojk. Bolnicam je treba naročiti, naj o vsaki epizodi povišane telesne temperature poročajo zdravstvenemu delavcu. Bolnice je treba spremljati za znake in simptome globoke venske tromboze (VTE) in pljučne embolije ter jih zdraviti, kot je medicinsko uteme- ljeno. Glede na stopnjo VTE bo morda treba spremeniti odmerek abemacikliba. Glede na povečanje vrednosti ALT ali AST je mogoče potrebna prilagoditev odmerka. Driska je najpogostej- ši neželeni učinek. Bolnice je treba ob prvem znaku tekočega blata začeti zdraviti z antidiaroiki, kot je loperamid, povečati vnos peroralnih tekočin in obvestiti zdravnika. Sočasni uporabi in- duktorjev CYP3A4 se je treba izogibati zaradi tveganja za zmanjšano učinkovitost abemacikliba. Bolnice z redkimi dednimi motnjami, kot so intoleranca za galaktozo, popolno pomanjkanje laktaze ali malapsorpcija glukoze/galaktoze, tega zdravila ne smejo jemati. Bolnice spremljajte glede pljučnih simptomov, ki kažejo na ILD/pnevmonitis, in jih ustrezno zdravite. Glede na stopnjo ILD/pnevmonitisa je morda potrebno prilagajanje odmerka abemacikliba. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Abemaciklib se primarno presnavlja s CYP3A4. Sočasna uporaba abemacikliba in zaviralcev CYP3A4 lahko poveča plazemsko koncentracijo abemacikliba. Uporabi močnih zaviralcev CYP3A4 sočasno z abemaciklibom se je treba izogibati. Če je močne zaviralce CYP3A4 treba dajati sočasno, je treba odmerek abemacikliba zmanjšati, nato pa bolnico skrbno spremljati glede toksičnosti. Pri bolnicah, zdravljenih z zmernimi ali šibkimi zaviralci CYP3A4, ni potrebno prilagajanje odmerka, vendar jih je treba skrbno spremljati za znake toksičnosti. Sočasni uporabi močnih induktorjev CYP3A4 (vključno, vendar ne omejeno na: karbamazepin, fenitoin, rifampicin in šentjanževko) se je treba izogibati zaradi tveganja za zmanjšano učinkovitost abemacikliba. Abemaciklib in njegovi glavni aktivni presnovki zavirajo prenašalce v ledvicah, in sicer kationski organski prenašalec 2 (OCT2) ter prenašalca MATE1. In vivo lahko pride do medsebojnega delovanja abemacikliba in klinično pomembnih substratov teh prenašalcev, kot je dofelitid ali kreatinin. Trenutno ni znano, ali lahko abemaciklib zmanjša učinkovitost sistemskih hormonskih kontraceptivov, zato se ženskam, ki uporabljajo sistemske hormonske kontraceptive, svetuje, da hkrati uporabljajo tudi mehansko metodo. Neželeni učinki: Najpogostejši neželeni učinki so driska, okužbe, nevtropenija, levko- penija, anemija, utrujenost, navzea, bruhanje in zmanjšanje apetita. Zelo pogosti: okužbe, nevtropenija, levkopenija, anemija, trombocitopenija, limfopenija, zmanjšanje apetita, glavobol, disgevzija, omotica, driska, bruhanje, navzea, stomatitis, alopecija, pruritus, izpuščaj, pireksija, utrujenost, povečana vrednost alanin-aminotransferaze, povečana vrednost aspartat-ami- notransferaze. Pogosti: povečano solzenje, venska trombembolija, ILD/pnevmonitis, dispepsija, spremembe na nohtih, suha koža, mišična šibkost. Občasni: febrilna nevtropenija. Rok upo- rabnosti 3 leta. Posebna navodila za shranjevanje Za shranjevanje zdravila niso potrebna posebna navodila. Imetnik dovoljenja za promet z zdravilom: Eli Lilly Nederland B.V., Papendorp- seweg 83, 3528BJ, Utrecht, Nizozemska. Datum prve odobritve dovoljenja za promet: 27. september 2018. Datum zadnje revizije besedila: 26.1.2023. Režim izdaje: Rp/Spec - Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. abemaciklib vsak dan dvakrat na dan DAJTE JI VEČ KOT UPANJE Od 24.3.2023 na Pozitivni listi zdravil P100* tudi za adjuvantno zdravljenje HR+, HER2- zgodnjega raka dojk2 Za lajšanje bolečine in oteklin v ustni in žrelu, ki so posledica radiomukozitisa Sestava: 1,5 mg/ml: 1 ml raztopine vsebuje 1,5 mg benzidaminijevega klorida, kar ustreza 1,34 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,255 mg benzidaminijevega klorida, kar ustreza 0,2278 mg benzidamina. Sestava 3 mg/ml: 1 ml raztopine vsebuje 3 mg benzidaminijevega klorida, kar ustreza 2,68 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,51 mg benzidaminijevega klorida, kar ustreza 0,4556 mg benzidamina. Terapevtske indikacije: Samozdravljenje: Lajšanje bolečine in oteklin pri vnetju v ustni votlini in žrelu, ki so lahko posledica okužb in stanj po operaciji. Po nasvetu in navodilu zdravnika: Lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa. Odmerjanje in način uporabe: Uporaba: 2- do 6-krat na dan (vsake 1,5 do 3 ure). Odmerjanje 1,5 mg/ml: Odrasli: 4 do 8 razprškov 2- do 6-krat na dan. Pediatrična populacija: Mladostniki, stari od 12 do 18 let: 4-8 razprškov 2- do 6-krat na dan. Otroci od 6 do 12 let: 4 razprški 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 4 kg telesne mase; do največ 4 razprške 2- do 6-krat na dan. Odmerjanje 3 mg/ml: Odrasli: 2 do 4 razprški 2- do 6-krat na dan. Pediatrična populacija: Mladostniki, stari od 12 do 18 let: 2 do 4 razprški 2- do 6-krat na dan. Otroci od 6 do 12 let: 2 razprška 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 8 kg telesne mase; do največ 2 razprška 2- do 6-krat na dan. Starejši bolniki, bolniki z jetrno okvaro in bolniki z ledvično okvaro: niso potrebni posebni previdnostni ukrepi. Trajanje zdravljenja ne sme biti daljše od 7 dni. Način uporabe: Za orofaringealno uporabo. Zdravilo se razprši v usta in žrelo. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Pri nekaterih bolnikih lahko resne bolezni povzročijo ustne/žrelne ulceracije. Če se simptomi v treh dneh ne izboljšajo, se mora bolnik posvetovati z zdravnikom ali zobozdravnikom, kot je primerno. Uporaba benzid- amina ni priporočljiva za bolnike s preobčutljivostjo na salicilno kislino ali druga nesteroidna protivnetna zdravila. Pri bolnikih, ki imajo ali so imeli bronhialno astmo, lahko 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 količina alkohola v zdravilu ne bo imela nobenih opaznih učinkov. To zdravilo vsebuje metilparahidroksibenzoat (E218). Lahko povzroči 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 povzročijo alergijske reakcije. Zdravilo z jakostjo 3 mg/ml vsebuje makrogolglicerol hidroksistearat 40. Lahko povzroči želodčne težave in drisko. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Študij medsebojnega delovanja niso izvedli. Nosečnost in dojenje: O uporabi benzidamina pri nosečnicah in doječih ženskah ni zadostnih podatkov. Uporaba zdravila med nosečnostjo in dojenjem ni priporočljiva. Vpliv na sposobnost vožnje in upravljanja strojev: Zdravilo v priporočenem odmerku nima vpliva na sposobnost vožnje in upravljanja strojev. Neželeni učinki: Neznana pogostnost (ni mogoče oceniti iz razpoložljivih podatkov): anafilaktične reakcije, preobčutljivostne reakcije, odrevenelost, laringospazem, suha usta, navzea in bruhanje, oralna hipestezija, angioedem, fotosenzitivnost, pekoč občutek v ustih. Neposredno po uporabi se lahko pojavi občutek odrevenelosti v ustih in v žrelu. Ta učinek se pojavi zaradi načina delovanja zdravila in po kratkem času izgine. Način in režim izdaje zdravila: BRp-Izdaja zdravila je brez recepta v lekarnah in specializiranih prodajalnah. Imetnik dovoljenja za promet: Aziende Chimiche Riunite Angelini Francesco – A.C.R.A.F. S.p.A., Viale Amelia 70, 00181 Rim, Italija Datum zadnje revizije besedila: 05. 04. 2022 Pred svetovanjem ali izdajo preberite celoten Povzetek glavnih značilnosti zdravila. Samo za strokovno javnost. Datum priprave informacije: april 2022 Odgovoren za trženje: Bonifar d.o.o. PR /B SI /B EN /2 02 2/ 01 7 Bistvene informacije iz Povzetka glavnih značilnosti zdravila Tantum Verde 1,5 mg/ml oralno pršilo, raztopina Tantum Verde 3 mg/ml oralno pršilo, raztopina (akalabrutinib) 100 mg kapsule CALQUENCE MOČ ZAUPANJA PODATKI NEPOSREDNE PRIMERJAVE: CALQUENCE V PRIMERJAVI Z IBRUTINIBOM2 ZNAČILNO NIŽJA POJAVNOST ATRIJSKE FIBRILACIJE/UNDULACIJE KATERE KOLI STOPNJE PRI CALQUENCE V PRIMERJAVI Z IBRUTINIBOM PRI BOLNIKIH S KLL, KI SO PREDHODNO PREJEMALI VSAJ ENO ZDRAVLJENJE. Primarni opazovani dogodek, neinferiorno preživetje brez napredovanja bolezni (PFS - progression-free survival), je bil dosežen. Dodatne informacije so na voljo pri družbi Astrazeneca UK Limited, Podružnica v Sloveniji, Verovškova 55, Ljubljana. Tel. (01) 51 35 600 Samo za strokovno javnost. Datum priprave besedila: april 2023 SI-3038 Nova generacija zaviralcev BTK za zdravljenje KLL Visoka selektivnost za BTK z omejeno zunajtarčno aktivnostjo1 SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Calquence 100 mg trde kapsule Calquence 100 mg filmsko obložene tablete SESTAVA: Ena trda kapsula vsebuje 100 mg akalabrutiniba. Ena filmsko obložena tableta vsebuje 100 mg akalabrutiniba (v obliki akalabrutinibijevega maleata). Akalabrutinib je selektiven zaviralec Brutonove tirozin-kinaze. INDIKACIJE: Zdravilo Calquence je kot monoterapija ali v kombinaciji z obinutuzumabom indicirano za zdravljenje odraslih bolnikov s predhodno nezdravljeno kronično limfocitno levkemijo. In kot monoterapija za zdravljenje odraslih bolnikov s kronično limfocitno levkemijo, ki so predhodno prejemali vsaj eno zdravljenje. ODMERJANJE IN NAČIN UPORABE: Zdravljenje s tem zdravilom mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje raka. Priporočeni odmerek je 100 mg akalabrutiniba dvakrat na dan. Zdravljenje je treba nadaljevati do napredovanja bolezni ali nesprejemljive toksičnosti. Informacija o priporočenih prilagoditve odmerka zdravila Calquence v primeru neželenih učinkov ≥ 3. stopnje in pri uporabi z zaviralci ali induktorji CYP3A in zdravili za zmanjšanje izločanja želodčne kisline najdete v povzetku glavnih značilnosti zdravila. Starejšim bolnikom odmerka ni treba prilagoditi. Kliničnih študij pri bolnikih z okvaro ledvic niso izvedli. Bolnike z blago