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 1 I P a g e s 1 -1 4 0 I M a r c h 2 0 2 3 march 2023 vol.57 no.1 Radiol Oncol 2023; 57(1): A. Mach 2023 Vol. 57 No. 1 Pages 1-140 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(1): B. Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217 SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369 Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. 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Indexed and abstracted by: • Baidu Scholar • Case • Chemical Abstracts Service (CAS) - CAplus • Chemical Abstracts Service (CAS) - SciFinder • CNKI Scholar (China National Knowledge Infrastructure) • CNPIEC - cnpLINKer • Dimensions • DOAJ (Directory of Open Access Journals) • EBSCO (relevant databases) • EBSCO Discovery Service • Embase • Genamics JournalSeek • Google Scholar • Japan Science and Technology Agency (JST) • J-Gate • Journal Citation Reports/Science Edition • JournalGuide • JournalTOCs • KESLI-NDSL (Korean National Discovery for Science Leaders) • Medline • Meta • Microsoft Academic • Naviga (Softweco) • Primo Central (ExLibris) • ProQuest (relevant databases) • Publons • PubMed • PubMed Central • PubsHub • QOAM (Quality Open Access Market) • ReadCube • Reaxys • SCImago (SJR) • SCOPUS • Sherpa/RoMEO • Summon (Serials Solutions/ProQuest) • TDNet • Ulrich's Periodicals Directory/ulrichsweb • WanFang Data • Web of Science - Current Contents/Clinical Medicine • Web of Science - Science Citation Index Expanded • WorldCat (OCLC) This journal is printed on acid- free paper On the web: ISSN 1581-3207 https://content.sciendo.com/raon http://www.radioloncol.com Radiol Oncol 2023; 57(1): C. review 1 Oral verrucous carcinoma: a diagnostic and therapeutic challenge Nejc Kristofelc, Nina Zidar, Primoz Strojan 12 Molecular profiling of rare thymoma using next-generation sequencing: meta-analysis Jelena Kostic Peric, Andja Cirkovic, Sanja Srzentic Drazilov, Natalija Samardzic, Vesna Skodric Trifunovic, Dragana Jovanovic, Sonja Pavlovic radiology 20 Multimodality CT imaging contributes to improving the diagnostic accuracy of solitary pulmonary nodules: a multi-institutional and prospective study Gaowu Yan, Hongwei Li, Xiaoping Fan, Jiantao Deng, Jing Yan, Fei Qiao, Gaowen Yan, Tao Liu, Jiankang Chen, Lei Wang, Yang Yang, Yong Li, Linwei Zhao, Anup Bhetuwal, Morgan A. McClure, Na Li, Chen Peng 35 Ultrasonography of peripheral nerve tumours: a case series Simon Podnar 42 Effects of dynamic contrast enhancement on transition zone prostate cancer in Prostate Imaging Reporting and Data System Version 2.1 Jiahui Zhang, Lili Xu, Gumuyang Zhang, Xiaoxiao Zhang, Xin Bai, Hao Sun, Zhengyu Jin experimental oncology 51 Pancreatic islets implanted in an irreversible electroporation generated extracellular matrix in the liver Yanfang Zhang, Yanpeng Lv, Yunlong Wang, Tammy T Chang, Boris Rubinsky clinical oncology 59 Estimating exposure to extremely low frequency magnetic fields near high-voltage power lines and assessment of possible increased cancer risk among Slovenian children and adolescents Tina Zagar, Blaz Valic, Tadej Kotnik, Sara Korat, Sonja Tomsic, Vesna Zadnik, Peter Gajsek 70 Comparison of CalliSpheres® microspheres drug-eluting beads and conventional transarterial chemoembolization in hepatocellular carcinoma patients: a randomized controlled trial Zhongxing Shi, Dongqing Wang, Tanrong Kang, Ru Yi, Liming Cui, Huijie Jiang contents contents Radiol Oncol 2023; 57(1): D. 80 Does concurrent gynaecological surgery affect infectious complications rate after mastectomy with implant-based reconstruction? Nina Pislar, Barbara Peric, Uros Ahcan, Romi Cencelj-Arnez, Janez Zgajnar, Andraz Perhavec 86 The spine and carina as a surrogate for target registration in cone-beam CT imaging verification in locally advanced lung cancer radiotherapy Jasna But-Hadzic, Karmen Strljic, Valerija Zager Marcius 95 Effects of gold fiducial marker implantation on tumor control and toxicity in external beam radiotherapy of prostate cancer Matthias Moll, Magdalena Weiß, Vladimir Stanisav, Alexandru Zaharie, Gregor Goldner 103 The five-year KRAS, NRAS and BRAF analysis results and treatment patterns in daily clinical practice in Slovenia in 1st line treatment of metastatic colorectal (mCRC) patients with RAS wild-type tumour (wtRAS) – a real- life data report 2013–2018 Tanja Mesti, Martina Rebersek, Janja Ocvirk 110 Association of OPRM1, MIR23B, and MIR107 genetic variability with acute pain, chronic pain and adverse effects after postoperative tramadol and paracetamol treatment in breast cancer Zala Vidic, Katja Goricar, Branka Strazisar, Nikola Besic, Vita Dolzan 121 Treatment of vulvar cancer recurrences with electrochemotherapy – a detailed analysis of possible causes for unsuccessful treatment Gregor Vivod, Tanja Jesenko, Gorana Gasljevic, Nina Kovacevic, Masa Bosnjak, Gregor Sersa, Sebastjan Merlo, Maja Cemazar 127 CT-guided 125I brachytherapy for hepatocellular carcinoma in high- risk locations after transarterial chemoembolization combined with microwave ablation: a propensity score-matched study Zixiong Chen, Xiaobo Fu, Zhenkang Qiu, Maoyuan Mu, Weiwei Jiang, Guisong Wang, Zhihui Zhong, Han Qi, Fei Gao erratum 140 Similar complication rates for irreversible electroporation and thermal ablation in patients with hepatocellular tumors Niklas Verloh, Isabel Jensch, Lukas Luerken, Michael Haimerl, Marco Dollinger, Philipp Renner, Philipp Wiggermann, Jens Martin Werner, Florian Zeman, Christian Stroszczynski, Lukas Philipp Beyer I slovenian abstracts contents Radiol Oncol 2023; 57(1): 1-11. doi: 10.2478/raon-2023-0015 1 review Oral verrucous carcinoma: a diagnostic and therapeutic challenge Nejc Kristofelc1, Nina Zidar2, Primoz Strojan3,4 1 Department of Otorhinolaryngology, General Hospital Dr. Franc Derganc Nova Gorica, Šempeter pri Gorici, Slovenia 2 Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Radiation Oncology, Institute of Oncology, Ljubljana, Slovenia 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(1): 1-11. Received 17 December 2022 Accepted 29 January 2023 Correspondence to: Nejc Krištofelc, M.D., Department of Otorhinolaryngology, General Hospital Dr. Franc Derganc Nova Gorica, Ulica padlih borcev 13 A, SI-5290 Šempeter pri Gorici, Slovenia. E-mail: nejc.kristofelc@bolnisnica-go.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. Verrucous carcinoma is a low-grade variant of squamous cell carcinoma with specific morphologic, cytokinetic and clinical features. Despite low mitotic activity and slow growth, it can infiltrate adjacent tissues in advanced stages but does not metastasize. The most frequently affected site is the oral cavity. The following article provides latest updates in the etiology, clinical presentation, diagnostics and treatment options in oral verrucous car- cinoma and discusses the existing dilemmas linked to this unique malignancy. Conclusions. Oral verrucous carcinoma must be differentiated from conventional squamous cell carcinoma due to its less aggressive behaviour with a more favourable prognosis. Close communication between clinician and patholo- gist is mandatory for making a correct diagnosis. Primary surgery with negative surgical margins seems to be the most successful treatment. However, management recommendations are not uniform since they are mostly based on case reports and small retrospective case series. Prospective and pooled multi-institutional studies are therefore needed. Key words: verrucous carcinoma; oral verrucous carcinoma; squamous cell carcinoma; diagnostics; differential diagnosis; treatment Introduction Head and neck cancer is the world’s seventh most common cancer with over 870,000 new cases in 2020. Lip and oral cavity malignancies accounted for almost half of them.1 More than 90% of oral cavity cancers arises from squamous epitheli- um.2 Verrucous carcinoma is a low-grade variant of squamous cell carcinoma (SCC) with specific morphologic, cytokinetic and clinical features.3 It is a locally aggressive tumour and does not me- tastasize to regional lymph nodes or to distant sites.4 In 1941, Friedell and Rosenthal first report- ed verrucous papillary lesions on the buccal mu- cosa in eight tobacco chewers.5 Seven years later, Ackerman described histopathologic and clinical features of this neoplasm. He defined it as a dis- tinct clinicopathologic entity and introduced a term »verrucous carcinoma«.6 Verrucous carcinoma most often arises on mu- cous membranes of the head and neck region with the oral cavity most commonly involved, particu- larly buccal mucosa, gum and tongue.3 Oral ver- rucous carcinoma accounts for 0.57-16.08% of oral squamous cell carcinoma (SCC)7–9 and is predomi- nantly seen in males with the reported mean age at diagnosis between 49 and 69.5 years.9–11 In a study