ali zmerno okvaro ledvic so zdravili v kliničnih študijah zdravila Calquence. Bolnikom z blago ali zmerno okvaro odmerka ni treba prilagoditi. Pri bolnikih s hudo okvaro ledvic se zdravilo Calquence lahko uporabi le, če koristi odtehtajo tveganje; te bolnike je treba skrbno spremljati glede znakov toksičnosti. Podatkov o bolnikih s hudo okvaro ledvic in bolnikih na dializi ni. Bolnikom z blago ali zmerno okvaro jeter odmerka ni treba prilagoditi. Vendar je treba bolnike z zmerno okvaro jeter natančno nadzorovati glede znakov toksičnosti. Zdravila Calquence ni priporočljivo uporabljati pri bolnikih s hudo okvaro jeter. Bolniki s hudimi srčno-žilnimi boleznimi niso bili vključeni v klinične študije zdravila Calquence. Varnost in učinkovitost zdravila Calquence pri otrocih in mladostnikih v starosti od 0 do 18 let nista bili dokazani. Zdravilo Calquence je namenjeno za peroralno uporabo. Kapsule ali tablete je treba zaužiti cele z vodo, vsak dan ob približno istem času, s hrano ali brez nje. Kapsul ali tablet se ne sme gristi, raztapljati ali odpirati. KONTRAINDIKACIJE: Preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI: Krvavitve: Pri bolnikih s hematološkimi malignomi, ki so bili zdravljeni z zdravilom Calquence v monoterapiji ali v kombinaciji z obinutuzumabom, so se pojavile večje krvavitve, vključno s krvavitvami v osrednjem živčevju in gastrointestinalnimi krvavitvami, v nekaterih primerih s smrtnim izidom. Ti dogodki so se pojavili tako pri bolnikih s trombocitopenijo kot tudi pri tistih brez nje. Na splošno pa so bile krvavitve manj hudi dogodki, ki so vključevali podplutbe in petehije. Bolniki, ki prejemajo antitrombotična zdravila, imajo večje tveganje za krvavitve. Varfarina ali drugih antagonistov vitamina K se ne sme uporabljati sočasno z zdravilom Calquence. Razmislite o koristih in tveganjih zadržanja zdravila Calquence vsaj 3 dni pred kirurškim posegom in po njem. Okužbe: Pri bolnikih s hematološkimi malignomi, ki so bili zdravljeni z zdravilom Calquence v monoterapiji ali v kombinaciji z obinutuzumabom, so se pojavile resne okužbe (bakterijske, virusne ali glivične), vključno s smrtnimi primeri. Te okužbe so se večinoma pojavile brez nevtropenije 3. ali 4. stopnje. Reaktivacija virusov: Pri bolnikih, ki so prejemali zdravilo Calquence, so poročali o primerih reaktivacije hepatitisa B. Med uporabo zdravila Calquence v okviru predhodnega ali sočasnega imunosupresivnega zdravljenja so poročali o primerih progresivne multifokalne levkoencefalopatije (PML), vključno s primeri s smrtnim izidom. Zdravniki morajo PML upoštevati v diferencialni diagnostiki pri bolnikih, ki imajo nove nevrološke, kognitivne ali vedenjske znake ali simptome, oziroma se jim ti znaki ali simptomi poslabšajo. V primeru suma na PML je treba zdravljenje z zdravilom Calquence prekiniti, dokler PML ni izključena. Citopenije: Pri bolnikih s hematološkimi malignomi, ki so bili zdravljeni z zdravilom Calquence v monoterapiji ali v kombinaciji z obinutuzumabom, so se med zdravljenjem pojavile citopenije 3. ali 4. stopnje, vključno z nevtropenijo, anemijo in trombocitopenijo. Drugi primarni malignomi: Pri bolnikih s hematološkimi malignomi, ki so bili zdravljeni z zdravilom Calquence v monoterapiji ali v kombinaciji z obinutuzumabom, so se pojavili drugi primarni malignomi, vključno s kožnim in nekožnim rakom. O kožnem raku so poročali pogosto. Atrijska fibrilacija: Pri bolnikih s hematološkimi malignomi, ki so bili zdravljeni z zdravilom Calquence v monoterapiji ali v kombinaciji z obinutuzumabom, se je pojavila atrijska fibrilacija/undulacija. MEDSEBOJNO DELOVANJE Z DRUGIMI ZDRAVILI: Sočasna uporaba močnih zaviralcev CYP3A in zdravila Calquence lahko poveča izpostavljenost akalabrutinibu in tako poveča tveganje za toksične učinke. Nasprotno pa lahko sočasna uporaba induktorjev CYP3A zmanjša izpostavljenost akalabrutinibu in tako pomeni tveganje za nezadostno učinkovitost. Sočasni uporabi z močnimi zaviralci CYP3A se je treba izogniti. Če bo uporaba teh zaviralcev kratkotrajna (npr. antiinfektivna zdravila do sedem dni), je treba zdravljenje z zdravilom Calquence prekiniti. V primeru uporabe zmernega zaviralca CYP3A je treba bolnike skrbno nadzorovati glede znakov toksičnosti. Sočasni uporabi z močnimi induktorji CYP3A4 se je treba izogibati zaradi tveganja za nezadostno učinkovitost. Tablete akalabrutiniba je mogoče jemati sočasno z zdravili za zmanjšanje izločanja želodčne kisline (zaviralci protonske črpalke, antagonisti receptorjev H2, antacidi), za razliko od kapsul akalabrutiniba, pri katerih je privzem z zdravili za zmanjšanje izločanja kisline moten. Ker zaviralci protonske črpalke učinkujejo dolgo časa, je mogoče, da časovna ločitev odmerkov zaviralcev protonske črpalke ne odpravi medsebojnega delovanja, zato se je treba sočasni uporabi s kapsulami akalabrutiniba izogniti. NEŽELENI UČINKI: Med bolniki, ki so prejemali zdravilo Calquence v monoterapiji, so bili najpogosteje poročani neželeni učinki katere koli stopnje okužba, glavobol, driska, podplutbe, mišično-skeletne bolečine, navzea, utrujenost, kašelj in izpuščaj. Najpogosteje poročani neželeni učinki ≥ 3. stopnje so bili okužba, levkopenija, nevtropenija in anemija. Med bolniki, ki so prejemali zdravilo Calquence v okviru kombiniranega zdravljenja, so bili najpogosteje poročani neželeni učinki katere koli stopnje okužba, mišično-skeletne bolečine, driska, glavobol, levkopenija, nevtropenija, kašelj, utrujenost, artralgija, navzea, omotica in zaprtje. Najpogosteje poročani neželeni učinki ≥ 3. stopnje so bili levkopenija, nevtropenija, okužba, trombocitopenija in anemija. Med bolniki, zdravljenimi z monoterapijo zdravila Calquence, so o prenehanju zdravljenja zaradi neželenih učinkih poročali pri 9,3 % bolnikov. Ti glavni neželeni učinki so vključevali pljučnico, trombocitopenijo in drisko. O zmanjšanju odmerka zaradi neželenih učinkov so poročali pri 4,2 % bolnikov. Ti glavni neželeni učinki so vključevali reaktivacijo hepatitisa B, sepso in drisko. Med bolniki, zdravljenimi z zdravilom Calquence v obliki kombiniranega zdravljenja, so o prenehanju zdravljenja zaradi neželenih učinkov poročali pri 10,8 % bolnikov. Ti glavni neželeni učinki so vključevali pljučnico, trombocitopenijo in drisko. O zmanjšanju odmerka zaradi neželenih učinkov so poročali pri 6,7 % bolnikov. Ti glavni neželeni učinki so vključevali nevtropenijo, drisko in bruhanje. POSEBNA NAVODILA ZA SHRANJEVANJE: Posebna navodila za shranjevanje niso potrebna. VRSTA IN VSEBINA OVOJNINE: Calquence 100 mg trde kapsule: Aluminij/aluminijski pretisni omoti s simboli sonca/lune, vsebujejo 6 ali 8 trdih kapsul. Škatle s 56 ali 60 kapsulami. Calquence 100 mg filmsko obložene tablete: Aluminij/aluminijska pakiranja pretisnih omotov s simboli sonca/lune, vsebujejo 8 ali 10 filmsko obloženih tablet. Škatle s 56 ali 60 tabletami. Na trgu morda ni vseh navedenih pakiranj. NAČIN IZDAJANJA ZDRAVILA: Rp/Spec - Predpisovanje in izdaja zdravila je le na recept s posebnim režimom. DATUM REVIZIJE BESEDILA: februar 2023 (SI-2882) IMETNIK DOVOLJENJA ZA PROMET: AstraZeneca AB, S-151 85, Sodertalje, Švedska Pred predpisovanjem, prosimo, preberite celoten povzetek glavnih značilnosti zdravila. Dodatne informacije so na voljo pri družbi AstraZeneca UK Limited, Podružnica v Sloveniji, Verovškova 55, Ljubljana. Reference: 1. Barf, T. et al. (2017). Acalabrutinib (ACP-196): a covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. Journal of Pharmacology and Experimental Therapeutics, 363(2), 240-252. 2. Byrd, J. C., Hillmen, P., Ghia, P., Kater, A. P., Chanan- Khan, A., Furman, R. R., ... & Jurczak, W. (2021, July). Acalabrutinib versus ibrutinib in previously treated chronic lymphocytic leukemia: results of the first randomized phase III trial. American Society of Clinical Oncology (ASCO). BTK = Brutonova tirozin-kinaza; KLL = kronična limfocitna levkemija Terapija prvega izbora za 2L v smernicah ESMO in NCCN4,5 Zdravilo ENHERTU se uporablja v monoterapiji in je v raziskavi DESTINY-Breast03 dokazalo neprimerljivo podaljšanje PFS v primerjavi s trenutnim standardom zdravljenja (T-DM1). Poročali so o primerih intersticijske pljučne bolezni (ILD) in pnevmonitisa. Za diagnozo je ključno prepoznavanje simptomov, zato je bolnike treba spremljati in pričeti z zdravljenjem ob prvih znakih ILD.1,2 Datum priprave materiala: maj 2023. Samo za strokovno javnost. SI-3059 ENHERTU® je registrirana blagovna znamka družbe Daiichi Sankyo Company, Limited. © 2023 Daiichi Sankyo Company, Ltd. in AstraZeneca Ltd. POSTAVLJA NOVE STANDARDE ZDRAVLJENJA RAZSEJANEGA HER2+ RAKA DOJK2 NEPRIMERLJIVO PREŽIVETJE* ODOBRENO PO VSAJ ENI PREDHODNI TERAPIJI NA PODLAGI ANTI-HER21 SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. 