by Koch et al., glottic larynx was the most frequently affected nonoral site.3 Other reported locations in the head and neck region affected by Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma2 verrucous carcinoma are nasal cavity, paranasal sinuses, nasopharynx, oesophagus and temporal bone.12-14 Verrucous carcinoma on the skin and mu- cosa of the anogenital region and extremities are described in the literature as well.15,16 Etiology The etiopathogenesis of oral verrucous carcinoma is not completely understood. As in conventional oral SCC, there is a strong association with alcohol consumption and inhaled as well as chewing to- bacco use. Other irritants to the oral mucosa such as betel nut chewing, poor oral hygiene, a poorly fit- ting dental prosthesis and earlier mucosal injuries or scars have also been described as risk factors in the development of oral verrucous carcinoma.9,17–19 There is growing evidence that oral microbiota and its imbalances may play a role in the etiology of oral cancers through activation of smoking and alcohol related carcinogens locally and chronic inflammation systemically.20 Human papillomavi- ruses (HPVs) have been considered as a possible etiologic factor in verrucous carcinoma, with the reported prevalence of HPV in verrucous carcino- ma ranging from 0% to 100%.21–23 However, using highly sensitive and specific molecular methods, it has been shown that HPVs are not associated with the etiopathogenesis of verrucous carcinoma of the head and neck. Furthermore, no evidence of tran- scriptionally active high-risk α-HPV was found in verrucous carcinoma by real-time polymerase chain reaction (RT-PCR) for HPV E6/E7 messenger ribonucleic acid (mRNA). It appears that verrucous carcinoma of the head and neck is not associated with infection with HPV.4,24,25 Clinical presentation Verrucous carcinoma is characterized by low mi- totic activity reflecting in slow growth7; hence it can take several years to reach the size that causes symptoms. Patients may report oral discomfort, difficulty chewing or swallowing, and bad breath. Pain usually indicates tumour invasion into the surrounding structures.26,27 Oral verrucous carcinoma typically appears as an exophytic broad-based lesion with a cauliflow- er-like warty surface28 as presented in Figure 1. Despite its slow growth, it can reach a significant size and infiltrate adjacent tissues such as muscles and bone.3 However, even when locally advanced, oral verrucous carcinoma has no tendency to me- tastasize to regional lymph nodes and distant sites.4 Cervical lymphadenopathy is commonly seen at initial clinical or radiological examination and is mostly considered reactive secondary to in- flammation at the tumour-stromal interface.3 Initial reports of neck metastasis in verrucous carcinoma were later attributed to incorrect patho- logic diagnosis or to a presence of foci of convention- al SCC of varying degree of differentiation within a verrucous carcinoma. The so-called hybrid ver- rucous carcinoma was first described by Batsakis et al. in 1982 in three verrucous lesions of the lar- ynx.29 Medina et al. later reported coexistence of verrucous carcinoma and conventional SCC in 20% of 104 patients with oral verrucous carcinoma.30 In contrast to the classic, histologically uniform ver- rucous carcinoma, a hybrid verrucous carcinoma is capable of metastasizing and must therefore be managed as a more common and aggressive con- ventional SCC.31 However, it is not possible to dif- ferentiate these lesions at clinical examination due to similar appearance. Moreover, examination of small tumour samples obtained with biopsy could be misleading as an invasive component is often missed at sampling.32 Gokavarapu et al. reported that 51% of cases preoperatively diagnosed as oral FIGURE 1. Verrucous carcinoma of the right buccal mucosa (clinical stage T2N0M0) in an 81-year-old male patient. He presented with a whitish exophytic tumour mass of the inner side of the right cheek and without suspicious lymph nodes on the neck. The lesion was noticed by the patient a month before initial examination, and it occasionally hurt, but he had no problems feeding. Due to associated diseases, he was treated with radiotherapy (55 Gy, 2.2 Gy/ fraction) and concurrent intravenous chemotherapy (vinblastine 2 mg, day 1; methotrexate 50 mg, day 2; bleomycin 15 mg, days 2 and 3). The patient died of injury 5.5 years after completion of treatment for verrucous carcinoma with no evidence of malignant disease in oral cavity. Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma 3 verrucous carcinoma or its benign precursors were actually hybrid lesions.33 Although multiple biop- sies from different areas of the tumour might be helpful to identify invasive component, surgical ex- cision and histopathological examination of whole resection specimen is needed for definitive diagno- sis.32 If hybrid verrucous carcinoma is recognized, the pathologist should quantitate each component of the tumour, define the degree of differentiation of the conventional SCC component and comment on depth of the tumour invasion, potential pres- ence of lymphatic or perineural invasion and the adequacy of the resection margins. These features help the clinicians to decide about adjuvant treat- ment options.31 Various mucosal abnormalities including ver- rucous hyperplasia and dysplasia are frequently found adjacent to the oral verrucous carcinoma supporting the view that verrucous carcinoma de- velops from precursor lesions.28 Patients with oral verrucous carcinoma are also at high risk of de- veloping metachronous second primary tumours. This can be explained with the concept of »field cancerization« proposed by Slaughter et al. who postulated that prolonged exposure of the upper aerodigestive tract to carcinogens leads to genomic instability even beyond the area of clinically and histopathologically evident mucosal changes.34 Diagnostics and differential diagnosis Diagnosis of oral verrucous carcinoma is based on a patient’s history, clinical manifestation and his- topathologic features of the lesion. However, estab- lishing the correct diagnosis is often difficult due to other oral lesions with often similar verrucous presentation and/or insufficient biopsy specimen as well.9 Medical history should include informa- tion on the duration of the growth of the lesion and potential etiologic factors (smoking, alcohol abuse). Computed tomography (CT) and/or magnetic reso- nance imaging (MRI) is helpful to determine local extent of the lesion with potential invasion to sur- rounding structures and to exclude tumour spread to regional lymph nodes.35 The clinician’s impression of a malignant lesion frequently does not match its benign nature de- scribed in the histopathology report. Therefore, bi- opsies are often repeated, which can significantly delay the start of a treatment.10,36 Close communi- cation between the clinician and the pathologist is therefore of the utmost importance.33 Microscopically, verrucous carcinoma con- sists of filiform projections lined by thick, well- differentiated keratinized squamous epithelium, composed of one to a few layers of basal cells, and multiplied, voluminous spinous cells lacking cy- tological atypia. It invades the underlying stroma with a well-defined, pushing margin.37 When oral verrucous carcinoma is highly suspicious by clini- cal appearance, it is recommended that the lesion is surgically excised if not too extensive.38 Lesions in oral cavity with a verrucous appear- ance may belong to a broad spectrum, extending from verrucous hyperplasia, proliferative verru- cous leukoplakia, oral squamous papilloma, oral verrucous carcinoma, hybrid oral verrucous carci- noma to conventional oral SCC with an exophytic growth pattern (Figure 2). It is difficult to distin- guish them clinically from each other; they may also coexist.39 Oral verrucous hyperplasia resembles oral ver- rucous carcinoma both clinically and histopatho- logically. It presents as a white elevated mucosal plaque or mass with exophytic verrucous surface. In oral verrucous hyperplasia and oral verrucous carcinoma, hyperplastic epithelium is superficial to adjacent normal mucosa, but in oral verrucous carcinoma, broad epithelial processes also extend deeper, exhibiting a pushing-border invasion into the underlying connective tissue but the basement membrane remains intact.40 Therefore, it was sug- gested that oral verrucous carcinoma can be best differentiated from oral verrucous hyperplasia with biopsies taken from the deep portion and the margin of the tumour where adjacent normal mucosa is evident to compare.33 Oral verrucous hyperplasia is an irreversible precancerous lesion that may transform into oral verrucous carcinoma. Wang et al. reported a 10% of malignant