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 hrčka) in je prek razcepljivega veznika na tetrapeptidni bazi kovalentno vezan na DXd, ki je derivat eksatekana in zavira- lec topoizomeraze I. Na vsako molekulo protitelesa je vezanih približno 8 molekul derukstekana. Pomožne snovi: L-histidin, L-histidinijev klorid monohidrat, saharoza, polisorbat 80. TERAPEVTSKE INDIKACIJE: Rak dojk: HER2-pozitiven rak dojk: 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 več shem zdravljenja na podlagi anti-HER2. Rak dojk z nizkim statusom HER2: Zdravilo Enhertu kot monoterapija je indicirano za zdravl- jenje odraslih bolnikov z neresektabilnim ali metastatskim rakom dojk z nizkim statusom HER2, ki so pred tem že prejeli kemoterapijo v prisotnosti metastaz ali pa se je pri njih bolezen ponovila med adjuvantno kemoterapijo ali znotraj 6 mesecev po njenem zaključku. Rak želod- ca: Zdravilo Enhertu v obliki monoterapije je indicirano za zdravljenje odraslih bolnikov z napredovalim HER2-pozitivnim adenokarcinomom želodca ali gastroezofagealnega prehoda, ki so pred tem že prejeli shemo na podlagi trastuzumaba. ODMERJANJE IN NAČIN UPORABE: Zdravilo Enhertu mora predpisati zdravnik in njegovo dajanje nadzorovati zdravstveni delavec, ki sta izkušena v uporabi zdravil proti raku. Za preprečitev napak, povezanih z zdravili, je pomembno, da preverite nalepke na vialah in se prepričate, 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 zaradi HER2-pozitivnega raka dojk, raka želod- ca ali gastroezofagealnega prehoda, morajo imeti dokumentiran HER2-pozitiven status tumorja, ki je opredeljen kot ocena 3 + na podlagi imunohistokemije (IHC) ali razmerje ≥ 2,0 na podlagi in situ hibridizacije (ISH) ali fluorescenčne in situ hibridizacije (FISH), ocenjeno z in vitro diagnostičnim (IVD) medicinskim pripomočkom z oznako CE. Bolniki, ki se zdravijo s trastuzumab derukstekanom zaradi raka dojk z nizkim statusom HER2, morajo imeti dokumentiran nizek status HER2 tumorja, ki je opredeljen kot ocena IHC 1+ ali IHC 2+/ISH-, ocenjeno z IVD z oznako CE. Če IVD z oznako CE ni na voljo, je treba status HER2 oceniti z drugim potrjenim testom Odmerjanje: Rak dojk: Priporočeni odmerek zdravila Enhertu je 5,4 mg/kg, ki se daje z intravensko infuzijo enkrat vsake 3 tedne (21-dnevni cikel) do napredovanja bolezni ali nespre- jemljive toksičnosti. Rak želodca: Priporočeni odmerek zdravila Enhertu je 6,4 mg/kg, ki se daje z intravensko infuzijo enkrat vsake 3 tedne (21-dnevni cikel) do napredovanja bolezni ali nesprejemljive toksičnosti. Začetni odmerek je treba dati z 90-minutno intravensko infuzijo. Če bolnik prejšnjo infuzijo dobro prenaša, se lahko naslednji odmerki zdravila Enhertu dajejo kot 30-minutne infuzije. Hitrost infundiranja zdravila Enhertu je treba zmanjšati ali infundiranje prekiniti, če se pri bolniku razvijejo simptomi, povezani z infuzijo. V primeru hudih reakcij na infuzijo je treba zdravilo Enhertu trajno ukiniti. Premedikacija: Zdravilo Enhertu je emetogeno, kar vključuje zapoznelo navzeo in/ali bruhanje. Pred vsakim odmerkom zdravila Enhertu je treba bolnike premedicirati s kombiniranim režimom dveh ali treh zdravil (npr. deksametazon z antagonistom receptorjev 5-HT3 in/ali antagonistom receptorjev NK1 ter drugimi zdravili, kot je indicirano) za preprečevanje navzee in bruhanja zaradi kemoterapije. Prilagajanje odmerka: Obvladovanje neželenih učinkov lahko zajema začasno prekinitev uporabe, zmanjšanje od- merka ali ukinitev zdravljenja z zdravilom Enhertu, skladno s smernicami, podanimi v povzetku glavnih značilnosti zdravila (preglednici 1 in 2). Po zmanjšanju odmerka zdravila Enhertu se odmerek ne sme več ponovno povečati. Načrt zmanjševanja odmerka: Priporočeni začetni odmerek je 5,4 mg/kg pri raku dojk oz. 6,4 mg/kg pri raku želodca; prvo zmanjšanje odmerka (4,4 mg/kg oz. 5,4 mg/kg), drugo zmanjšanje odmerka (3,2 mg/kg oz. 4,4 mg/kg), pri potrebi po nadaljnjem zmanjšanju odmerka ukinite zdravljenje. Prosimo, glejte celoten povzetek glavnih značilnosti zdravila Enhertu za prilagajanje odmerka zaradi neželenih učinkov: intersticijska pljučna bolezen (IPB)/pnevmonitis (asimptomatska IPB/asimptomatski pnevmonitis (stopnja 1), simptomatska IPB/simptomatski pnevmonitis (stopnja 2 ali višja)), nevtropenija (stopnja 3 (manj kot 1,0-0,5 × 109/l), stopnja 4 (manj kot 0,5 × 109/l)), febrilna nevtropenija (absolutno število nevtrofilcev manj kot 1,0 × 109/l in telesna temperatura, višja od 38,3 °C, ali telesna temperatura 38 °C ali višja, ki vztraja več kot eno uro), zmanjšan iztisni delež levega prekata (LVEF) (LVEF več kot 45 % in absolutno zmanjšanje glede na izhodiščno vrednost za 10 % do 20 %; LVEF 40 % do 45 %; LVEF manj kot 40 % ali absolutno zmanjšanje glede na izhodiščno vrednost za več kot 20 %; simptomatično kongestivno srčno popuščanje). Zakasnjen ali izpuščen odmerek: Če se načrtovani odmerek zakasni ali izpusti, ga je treba dati takoj, ko je mogoče, brez čakanja na naslednji načrtovani cikel. Časovni načrt 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: Starejši: Pri bolnikih, starih 65 let ali starejših, prilagajanje odmerka zdravila Enhertu ni potrebno. Podatki pri bolnikih, starih ≥ 75 let, so omejeni. Okvara ledvic: Prilagajanje odmerka pri bolnikih z blago (očistek kreati- nina [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 ali končno ledvično odpovedjo ni mogoče opredeliti, ker je bila huda okvara ledvic v kliničnih študijah izključitveni kriterij. Pri bolnikih z zmerno okvaro ledvic so opazili višjo pogostnost IPB stopnje 1 in 2/pnevmonitisa, ki sta vodila do zvečanja števila prekinitev zdravljenja. Pri bolnikih z zmerno okvaro ledvic v izhodišču, ki so prejemali zdravilo Enhertu 6,4 mg/kg, so ugotovili večjo pogostnost resnih neželenih učinkov kot pri tistih z normalnim delovanjem ledvic. Bolnike z zmerno ali hudo okvaro ledvic je treba natančno spremljati glede neželenih učinkov, vključno z IPB/pnevmonitisom. Okvara jeter: Pri bolnikih, ki imajo celokupni bilirubin ≤ 1,5-kratnik 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 > 1,5-kratnik ZMN, ne glede na vrednost AST, ni mogoče opredeliti zaradi pomanjkanja podatkov. Zato je treba te bolnike natančno spremljati. Način uporabe: Zdravilo Enhertu je za intravensko uporabo. Zdravstveni delavec ga mora rekonstituirati in razredčiti. Treba ga je dati z intravenskim infundiranjem. Zdravilo Enhertu se ne sme dati kot hitro intra- vensko injekcijo ali bolus. KONTRAINDIKACIJE: Preobčutljivost na učinkovino ali katero koli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Intersticijska pljučna bolezen/pnevmonitis: Pri zdravilu Enhertu so poročali o primerih intersticijske pljučne bolezni (IPB) in/ali pnevmonitisa. Nekateri primeri so bili smrtni. Bolnikom je treba naročiti, naj takoj poročajo o kašlju, dispneji, zvišani telesni temperaturi in/ali katerih koli novih dihalnih simptomih ali poslabšanju obstoječih. Bolnike je treba spremljati glede znakov in simptomov IPB/pnevmo- nitisa. Dokaze za IPB/pnevmonitis je treba takoj proučiti. Bolnike s sumom na IPB/pnevmonitis je treba oceniti z radiografskimi posnetki, najbolje z računalniško tomografijo (CT). Treba je razmisliti o posvetu s pulmologom. Nevtropenija: V kliničnih študijah z zdravilom Enhertu so poročali o primerih nevtropenije, vključno s primeri febrilne nevtropenije s smrtnim izidom. Pred uvedbo zdravila Enhertu in pred vsakim odmerkom ter vsakič, ko je klinično indicirano, je treba preveriti celotno krvno sliko. Morda bo treba začasno prekiniti dajanje zdravila Enhertu ali zmanjšati odmerek, odvisno od tega, kako huda je nevtropenija. Zmanjšanje iztisnega deleža levega prekata: Pri zdravljenjih anti-HER2 so poročali o zmanjšanem iztisnem deležu levega prekata (LVEF). Pred uvedbo zdravljenja z zdravilom Enhertu in v rednih intervalih med njim (v skladu s kliničnimi indikacijami) je treba izvesti standardne preiskave delovanja srca (ehokardiografija ali slikanje MUGA) za oceno LVEF. Zmanjšanje LVEF je treba obvladovati s prekinitvami zdravljenja. Zdravljenje z zdravilom Enhertu je treba trajno ukiniti, če se potrdi LVEF manj kot 40 % ali absolutno zmanjšanje glede na izhodiščno vrednost za več kot 20 %. Zdravilo Enhertu je treba trajno ukiniti pri bolnikih s simptomatskim kongestivnim srčnim popuščanjem. Embrio-fetalna toksičnost: Zdravilo Enhertu lahko ima škodljiv vpliv na plod, če se da noseč- nici. Pri ženskah v rodni dobi je treba pred uvedbo zdravljenja z zdravilom Enhertu preveriti status nosečnosti. Bolnice je treba seznaniti z možnimi tveganji za plod. Ženskam v rodni dobi je treba svetovati, da uporabljajo učinkovito 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 učinkovito 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 sočasnem dajanju trastuzumab derukstekana z zdravili, ki so zaviralci CYP3A ali OATP1B ali prenašalcev P-gp, odmerka ni treba prilagajati. PLODNOST, NOSEČNOST IN DOJENJE: Nosečnost: Dajanje zdravila Enhertu nosečnicam se ne priporoča. Bolnice je treba seznaniti z možnimi tveganji za plod, preden zanosijo. Ženske, ki zanosijo, se morajo takoj obrniti na zdravnika. Če ženska zanosi med zdravljenjem z zdravilom Enhertu ali v obdobju 7 mesecev po zadnjem odmerku zdravila Enhertu, se priporoča natančno spremljanje. Dojenje: Ni znano, ali se trastuzumab derukstekan izloča v materino mleko. Humani IgG se izloča v materino mleko in potencial za absorpcijo in resne neželene učinke na dojenčka ni znan. Zato ženske ne smejo dojiti med zdravljenjem z zdravilom Enhertu in še 7 mesecev po zadnjem odmerku. Odločiti se je treba med prenehanjem dojenja in prenehanjem zdravljenja z zdravilom Enhertu, pri čemer je treba pretehtati prednosti dojenja za otroka in prednosti zdravljenja za mater. Plodnost: Namenskih študij plodnosti s trastuzumab derukstekanom niso izvedli. Ni znano, ali so trastuzumab derukstekan ali njegovi presnovki prisotni v semenski tekočini. Pred začetkom 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 UČINKI: Zdravilo Enhertu 5,4 mg/ kg: Združeno varnostno populacijo so ocenili pri bolnikih, ki so v kliničnih študijah dobili vsaj en odmerek 5,4 mg/kg zdravila Enhertu (N = 944) zaradi različnih vrst tumorjev. Mediani čas trajanja zdravljenja v tej združeni populaciji je bil 39,6 meseca (razpon: 0,2-37,9 meseca). Zelo pogosti: okužba zgornjih dihal, anemija, nevtropenija, trombocitopenija, levkopenija, zmanjšan apetit, hipokaliemija, glavobol, omotica, intersticijska pljučna bolezen, kašelj, dispneja, epistaksa, navzea, bruhanje, driska, zaprtje, bolečina v trebuhu, stomatitis, dispepsija, zvišane transaminaze, alopecija, izpuščaj, mišično-skeletna bolečina, utrujenost, pireksija, zmanjšanje telesne mase, zmanjšan iztisni delež. Pogosti: pljučnica, limfopenija, febrilna nevtropenija, dehidracija, disgevzija, zamegljen vid, abdominalna distenzija, flatulenca, gastritis, pruritus, hiperpigmentacija kože, perifermni edem, zvišana alkalna fosfataza v krvi, zvišan bilirubin v krvi, zvišan kreatinin v krvi, reakcije povezane z infuzijo. Zdravilo Enhertu 6,4 mg/kg: Združeno varnostno populacijo so ocenili za bolnike, ki so v kliničnih študijah dobili vsaj en odmerek 6,4 mg/kg zdravila Enhertu (N = 619) zaradi različnih vrst tumorjev. Mediani čas trajanja zdravljenja v tej združeni populaciji je bil 5,6 meseca (razpon: 0,7-41,0 meseca). Zelo pogosti: okužba zgornjih dihal, pljučnica, anemija, nevtropenija, trombocitopenija, levkopenija, limfopeni- ja, zmanjšan apetit, hipokaliemija, glavobol, disgevzija, intersticijska pljučna bolezen, kašelj, navzea, bruhanje, driska, zaprtje, bolečina v trebuhu, stomatitis, zvišane transaminaze, alopecija, mišično-skeletna bolečina, utrujenost, pireksija, perifermni edem, zmanjšanje telesne mase, zmanjšan iztisni delež. Pogosti: febrilna nevtropenija, dehidracija, omotica, zamegljen vid, dispneja, epistaksa, dispepsija, izpuščaj, pruritus, hiperpigmentacija kože, 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 München, Nemčija DATUM ZADNJE REVIZIJE BESEDILA: 23. 1. 2023 (SI-2799) REŽIM PREDPISOVANJA IN IZDAJE: H Prosimo, da pred predpisovanjem preberete celoten povzetek glavnih značilnosti 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) ** tveganje za napredovanje bolezni ob zdravljenju z zdravilom ENHERTU v primerjavi s T-DM1 (HR: 0,33; 95 % IZ: 0,26-0,43; p<0,000001, ključni opazovani dogodek raziskave: PFS glede na BICR). Mediani PFS je znašal 28,8 mesecev pri bolnikih, ki so prejemali ENHERTU, v primerjavi s 6,8 mesecev pri bolnikih zdravljenih s T-DM13 PFS - preživetje brez napredovanja bolezni, mPFS - mediano preživetje brez napredovanja bolezni, T-DM1 - trastuzumab emtanzin, BICR - ocena slepega neodvisnega centralnega pregleda, IZ - interval zaupanja, HR - razmerje ogroženosti Literatura: 1. Povzetek glavnih značilnosti zdravila ENHERTU, 23. 1. 2023, 2. Cortes J et al; Trastuzumab Deruxtecan versus Trastuzumab Emtansine for Breast Cancer; NEJM 2022;386(12):1143-1154, 3. Hurvitz SA et al, Presented at SABCS 2022, 6-10.12.2022 in San Antonio. Presentation ID: GS2-02, 4. Gennari A et al: Ann Oncol 2021;32(12): 1475-1495 5. NCCN guidelines Breast Cancer, v2.2023, https://www.nccn.org/guidelines/category_1, dostopano 10.3.2023. Okrajšave: MSI-H - visoka mikrosatelitska nestabilnost; dMMR - pomankljivo popravljanje neujemanja pri podvojevanju DNA; 1L - prva linija zdravljenja; TNRD - trojno negativni rak dojk Referenci: 1. www.zzzs.si; https://www.zzzs.si/zzzs-api/e-gradiva/vsa-gradiva/?vrsta=BR3A2Q326 (22.12.2022) 2. KEYTRUDA SPC Ime zdravila: KEYTRUDA 25 mg/ml koncentrat za raztopino za infundiranje vsebuje pembrolizumab. Terapevtske indikacije: Zdravilo KEYTRUDA je kot samostojno zdravljenje indicirano za zdravljenje: odraslih in mladostnikov, starih 12 let ali več, z napredovalim (neoperabilnim ali metastatskim) melanomom; za adjuvantno zdravljenje odraslih in mladostnikov, starih 12 let ali več, z melanomom v stadiju IIB, IIC ali III, in sicer po popolni kirurški odstranitvi; metastatskega nedrobnoceličnega pljučnega raka (NSCLC) v prvi liniji zdravljenja pri odraslih, ki imajo tumorje z ≥ 50 % izraženostjo PD-L1 (TPS) in brez pozitivnih tumorskih mutacij EGFR ali ALK; lokalno napredovalega ali metastatskega NSCLC pri odraslih, ki imajo tumorje z ≥ 1 % izraženostjo PD-L1 (TPS) in so bili predhodno zdravljeni z vsaj eno shemo kemoterapije, bolniki s pozitivnimi tumorskimi mutacijami EGFR ali ALK so pred prejemom zdravila KEYTRUDA morali prejeti tudi tarčno zdravljenje; odraslih in pediatričnih bolnikov, starih 3 leta ali več, s ponovljenim ali neodzivnim klasičnim Hodgkinovim limfomom (cHL), pri katerih avtologna presaditev matičnih celic (ASCT) ni bila uspešna, ali po najmanj dveh predhodnih zdravljenjih kadar ASCT ne pride v poštev kot možnost zdravljenja; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, predhodno zdravljenih s kemoterapijo, ki je vključevala platino; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, ki niso primerni za zdravljenje s kemoterapijo, ki vsebuje cisplatin in imajo tumorje z izraženostjo PD-L1 ≥ 10, ocenjeno s kombinirano pozitivno oceno (CPS); ponovljenega ali metastatskega ploščatoceličnega raka glave in vratu (HNSCC) pri odraslih, ki imajo tumorje z ≥ 50 % izraženostjo PD-L1 (TPS), in pri katerih je bolezen napredovala med zdravljenjem ali po zdravljenju s kemoterapijo, ki je vključevala platino; za adjuvantno zdravljenje odraslih z rakom ledvičnih celic s povišanim tveganjem za ponovitev bolezni po nefrektomiji, ali po nefrektomiji in kirurški odstranitvi metastatskih lezij, za zdravljenje odraslih z MSI-H (microsatellite instability-high) ali dMMR (mismatch repair de cient) kolorektalnim rakom v naslednjih terapevtskih okoliščinah: prva linija zdravljenja metastatskega kolorektalnega raka; zdravljenje neoperabilnega ali metastatskega kolorektalnega raka po predhodnem kombiniranem zdravljenju, ki je temeljilo na  uoropirimidinu; in za zdravljenje MSI-H ali dMMR tumorjev pri odraslih z: napredovalim ali ponovljenim rakom endometrija, pri katerih je bolezen napredovala med ali po predhodnem zdravljenju, ki je vključevalo platino, v katerih koli terapevtskih okoliščinah, in ki niso kandidati za kurativno operacijo ali obsevanje; neoperabilnim ali metastatskim rakom želodca, tankega črevesa ali žolčnika in žolčnih vodov, pri katerih je bolezen napredovala med ali po vsaj enem predhodnem zdravljenju. Zdravilo KEYTRUDA je kot samostojno zdravljenje ali v kombinaciji s kemoterapijo s platino in 5- uorouracilom (5-FU) indicirano za prvo linijo zdravljenja metastatskega ali neoperabilnega ponovljenega ploščatoceličnega raka glave in vratu pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 1. Zdravilo KEYTRUDA je v kombinaciji s pemetreksedom in kemoterapijo na osnovi platine indicirano za prvo linijo zdravljenja metastatskega neploščatoceličnega NSCLC pri odraslih, pri katerih tumorji nimajo pozitivnih mutacij EGFR ali ALK; v kombinaciji s karboplatinom in bodisi paklitakselom bodisi nab-paklitakselom je indicirano za prvo linijo zdravljenja metastatskega ploščatoceličnega NSCLC pri odraslih; v kombinaciji z aksitinibom ali v kombinaciji z lenvatinibom je indicirano za prvo linijo zdravljenja napredovalega raka ledvičnih celic (RCC) pri odraslih; v kombinaciji s kemoterapijo s platino in  uoropirimidinom je indicirano za prvo linijo zdravljenja lokalno napredovalega neoperabilnega ali metastatskega raka požiralnika ali HER-2 negativnega adenokarcinoma gastroezofagealnega prehoda pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 10; v kombinaciji s kemoterapijo za neoadjuvantno zdravljenje, in v nadaljevanju kot samostojno adjuvantno zdravljenje po kirurškem posegu, je indicirano za zdravljenje odraslih z lokalno napredovalim trojno negativnim rakom dojk ali trojno negativnim rakom dojk v zgodnjem stadiju z visokim tveganjem za ponovitev bolezni; v kombinaciji s kemoterapijo je indicirano za zdravljenje lokalno ponovljenega neoperabilnega ali metastatskega trojno negativnega raka dojk pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 10 in predhodno niso prejeli kemoterapije za metastatsko bolezen; v kombinaciji z lenvatinibom je indicirano za zdravljenje napredovalega ali ponovljenega raka endometrija (EC) pri odraslih z napredovalo boleznijo med ali po predhodnem zdravljenju s kemoterapijo, ki je vključevala platino, v katerih koli terapevtskih okoliščinah, in ki niso kandidati za kurativno operacijo ali obsevanje; v kombinaciji s kemoterapijo, z bevacizumabom ali brez njega, je indicirano za zdravljenje persistentnega, ponovljenega ali metastatskega raka materničnega vratu pri odraslih bolnicah, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 1. Odmerjanje in način uporabe: Testiranje PD-L1: Če je navedeno v indikaciji, je treba izbiro bolnika za zdravljenje z zdravilom KEYTRUDA na podlagi izraženosti PD-L1 tumorja potrditi z validirano preiskavo. Testiranje MSI/MMR: Če je navedeno v indikaciji, je treba izbiro bolnika za zdravljenje z zdravilom KEYTRUDA na podlagi MSI-H/dMMR statusa tumorja potrditi z validirano preiskavo. Odmerjanje: Priporočeni odmerek zdravila KEYTRUDA pri odraslih je bodisi 200 mg na 3 tedne ali 400 mg na 6 tednov, apliciran z intravensko infuzijo v 30 minutah. Priporočeni odmerek zdravila KEYTRUDA za samostojno zdravljenje pri pediatričnih bolnikih s cHL, starih 3 leta ali več, ali bolnikih z melanomom, starih 12 let ali več, je 2 mg/kg telesne mase (do največ 200 mg) na 3 tedne, apliciran z intravensko infuzijo v 30 minutah. Za uporabo v kombinaciji glejte povzetke glavnih značilnosti zdravil sočasno uporabljenih zdravil. Če se uporablja kot del kombiniranega zdravljenja skupaj z intravensko kemoterapijo, je treba zdravilo KEYTRUDA aplicirati prvo. Bolnike je treba zdraviti do napredovanja bolezni ali nesprejemljivih toksičnih učinkov (in do maksimalnega trajanja zdravljenja, če je le to določeno za indikacijo). Pri adjuvantnem zdravljenju melanoma ali RCC je treba zdravilo uporabljati do ponovitve bolezni, pojava nesprejemljivih toksičnih učinkov oziroma mora zdravljenje trajati do enega leta. Za neoadjuvantno in adjuvantno zdravljenje TNBC morajo bolniki neoadjuvantno prejeti zdravilo KEYTRUDA v kombinaciji s kemoterapijo, in sicer 8 odmerkov po 200 mg na 3 tedne ali 4 odmerke po 400 mg na 6 tednov, ali do napredovanja bolezni, ki izključuje de nitivni kirurški poseg, ali do pojava nesprejemljivih toksičnih učinkov, čemur sledi adjuvantno zdravljenje z zdravilom KEYTRUDA kot samostojnim zdravljenjem, in sicer 9 odmerkov po 200 mg na 3 tedne ali 5 odmerkov po 400 mg na 6 tednov ali do ponovitve bolezni ali pojava nesprejemljivih toksičnih učinkov. Bolniki, pri katerih pride do napredovanja bolezni, ki izključuje de nitivni kirurški poseg, ali do nesprejemljivih toksičnih učinkov povezanih z zdravilom KEYTRUDA kot neoadjuvantnim zdravljenjem v kombinaciji s kemoterapijo, ne smejo prejeti zdravila KEYTRUDA kot samostojnega zdravljenja za adjuvantno zdravljenje. Če je aksitinib uporabljen v kombinaciji s pembrolizumabom, se lahko razmisli o povečanju odmerka aksitiniba nad začetnih 5 mg v presledkih šest tednov ali več. V primeru uporabe v kombinaciji z lenvatinibom je treba zdravljenje z enim ali obema zdraviloma prekiniti, kot je primerno. Uporabo lenvatiniba je treba zadržati, odmerek zmanjšati ali prenehati z uporabo, v skladu z navodili v povzetku glavnih značilnosti zdravila za lenvatinib, in sicer za kombinacijo s pembrolizumabom. Pri bolnikih starih ≥ 65 let, bolnikih z blago do zmerno okvaro ledvic, bolnikih z blago ali zmerno okvaro jeter prilagoditev odmerka ni potrebna. Odložitev odmerka ali ukinitev zdravljenja: Zmanjšanje odmerka zdravila KEYTRUDA ni priporočljivo. Za obvladovanje neželenih učinkov je treba uporabo zdravila KEYTRUDA zadržati ali ukiniti, prosimo, glejte celoten Povzetek glavnih značilnosti zdravila. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Povzetek posebnih opozoril, previdnostnih ukrepov, interakcij in neželenih učinkov: Imunsko pogojeni neželeni učinki (pnevmonitis, kolitis, hepatitis, nefritis, endokrinopatije, neželeni učinki na kožo in drugi): Pri bolnikih, ki so prejemali pembrolizumab, so se pojavili imunsko pogojeni neželeni učinki, vključno s hudimi in smrtnimi primeri. Večina imunsko pogojenih neželenih učinkov, ki so se pojavili med zdravljenjem s pembrolizumabom, je bila reverzibilnih in so jih obvladali s prekinitvami uporabe pembrolizumaba, uporabo kortikosteroidov in/ali podporno oskrbo. Pojavijo se lahko tudi po zadnjem odmerku pembrolizumaba in hkrati prizadanejo več organskih sistemov. V primeru suma na imunsko pogojene neželene učinke je treba poskrbeti za ustrezno oceno za potrditev etiologije oziroma izključitev drugih vzrokov. Glede na izrazitost neželenega učinka je treba zadržati uporabo pembrolizumaba in uporabiti kortikosteroide – za natančna navodila, prosimo, glejte Povzetek glavnih značilnosti zdravila Keytruda. Zdravljenje s pembrolizumabom lahko poveča tveganje za zavrnitev pri prejemnikih presadkov čvrstih organov. Pri bolnikih, ki so prejemali pembrolizumab, so poročali o hudih z infuzijo povezanih reakcijah, vključno s preobčutljivostjo in ana laksijo. Pembrolizumab se iz obtoka odstrani s katabolizmom, zato presnovnih medsebojnih delovanj zdravil ni pričakovati. Uporabi sistemskih kortikosteroidov ali imunosupresivov pred uvedbo pembrolizumaba se je treba izogibati, ker lahko vplivajo na farmakodinamično aktivnost in učinkovitost pembrolizumaba. Vendar pa je kortikosteroide ali druge imunosupresive mogoče uporabiti za zdravljenje imunsko pogojenih neželenih učinkov. Kortikosteroide je mogoče uporabiti tudi kot premedikacijo, če je pembrolizumab uporabljen v kombinaciji s kemoterapijo, kot antiemetično pro lakso in/ali za ublažitev neželenih učinkov, povezanih s kemoterapijo. Ženske v rodni dobi morajo med zdravljenjem s pembrolizumabom in vsaj še 4 mesece po zadnjem odmerku pembrolizumaba uporabljati učinkovito kontracepcijo, med nosečnostjo in dojenjem se ga ne sme uporabljati. Varnost pembrolizumaba pri samostojnem zdravljenju so v kliničnih študijah ocenili pri 7.631 bolnikih, ki so imeli različne 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 čas opazovanja znašal 8,5 meseca (v razponu od 1 dneva do 39 mesecev), najpogostejši neželeni učinki zdravljenja s pembrolizumabom pa so bili utrujenost (31 %), diareja (22 %) in navzea (20 %). Večina poročanih neželenih učinkov pri samostojnem zdravljenju je bila po izrazitosti 1. ali 2. stopnje. Najresnejši neželeni učinki so bili imunsko pogojeni neželeni učinki in hude z infuzijo povezane reakcije. Pojavnost imunsko pogojenih neželenih učinkov pri uporabi pembrolizumaba samega za adjuvantno zdravljenje (n = 1.480) je znašala 36,1 % za vse stopnje in 8,9 % od 3. do 5. stopnje, pri metastatski bolezni (n = 5.375) pa 24,2 % za vse stopnje in 6,4 % od 3. do 5. stopnje. Pri adjuvantnem zdravljenju niso zaznali nobenih novih imunsko pogojenih neželenih učinkov. Varnost pembrolizumaba pri kombiniranem zdravljenju s kemoterapijo so ocenili pri 3.123 bolnikih z različnimi vrstami raka, ki so v kliničnih študijah prejemali pembrolizumab v odmerkih 200 mg, 2 mg/ kg telesne mase ali 10 mg/kg telesne mase na vsake 3 tedne. V tej populaciji bolnikov so bili najpogostejši neželeni učinki naslednji: anemija (55 %), navzea (54 %), utrujenost (38 %), nevtropenija (36 %), zaprtost (35 %), alopecija (35 %), diareja (34 %), bruhanje (28 %) in zmanjšanje apetita (27 %). Pojavnost neželenih učinkov 3. do 5. stopnje je pri bolnikih z NSCLC pri kombiniranem zdravljenju s pembrolizumabom znašala 67 % in pri zdravljenju samo s kemoterapijo 66 %, pri bolnikih s HNSCC pri kombiniranem zdravljenju s pembrolizumabom 85 % in pri zdravljenju s kemoterapijo v kombinaciji s cetuksimabom 84 %, pri bolnikih z rakom požiralnika pri kombiniranem zdravljenju s pembrolizumabom 86 % in pri zdravljenju samo s kemoterapijo 83 %, pri bolnikih s TNBC pri kombiniranem zdravljenju s pembrolizumabom 80 % in pri zdravljenju samo s kemoterapijo 77 % in pri bolnicah z rakom materničnega 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 kliničnih študijah prejemali 200 mg pembrolizumaba na 3 tedne skupaj s 5 mg aksitiniba dvakrat na dan ali z 20 mg lenvatiniba enkrat na dan, kot je bilo ustrezno. V teh populacijah bolnikov so bili najpogostejši neželeni učinki diareja (58 %), hipertenzija (54 %), hipotiroidizem (46 %), utrujenost (41 %), zmanjšan apetit (40 %), navzea (40 %), artralgija (30 %), bruhanje (28 %), zmanjšanje telesne mase (28 %), disfonija (28 %), bolečine v trebuhu (28 %), proteinurija (27 %), sindrom palmarno-plantarne eritrodizestezije (26 %), izpuščaj (26 %), stomatitis (25 %), zaprtost (25 %), mišično-skeletna bolečina (23 %), glavobol (23 %) in kašelj (21 %). Neželenih učinkov od 3. do 5. stopnje je bilo pri bolnikih z RCC med uporabo pembrolizumaba v kombinaciji z aksitinibom ali lenvatinibom 80 % in med uporabo sunitiniba samega 71 %. Pri bolnicah z EC je bilo neželenih učinkov od 3. do 5. stopnje med uporabo pembrolizumaba v kombinaciji z lenvatinibom 89 % in med uporabo kemoterapije same 73 %. Za celoten seznam neželenih učinkov, prosimo, glejte celoten Povzetek glavnih značilnosti zdravila. Za dodatne informacije o varnosti v primeru uporabe pembrolizumaba v kombinaciji glejte povzetke glavnih značilnosti zdravila za posamezne komponente kombiniranega zdravljenja. Način in režim izdaje zdravila: H – Predpisovanje in izdaja zdravila je le na recept, zdravilo se uporablja samo v bolnišnicah. Imetnik dovoljenja za promet z zdravilom: Merck Sharp & Dohme B.V. , Waarderweg 39, 2031 BN Haarlem, Nizozemska. Merck Sharp & Dohme inovativna zdravila d.o.o., Ameriška ulica 2, 1000 Ljubljana, tel: +386 1/ 520 42 01, fax: +386 1/ 520 43 50; Pripravljeno v Sloveniji, 01/2023; SI-KEY-00501 EXP: 01/2025 Samo za strokovno javnost. H - Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. 5 NA NOVO razvrščenih indikacij:1 . . . (pembrolizumab, MSD)  MELANOM, adjuvantno zdravljenje melanoma v stadiju IIB/IIC2  RAK LEDVIČNIH CELIC, adjuvantno zdravljenje po nefrektomiji2  KOLOREKTALNI RAK, z MSI-H ali dMMR, metastatski, samostojno zdravljenje v 1L2  TROJNO NEGATIVNI RAK DOJK: – v kombinaciji s kemoterapijo za neoadjuvantno, v nadaljevanju samostojno adjuvantno zdravljenje lokalno napredovalega TNRD ali TNRD v zgodnjem stadiju z visokim tveganjem za ponovitev bolezni2 – v kombinaciji s kemoterapijo za zdravljenje lokalno ponovljenega neoperabilnega ali metastatskega TNRD z PD-L1 CPS ≥ 102 Ime zdravila: Polivy 30 mg prašek za koncentrat za raztopino za infundiranje in Polivy 140 mg prašek za koncentrat za raztopino za infundiranje Kakovostna in količinska sestava: Polivy 30 mg: Ena viala s praškom za koncentrat za raztopino za infundiranje vsebuje 30 mg polatuzumaba vedotina. Po rekonstituciji en mililiter vsebuje 20 mg polatuzumaba vedotina. Polivy 140 mg: Ena viala s praškom za koncentrat za raztopino za infundiranje vsebuje 140 mg polatuzumaba vedotina. Po rekonstituciji en mililiter vsebuje 20 mg polatuzumaba vedotina. Polatuzumab vedotin je konjugat protitelesa in zdravila, sestavljen iz antimitotičnega sredstva monometil-avristatina E (MMAE), kovalentno konjugiranega na monoklonsko protitelo, ki je usmerjeno proti CD79b. Terapevtske indikacije: Zdravilo Polivy je v kombinaciji z rituksimabom, ciklofosfamidom, doksorubicinom in prednizonom (R-CHP) indicirano za zdravljenje odraslih bolnikov s predhodno nezdravljenim difuznim velikoceličnim limfomom B (DVCLB). Zdravilo Polivy je v kombinaciji z bendamustinom in rituksimabom indicirano za zdravljenje odraslih bolnikov z difuznim velikoceličnim limfomom B (DVCLB), ki se na prejšnje zdravljenje niso odzvali ali pa se je bolezen pri njih ponovila in niso primerni za presaditev krvotvornih matičnih celic. Odmerjanje in način uporabe: Odmerjanje: Difuzni velikocelični limfom B Predhodno nezdravljeni bolniki: Priporočeni odmerek zdravila Polivy je 1,8 mg/kg v intravenski infuziji na 21 dni v kombinaciji z rituksimabom, ciklofosfamidom, doksorubicinom in prednizonom (R-CHP) v obdobju 6 ciklov. Bolniki s ponovljeno boleznijo ali bolniki, neodzivni na zdravljenje: Priporočeni odmerek zdravila Polivy je 1,8 mg/kg v intravenski infuziji na 21 dni v kombinaciji z bendamustinom in rituksimabom v trajanju 6 ciklov. Način uporabe: Zdravilo Polivy je namenjeno intravenski uporabi. Začetni odmerek zdravila Polivy je treba dati v 90-minutni intravenski infuziji. Bolnike je treba med infundiranjem in vsaj še 90 minut po končani infuziji začetnega odmerka nadzorovati glede reakcij, povezanih z infundiranjem in preobčutljivostnih reakcij. Če je bolnik prejšnje infundiranje dobro prenesel, je mogoče nadaljnje odmerke zdravila Polivy dati v 30-minutni infuziji, bolnika pa je treba nadzirati med infundiranjem in vsaj še 30 minut po končani infuziji. Zdravilo Polivy je treba rekonstituirati in razredčiti z upoštevanjem aseptičnega postopka in pod nadzorom zdravstvenega delavca. Treba ga je aplicirati v intravenski infuziji po namenski infuzijski liniji, opremljeni s sterilnim nepirogenim filtrom, ki malo veže beljakovine in s katetrom. Zdravila Polivy se ne sme aplicirati kot hiter intravenski odmerek ali bolus. Previdnostni ukrepi, potrebni pred ravnanjem z zdravilom ali dajanjem zdravila: Zdravilo Polivy vsebuje citotoksično komponento, ki je kovalentno vezana na monoklonsko protitelo. Upoštevajte ustrezen postopek za ravnanje in odstranjevanje. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Sledljivost: Z namenom izboljšanja sledljivosti bioloških zdravil je treba jasno zabeležiti ime in številko serije uporabljenega zdravila. Mielosupresija: Pri bolnikih, zdravljenih z zdravilom Polivy, so poročali o resni in hudi nevtropeniji in febrilni nevtropeniji že v prvem ciklu zdravljenja. Treba je razmisliti o profilaktični uporabi granulocitnega rastnega dejavnika (G-CSF), saj je bila med kliničnim razvojem zdravila potrebna njegova uporaba. Med uporabo zdravila Polivy se lahko pojavi tudi trombocitopenija ali anemija 3. ali 4. stopnje. Pred vsakim odmerkom zdravila Polivy je treba kontrolirati celotno krvno sliko. Pri bolnikih z nevtropenijo in/ali trombocitopenijo 3. ali 4. stopnje je treba pretehtati opravljanje pogostejših laboratorijskih kontrol in/ali odložitev ali ukinitev uporabe zdravila Polivy. Periferna nevropatija: Pri bolnikih, zdravljenih z zdravilom Polivy, so poročali o periferni nevropatiji že v prvem ciklu zdravljenja; tveganje se povečuje z zaporednimi odmerki. Bolnikom z že obstoječo periferno nevropatijo se nevropatija lahko poslabša. Bolnike je treba nadzorovati glede simptomov periferne nevropatije. Bolnikom, pri katerih se pojavi novonastala periferna nevropatija ali se poslabša obstoječa periferna nevropatija, bo morda treba odmerek zdravila Polivy odložiti, zmanjšati ali uporabo zdravila Polivy ukiniti. Okužbe: Pri bolnikih, zdravljenih z zdravilom Polivy, so poročali o resnih, življenje ogrožajočih ali smrtnih okužbah, vključno z oportunističnimi okužbami. Poročali so tudi o ponovni aktivaciji latentnih okužb. Bolnike je treba med zdravljenjem skrbno nadzorovati glede znakov bakterijskih, glivnih ali virusnih okužb; če se pojavijo znaki in simptomi okužbe, mora bolnik poiskati zdravniško pomoč. Razmisliti je treba o profilaksi z zdravili proti okužbam v celotnem obdobju zdravljenja z zdravilom Polivy. Zdravila Polivy se ob prisotnosti aktivne hude okužbe ne sme aplicirati. Pri bolnikih, pri katerih se pojavijo resne okužbe, je treba ukiniti zdravilo Polivy in sočasno kemoterapijo. Imunizacija: Sočasno z zdravljenjem se bolnikom ne sme dajati živih in živih oslabljenih cepiv. Progresivna multifokalna levkoencefalopatija (PML): Med zdravljenjem z zdravilom Polivy so poročali o pojavu PML. Bolnike je treba skrbno nadzorovati glede novonastalih nevroloških, kognitivnih ali vedenjskih sprememb, ki nakazujejo na PML, oziroma glede poslabšanja takšnih sprememb. V primeru suma na PML je treba uporabo zdravila Polivy in morebitne sočasne kemoterapije odložiti, v primeru potrjene diagnoze pa ukiniti. Sindrom razpada tumorja: Bolniki z velikim tumorskim bremenom in hitro proliferirajočim tumorjem imajo lahko večje tveganje za sindrom razpada tumorja. Pred zdravljenjem z zdravilom Polivy je treba uporabiti ustrezne profilaktične ukrepe v skladu z lokalnimi smernicami. Bolnike je treba med zdravljenjem z zdravilom Polivy skrbno spremljati glede pojava sindroma razpada tumorja. Reakcije, povezane z infundiranjem: Zdravilo Polivy lahko povzroči reakcije, povezane z infundiranjem, vključno s hudimi primeri. Zapoznele reakcije, povezane z infundiranjem, so se pojavile tudi 24 ur po prejetju zdravila Polivy. Pred zdravilom Polivy je treba aplicirati antihistaminik in antipiretik in skrbno spremljati bolnike ves čas infundiranja. Če se pojavi reakcija, povezana z infundiranjem, infundiranje prekinite in ustrezno ravnajte oziroma zdravite. Embrio-fetalna toksičnost: Glede ne mehanizem delovanja in rezultate predkliničnih študij lahko pri nosečnici uporabljeno zdravilo Polivy škoduje plodu. Nosečnicam je treba svetovati glede tveganja za plod. Ženskam v rodni dobi je treba svetovati, naj uporabljajo učinkovito kontracepcijo med zdravljenjem z zdravilom Polivy in še vsaj 9 mesecev po zadnjem odmerku. Moškim, ki imajo partnerke v rodni dobi, je treba svetovati, naj uporabljajo učinkovito kontracepcijo med zdravljenjem z zdravilom Polivy in še vsaj 6 mesecev po zadnjem odmerku. Plodnost: Za moške, ki se bodo zdravili z zdravilom Polivy, je priporočljivo pred zdravljenjem shraniti seme. Starejši bolniki: Klinične študije z zdravilom Polivy niso vključevale zadostnega števila bolnikov, starih 65 let ali več, da bi lahko določili, ali se odzovejo drugače od mlajših bolnikov. Hepatotoksičnost: Pri bolnikih, zdravljenih z zdravilom Polivy, so se pojavili resni primeri hepatotoksičnosti. Predhodna bolezen jeter, izhodiščno zvišani jetrni encimi in uporaba sočasnih zdravil lahko tveganje povečajo. Kontrolirati je treba raven jetrnih encimov in bilirubina. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Namenskih kliničnih študij medsebojnega delovanja zdravil s polatuzumabom vedotinom pri človeku niso izvedli. Pri sočasnem zdravljenju z zaviralcem CYP3A4 je potrebna previdnost. Bolnike, ki sočasno prejemajo močne zaviralce CYP3A4, je treba skrbneje nadzorovati glede znakov toksičnosti. Močni induktorji CYP3A4 lahko zmanjšajo izpostavljenost nekonjugiranemu MMAE. Sočasna uporaba polatuzumaba vedotina ne vpliva na farmakokinetiko rituksimaba, bendamustina, ciklofosfamida in doksorubicina. Neželeni učinki: Seznam neželenih učinkov pri bolnikih, zdravljenih z zdravilom Polivy v kliničnih preskušanjih: Zelo pogosti: pljučnica, okužba zgornjih dihal, febrilna nevtropenija, nevtropenija, trombocitopenija, anemija, levkopenija, hipokaliemija, zmanjšan apetit, periferna nevropatija, kašelj, diareja, navzea, zaprtost, bruhanje, mukozitis, bolečine v trebuhu, alopecija, zvišana telesna temperatura, utrujenost, astenija, zmanjšanje telesne mase in z infundiranjem povezana reakcija. Pogosti: sepsa, okužba s herpesvirusom, okužba s citomegalovirusom, okužba sečil, limfopenija, pancitopenija, hipokalciemija, hipoalbuminemija, omotica, pnevmonitis, dispneja, srbenje, okužbe kože, izpuščaj, suha koža, artralgija, mialgija, periferni edem, mrzlica, zvišanje transaminaz, zvišanje lipaze in hipofosfatemija. Poročanje o domnevnih neželenih učinkih: Poročanje o domnevnih neželenih učinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoča namreč stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da poročajo o katerem koli domnevnem neželenem učinku zdravila na: Javna agencija Republike Slovenije za zdravila in medicinske pripomočke, Sektor za farmakovigilanco, Nacionalni center za farmakovigilanco, Slovenčeva ulica 22, SI-1000 Ljubljana, Tel: +386 (0)8 2000 500, Faks: +386 (0)8 2000 510, e-pošta: h-farmakovigilanca@jazmp.si, spletna stran: www.jazmp.si. Za zagotavljanje sledljivosti zdravila je pomembno, da pri izpolnjevanju obrazca o domnevnih neželenih učinkihzdravila navedete številko serije biološkega zdravila. Režim izdaje zdravila: H. Imetnik dovoljenja za promet: Roche Registration GmbH, Emil-Barell-Strasse 1, 79639 Grenzach-Wyhlen, Nemčija. Verzija: 1.0/22 DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o., Stegne 13G, 1000 Ljubljana M-SI-00000784(v1.0) Pripravljeno: februar 2023 SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA POLIVY Samo za strokovno javnost Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Kako poročati o neželenih učinkih, si poglejte skrajšani povzetek glavnih značilnosti zdravila pod ‚‘Poročanje o domnevnih neželenih učinkih‘‘. ZDRAVILO ZA ZDRAVLJENJE DIFUZNEGA VELIKOCELIČNEGA LIMFOMA B V PRVI LINIJI1 Klinična korist Indikacija POLIVY (polatuzumab vedotin) je v kombinaciji z rituksimabom, ciklofosfamidom, doksorubicinom in prednizonom indiciran za zdravljenje odraslih bolnikov s predhodno nezdravljenim difuznim velikoceličnim limfomom B (DVCLB). manjše tveganje za napredovanje bolezni, ponovitev bolezni ali smrt v primerjavi s kombinacijo R-CHOP.1 DVCLB = difuzni velikocelični limfom B; R-CHOP = rituksimab + ciklofosfamid + doksorubicin + vinkristin + prednizon. Reference: 1. Povzetek glavnih značilnosti zdravila POLIVY. Dostopano (19.12.2022) na https://www.ema.europa.eu/en/documents/product-information/polivy-epar-product-information_sl.pdf VEČ ČASA za več trenutkov, ki štejejo Zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih bolnikov z metastatskim rakom želodca vključno z adenokarcinomom gastro‑ezofagealnega prehoda, ki so bili predhodno že zdravljeni z najmanj dvema sistemskima režimoma zdravljenja za napredovalo bolezen.1 Podaljša celokupno preživetje v 3. liniji zdravljenja bolnikov z mCRC in mGC2,3 Zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (KRR), ki so bili predhodno že zdravljeni ali niso primerni za zdravljenja, ki so na voljo. Ta vključujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF – Vascular Endothelial Growth Factor) in zaviralci receptorjev za epidermalni rastni dejavnik (EGFR – Epidermal Growth Factor Receptor).1 trifluridin/tipiracil Literatura: 1. Povzetek glavnih značilnosti zdravila Lonsurf, december 2020. 2. Mayer R et al. N Engl J Med. 2015;372:1909‑19. 3. Shitara K et al. Lancet Oncol. 2018;19:1437‑1448. Družba Servier ima licenco družbe Taiho za zdravilo Lonsurf®. Pri globalnem razvoju zdravila sodelujeta obe družbi in ga tržita na svojih določenih področjih. Skrajšan povzetek glavnih značilnosti 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 vključujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF ‑ Vascular Endothelial Growth Factor) in zaviralci receptorjev za epidermalni rastni dejavnik (EGFR ‑ Epidermal Growth Factor Receptor). Rak želodca ‑ v monoterapiji za zdravljenje odraslih bolnikov z metastatskim rakom želodca vključno z adenokarcinomom gastro‑ezofagealnega prehoda, ki so bili predhodno že zdravljeni z najmanj dvema sistemskima režimoma zdravljenja za napredovalo bolezen. ODMERJANJE IN NAČIN UPORABE*: Priporočeni začetni odmerek zdravila Lonsurf pri odraslih je 35 mg/m2/odmerek peroralno dvakrat dnevno na 1. do 5. dan in 8. do 12. dan vsakega 28‑dnevnega cikla zdravljenja, najpozneje 1 uro po zaključku jutranjega in večernega obroka (20 mg/m2/odmerek dvakrat dnevno pri bolnikih s hudo ledvično okvaro). Odmerek, izračunan glede na telesno površino, ne sme preseči 80 mg/odmerek. Možne prilagoditve odmerka glede na varnost in prenašanje zdravila: dovoljena so zmanjšanja odmerka na najmanjši odmerek 20 mg/m2 dvakrat dnevno (oz. 15 mg/m2 dvakrat dnevno pri bolnikih s hudo ledvično okvaro). Potem ko je bil odmerek zmanjšan, povečanje ni dovoljeno. KONTRAINDIKACIJE*: Preobčutljivost na učinkovini ali katero koli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI*: Supresija kostnega mozga: Pred uvedbo zdravljenja in po potrebi za spremljanje toksičnosti zdravila, najmanj pred vsakim ciklom zdravljenja, je treba pregledati celotno krvno sliko. Zdravljenja ne smete začeti, če je absolutno število nevtrofilcev < 1,5 x 109/l, če je število trombocitov < 75 x 109/l ali če se je pri bolniku zaradi predhodnih zdravljenj pojavila klinično pomembna nehematološka toksičnost 3. ali 4. stopnje, ki še traja. Bolnike je treba skrbno spremljati zaradi morebitnih okužb, uvesti je treba ustrezne ukrepe, kot je klinično indicirano. Toksičnost za prebavila: Potrebna je uporaba antiemetikov, antidiaroikov ter drugih ukrepov, kot je klinično indicirano. Če je potrebno, prilagodite odmerke. Ledvična okvara: Uporaba zdravila ni priporočljiva pri bolnikih s končno stopnjo ledvične okvare. Bolnike z ledvično okvaro je potrebno med zdravljenjem skrbno spremljati; bolnike z zmerno ali hudo ledvično okvaro je treba zaradi hematološke toksičnosti bolj pogosto spremljati. Jetrna okvara: Uporaba zdravila Lonsurf pri bolnikih z obstoječo zmerno ali hudo jetrno okvaro ni priporočljiva. Proteinurija: Pred začetkom zdravljenja in med njim je priporočljivo spremljanje proteinurije z urinskimi testnimi lističi. Pomožne snovi: Zdravilo vsebuje laktozo. INTERAKCIJE*: 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*. NOSEČNOST IN DOJENJE*: Ni priporočljivo. KONTRACEPCIJA*: Ženske in moški morajo uporabljati zelo učinkovite metode kontracepcije med zdravljenjem in do 6 mesecev po zaključku zdravljenja. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA STROJEV*: Med zdravljenjem se lahko pojavijo utrujenost, omotica ali splošno slabo počutje. NEŽELENI UČINKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, zmanjšan apetit, diareja, navzea, bruhanje, utrujenost. Pogosti: okužba spodnjih dihal, febrilna nevtropenija, limfopenija, hipoalbuminemija, disgevzija, periferna nevropatija, dispneja, bolečina v trebuhu, zaprtje, stomatitis, bolezni ustne votline, hiperbilirubinemija, sindrom palmarne plantarne eritrodisestezije, izpuščaj, alopecija, pruritus, suha koža, proteinurija, pireksija, edem, vnetje sluznice, splošno slabo počutje, zvišanje jetrnih encimov, zvišanje alkalne fosfataze v krvi, zmanjšanje telesne mase. Občasni: septični šok, infekcijski enteritis, pljučnica, okužba žolčevoda, gripa, okužba sečil, gingivitis, herpes zoster, tinea pedis, okužba s kandido, bakterijska okužba, okužba, nevtropenična sepsa, okužba zgornjih dihal, konjunktivitis, bolečina zaradi raka, pancitopenija, granulocitopenija, monocitopenija, eritropenija, levkocitoza, monocitoza, dehidracija, hiperglikemija, hiperkaliemija, hipokaliemija, hipofosfatemija, hipernatriemija, hiponatriemija, hipokalciemija, protin, anksioznost, nespečnost, nevrotoksičnost, disestezija, hiperestezija, hipoestezija, sinkopa, parestezija, pekoč občutek, letargija, omotica, glavobol, zmanjšana ostrina vida, zamegljen vid, diplopija, katarakta, suho oko, vrtoglavica, neugodje v ušesu, angina pektoris, aritmija, palpitacije, embolija, hipertenzija, hipotenzija, vročinski oblivi, pljučna embolija, plevralni izliv, izcedek iz nosu, disfonija, orofaringealna bolečina, epistaksa, kašelj, hemoragični enterokolitis, krvavitev v prebavilih, akutni pankreatitis, ascites, ileus, subileus, kolitis, gastritis, refluksni gastritis, ezofagitis, moteno praznjenje želodca, abdominalna distenzija, analno vnetje, razjede v ustih, dispepsija, gastroezofagealna refluksna bolezen, proktalgija, bukalni polip, krvavitev dlesni, glositis, parodontalna bolezen, bolezen zob, siljenje na bruhanje, flatulenca, slab zadah, hepatotoksičnost, razširitev žolčnih vodov, luščenje kože, urtikarija, preobčutljivostne reakcije na svetlobo, eritem, akne, hiperhidroza, žulj, bolezni nohtov, otekanje sklepov, artralgija, bolečina v kosteh, mialgija, mišično‑skeletna bolečina, mišična oslabelost, mišični krči, bolečina v okončinah, ledvična odpoved, neinfektivni cistitis, motnje mikcije, hematurija, levkociturija, motnje menstruacije, poslabšanje splošnega zdravstvenega stanja, bolečina, občutek spremembe telesne temperature, kseroza, nelagodje, zvišanje kreatinina v krvi, podaljšanje intervala QT na elektrokardiogramu, povečanje mednarodnega umerjenega razmerja (INR), podaljšanje aktiviranega parcialnega tromboplastinskega časa (aPTČ), zvišanje sečnine v krvi, zvišanje laktatne dehidrogenaze v krvi, znižanje celokupnih proteinov, zvišanje C‑reaktivnega proteina, zmanjšan hematokrit. Post-marketinške izkušnje: intersticijska bolezen pljuč. PREVELIKO ODMERJANJE*: Neželeni učinki, o katerih so poročali v povezavi s prevelikim odmerjanjem, so bili v skladu z uveljavljenim varnostnim profilom. Glavni pričakovani zaplet prevelikega odmerjanja je supresija kostnega mozga. FARMAKODINAMIČNE LASTNOSTI*: Farmakoterapevtska skupina: zdravila z delovanjem na novotvorbe, antimetaboliti, oznaka ATC: L01BC59. Zdravilo Lonsurf sestavljata antineoplastični timidinski nukleozidni analog, trifluridin, in zaviralec timidin‑fosforilaze (TPaze), tipiracilijev klorid. Po privzemu v rakave celice timidin‑kinaza fosforilira trifluridin. Ta se v celicah nato presnovi v substrat deoksiribonukleinske kisline (DNA), ki se vgradi neposredno v DNA ter tako preprečuje celično proliferacijo. TPaza hitro razgradi trifluridin in njegova presnova po peroralni uporabi je hitra zaradi učinka prvega prehoda, zato je v zdravilo vključen zaviralec TPaze, tipiracilijev klorid. PAKIRANJE*: 20 filmsko obloženih tablet. NAČIN PREDPISOVANJA IN IZDAJE ZDRAVILA: Rp/Spec. Imetnik dovoljenja za promet: Les Laboratoires Servier, 50, rue Carnot, 92284 Suresnes cedex, Francija. Številka dovoljenja za promet z zdravilom: EU/1/16/1096/001 (Lonsurf 15 mg/6,14 mg), EU/1/16/1096/004 (Lonsurf 20 mg/8,19 mg). Datum zadnje revizije besedila: december 2020. *Pred predpisovanjem preberite celoten povzetek glavnih značilnosti zdravila. Celoten povzetek glavnih značilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., Podmilščakova ulica 24, 1000 Ljubljana, tel: 01 563 48 11, www.servier.si. LON AD1 C1 2022‑23. Samo za strokovno javnost. Datum priprave informacije: oktober 2022. 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). The Editorial Board requires that the paper has not been published or submitted for publication elsewhere; the authors are responsible for all statements in their papers. Accepted cannot be published elsewhere without the written permission of the editors. Submission of the manuscript The manuscript written in English should be submitted to the journal via online submission system Editorial Manager avail- able for this journal at: www.radioloncol.com. 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In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. doi: 10.1038/bjc.1981.71 Chapman S, Nakielny R. A guide to radiological procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology of macrophage. New York: Academic Press; 1976. p. 45-74. Authorization for the use of human subjects or experimental animals When reporting experiments on human subjects, authors should state whether the procedures followed the Helsinki Declaration. Patients have the right to privacy; therefore, the identifying information (patient’s names, hospital unit numbers) should not be published unless it is essential. In such cases the patient’s informed consent for publication is needed, and should appear as an appropriate statement in the article. Institutional approval and Clinical Trial registration number is re- quired. 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Open access Papers are published electronically as open access on https://content.sciendo.com/raon, also papers accepted for publication as E-ahead of print. instructions palbociklib Ustvarjen za mRD HR+/HER2- = pozitiven na estrogenske receptorje in negativen na receptorje humanega epidermalnega rastnega faktorja 2; mRD = metastatski rak dojke Zdravilo IBRANCE že več kot 8 let pomaga do trenutkov, kot so ti.2-5 Zdravilo IBRANCE, ki ga podpirajo obširne izkušnje iz klinične in vsakodnevne dejanske prakse ter dokazana klinična učinkovitost in dobro prenašanje,1,4,6-35 vašim bolnicam pomaga, da najbolje izkoristijo vsak dan.4,5,36,37 DRAGOCENI TRENUTKI BOLNIKA SO MERILO VAŠEGA USPEHA. OMOGOČA JIH ZDRAVILO IBRANCE. ZA ODRASLE BOLNIKE S HR+/HER2- mRD1 PP-IBR-SVN-0011 Datum priprave: maj 2023. Samo za strokovno javnost. Pfizer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J. F. Kennedy, L-1855 Pfizer, podružnica Ljubljana, Letališka cesta 29a, Ljubljana BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA IBRANCE 75 mg, 100 mg, 125 mg trde kapsule(1) IBRANCE 75 mg, 100 mg, 125 mg filmsko obložene tablete(2) Sestava in oblika zdravila: (1) Ena trda kapsula vsebuje 75 mg, 100 mg ali 125 mg palbocikliba in 56 mg, 74 mg ali 93 mg laktoze (v obliki monohidrata). (2) Ena filmsko obložena tableta vsebuje 75 mg, 100 mg ali 125 mg palbocikliba. Indikacije: Zdravljenje lokalno napredovalega ali metastatskega na hormonske receptorje (HR – Hormone Receptors) pozitivnega in na receptorje humanega epidermalnega rastnega faktorja 2 (HER2 – Human Epidermal growth factor Receptor 2) negativnega raka dojk: v kombinaciji z zaviralcem aromataze ali v kombinaciji s fulvestrantom pri ženskah, ki so prejele predhodno endokrino zdravljenje. Pri ženskah v pred- in perimenopavzi je treba endokrino zdravljenje kombinirati z agonistom gonadoliberina. Odmerjanje in način uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. Priporočeni odmerek je 125 mg enkrat na dan 21 zaporednih dni, sledi 7 dni brez zdravljenja (shema 3/1), celotni cikel traja 28 dni. Zdravljenje je treba nadaljevati, dokler ima bolnik od zdravljenja klinično korist ali dokler se ne pojavi nesprejemljiva toksičnost. Pri sočasnem dajanju s palbociklibom je treba zaviralec aromataze dajati v skladu s shemo odmerjanja, ki je navedena v Povzetku glavnih značilnosti zdravila (PGZZ). Pri sočasnem dajanju s palbociklibom je priporočeni odmerek fulvestranta 500 mg intramuskularno 1., 15. in 29. dan ter nato enkrat na mesec, glejte PGZZ za fulvestrant. Prilagajanja odmerkov: Za prilagajanja odmerkov zaradi hematološke toksičnosti glejte preglednico 2, zaradi nehematološke toksičnosti pa preglednico 3 v PGZZju. Pri bolnikih s hudo intersticijsko boleznijo pljuč (ILD)/pnevmonitisom je treba zdravljenje trajno prekiniti. Posebne skupine bolnikov: Starejši: Prilagajanje odmerka ni potrebno. Okvara jeter ali ledvic: Pri bolnikih z blago ali zmerno okvaro jeter ali blago, zmerno ali hudo okvaro ledvic prilagajanje odmerka ni potrebno. Pri bolnikih s hudo okvaro jeter je priporočeni odmerek 75 mg enkrat na dan po shemi 3/1. Pediatrična populacija: Varnost in učinkovitost pri otrocih in mladostnikih, starih < 18 let, nista bili dokazani. Način uporabe: Peroralna uporaba. (1) Jemanje s hrano, priporočljivo z obrokom. (2) Tablete se lahko jemlje s hrano ali brez nje. (1, 2) Ne smemo jemati z grenivko ali grenivkinim sokom. Kapsule oz. tablete zdravila je treba pogoltniti cele. Kontraindikacije: Preobčutljivost na učinkovino ali katerokoli pomožno snov. Uporaba pripravkov s šentjanževko. Posebna opozorila in previdnostni ukrepi: Ženske v pred- in perimenopavzi: Kadar zdravilo uporabljamo v kombinaciji z zaviralcem aromataze je obvezna ovarijska ablacija ali supresija z agonistom gonadoliberina. Hematološke bolezni: Pri nevtropeniji stopnje 3 ali 4 je priporočljiva prekinitev odmerjanja, zmanjšanje odmerka ali odložitev začetka ciklov zdravljenja, bolnike pa je treba ustrezno spremljati. ILD/pnevmonitis: Pri bolnikih se lahko pojavita huda, življenjsko ogrožajoča ali smrtna ILD in/ali pnevmonitis, kadar zdravilo jemljejo v kombinaciji z endokrinim zdravljenjem. Bolnike je treba spremljati glede pljučnih simptomov, ki kažejo na ILD/pnevmonitis (npr. hipoksija, kašelj, dispneja), in pri pojavu novih ali poslabšanju respiratornih simptomov oz. sumu na ILD/pnevmonitis zdravljenje prekiniti. Okužbe: Zdravilo lahko poveča nagnjenost k okužbam, zato je bolnike treba spremljati glede znakov in simptomov okužbe ter jih ustrezno zdraviti. Okvara jeter ali ledvic: Pri bolnikih z zmerno ali hudo okvaro jeter ali ledvic je treba zdravilo uporabljati previdno in skrbno spremljati znake toksičnosti. (1) Laktoza: Vsebuje laktozo. Bolniki z redko dedno intoleranco za galaktozo, odsotnostjo encima laktaze ali malabsorpcijo glukoze-galaktoze ne smejo jemati tega zdravila. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Učinki drugih zdravil na farmakokinetiko palbocikliba: Zaviralci CYP3A: Sočasni uporabi močnih zaviralcev CYP3A, med drugim klaritromicina, indinavirja, itrakonazola, ketokonazola, lopinavirja/ritonavirja, nefazodona, nelfinavirja, posakonazola, sakvinavirja, telaprevirja, telitromicina, vorikonazola in grenivke ali grenivkinega soka, se je treba izogibati. Induktorji CYP3A: Sočasni uporabi močnih induktorjev CYP3A, med drugim karbamazepina, enzalutamida, fenitoina, rifampicina in šentjanževke, se je treba izogibati. Učinek zdravil za zmanjševanje kisline: (1) Če palbociklib zaužijemo s hrano, klinično pomembnega učinka na izpostavljenost palbociklibu ni pričakovati. (2) Klinično pomembnega učinka na izpostavljenost palbociklibu ni pričakovati. Učinki palbocikliba na farmakokinetiko drugih zdravil: Pri sočasni uporabi bo morda treba zmanjšati odmerek občutljivih substratov CYP3A z ozkim terapevtskim indeksom (npr. alfentanil, ciklosporin, dihidroergotamin, ergotamin, everolimus, fentanil, pimozid, kinidin, sirolimus in takrolimus), saj IBRANCE lahko poveča izpostavljenost tem zdravilom. Študije in vitro s prenašalci: Palbociklib lahko zavira prenos, posredovan s P-gp v prebavilih in beljakovino odpornosti pri raku dojk (BCRP). Uporaba palbocikliba z zdravili, ki so substrati P-gp (npr. digoksin, dabigatran, kolhicin) ali BCRP (npr. pravastatin, rosuvastatin, sulfasalazin) lahko poveča njihov terapevtski učinek in neželene učinke. Palbociklib lahko zavira privzemni prenašalec organskih kationov OCT1. Plodnost, nosečnost in dojenje: Med zdravljenjem in vsaj 3 tedne (ženske) oziroma 14 tednov (moški) po koncu zdravljenja je treba uporabljati ustrezne kontracepcijske metode. Zdravila ne uporabljajte pri nosečnicah in ženskah v rodni dobi, ki ne uporabljajo kontracepcije. Bolnice, ki prejemajo palbociklib, ne smejo dojiti. Zdravljenje s palbociklibom lahko ogrozi plodnost pri moških. Pred začetkom zdravljenja naj moški zato razmislijo o hrambi sperme. Vpliv na sposobnost vožnje in upravljanja s stroji: Ima blag vpliv na sposobnost vožnje in upravljanja strojev. Potrebna je previdnost. Neželeni učinki: Zelo pogosti: okužbe, nevtropenija, levkopenija, anemija, trombocitopenija, pomanjkanje teka, stomatitis, navzea, diareja, bruhanje, izpuščaj, alopecija, suha koža, utrujenost, astenija, pireksija, povečane vrednosti ALT/AST. Način in režim izdaje: Rp/Spec - Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Imetnik dovoljenja za promet: Pfizer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. Datum zadnje revizije besedila: 30.03.2023 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. Literatura: 1. Povzetek glavnih značilnosti zdravila Ibrance, 30.3.2023. 2. Beaver JA, et al. Clin Cancer Res. 2015;21(21):4760-4766. 3. George MA, et al. Front Oncol. 2021;11:693104. 4. Rugo H, et al. Breast Cancer Res Treat. 2019;174(3):719-729. 5. Rugo HS, et al. Ann Oncol. 2018;29:888-894. 6. Cristofanilli M, et al. Clin Cancer Res. 2022;28(16):3433-3442. 7. Xu B, et al. Eur J Cancer. 2022;175:236-245. 8. Finn RS, et al. N Engl J Med. 2016;375(20):1925- 1936. 9. Gelmon K, et al. Breast. 2021;59:321-326. 10. Rugo HS, et al. Eur J Cancer. 2018;101:123-133. 11. Turner NC, et al. Ann Oncol. 2018;29(3):669-680. 12. Cristofanilli F, et al. Lancet Oncol. 2016;17(4):425-439. 13. Harbeck N, et al. Future Oncol. 2021;17(16):2107-2122. 14. Goyal RK, et al. Cancer. 2023 Feb 9. Epub ahead of print. 15. Richardson D, et al. Breast Cancer Res Treat. 2021;187(1):113-124. 16. De Michele A, et al. Breast Cancer Res. 2021;23:37. 17. Rugo HS, et al. NPJ Breast Cancer. 2022;8(1):114. 18. Taylor-Stokes G, et al. Breast. 2019;43:22-27. 19. Waller J, et al. J Glob Oncol. 2019;5:JGO1800239. 20. Mycock K, et al. Future Oncol. 2022;18:349-362. 21. Mycock K, et al. Curr Oncol. 2021;28:678-688. 22. Mycock K, et al. Cancer Treat Res Commun. 2022;32:100573. Epub 6 May 2022. 23. Mycock K, et al. Clin Ther. 2022;44(12):1588-1601. 24. Kraus AL, et al. Clin Pharmacol Ther. 2022;111(1):302-309. 25. Rugo HS, et al. SABCS 2022; Abstract P3-01-15. 26. De Laurentiis M, et al. SABCS 2019; Poster P3-11-25. 27. Caillet P. ASCO 2021; Oral presentation 1012. 28. Tripathy D, et al. ESMO 2022; Poster 251P. 29. Karuturi MS, et al. ESMO BC 2022; Poster 190P. 30. Blum J, et al. SABCS 2021; Abstract P1-18-29. 31. Finn R, et al. ASCO 2022; Oral presentation. 32. Finn R, et al. Oncologist. 2021;26:e749-e755. 33. Diéras V, et al. Oncologist. 2019;24(12):1514-1525. 34. Verma S, et al. Oncologist. 2016;21(10):1165-1175. 35. Harbeck N, et al. Ann Oncol. 2016;27(6):1047-1054. 36. Rocque G, et al. ESMO 2022; Poster 266P. 37. Karuturi M, et al. SABCS 2021; Poster P1-18-25. R a d io lo g y a n d O n c o lo g y I V o lu m e 5 7 I N u m b e r 2 I P a g e s 1 4 1 -2 7 8 I J u n e 2 0 2 3 june 2023 vol.57 no.2