transfor- mation rate in their series of 60 oral verrucous hyperplasia cases. Thus, once diagnosed, oral ver- rucous hyperplasia should be treated as oral ver- rucous carcinoma.41 Proliferative verrucous leukoplakia is an ag- gressive form of nonhomogeneous multifocal oral leukoplakia characterized by a progressive clinical course with changing clinical and histopathologic features. It is more commonly seen among elderly women. Although etiology of proliferative verru- cous leukoplakia remains unclear, it seems that consumption of tobacco and alcohol does not play a role.42 Proliferative verrucous leukoplakia usu- ally begins as a single white mucosal plaque that eventually becomes multifocal with exophytic, verrucous or erythematous appearance. Its de- Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma4 scription includes a histopathological continuum ranging from benign hyperkeratosis to lesions with increasing degree of dysplasia. Therefore, proliferative verrucous leukoplakia has no specific histological features and microscopic findings de- pend on the histopathologic stage of proliferative verrucous leukoplakia.43 According to the World Health Organization, proliferative verrucous leu- koplakia is a potentially malignant disorder with the highest rate of malignant transformation either to oral verrucous carcinoma or conventional oral SCC.42 In addition, several authors proposed dif- ferent clinical and histopathological criteria for di- agnosing proliferative verrucous leukoplakia.44,45 In the meta-analysis by Palaia et al. which included 22 studies with a total of 699 proliferative verru- cous leukoplakia patients, a malignant transfor- mation rate of proliferative verrucous leukoplakia was 45.8%.46 Thus, once proliferative verrucous leukoplakia is confirmed, active therapy should be undertaken, such as surgery, laser ablation, photo- dynamic therapy or radiotherapy.47 However, pro- liferative verrucous leukoplakia responds poorly to various treatment modalities and its recurrence rate is high, even after surgical removal.48 Squamous cell papilloma (SCP) in the oral cav- ity appears as a pink to white mucosal exophytic lesion with a warty or granular surface. It is most commonly caused by HPV type 6 and type 11, tends to progress slowly and has a very low risk of becoming malignant. SCP is possible to differenti- ate from oral verrucous carcinoma microscopical- ly. In contrast to oral verrucous carcinoma which shows epithelial processes with downgrowth into the underlying connective tissue, SCP presents with long, thin and finger-like projections, extend- ing above the mucosal surface. Each of these pro- jections is lined by stratified squamous epithelium and contains a central connective tissue core.49 Conventional oral SCC most commonly presents as an ulcerated mucosal lesion with necrotic cen- tral area, surrounded by irregular raised and indu- rated borders. However, an exophytic growth with a smooth, ulcerated or verrucous surface may also be seen.50,51 In comparison to oral verrucous carci- noma, conventional oral SCC is histopathologically marked by a greater degree of atypia and mitotic activity of the tumour cells and invasion beyond the basement membrane.31 It grows more rapidly, frequently metastasizes to the regional and distant sites and has a worse prognosis.8 Oral verrucous carcinoma with foci of conventional SCC can be found at histopathological examination, which dic- tates treatment choices and prognosis.31 FIGURE 2. Histopathology images of oral verrucous lesions. Squamous cell papilloma (A) exophytic lesion, composed of finger-like projections, lined by non-keratinizing stratified squamous epithelium and a central connective tissue core. Verrucous hyperplasia (B) exophytic lesion, composed of hyperplastic keratinizing squamous epithelium with no invasion into the underlying stroma. Verrucous carcinoma (C) exophytic tumour, resembling verrucous hyperplasia, but with invasive growth, consisting of broad epithelial islands and processes, with no atypia, exhibiting a pushing-border into the underlying stroma. A B C Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma 5 Molecular biomarkers The development of oral verrucous carcinoma is modulated by genetic predisposition and environ- mental influences resulting in a wide range of ge- netic and epigenetic alterations that can be detect- ed by various tumour markers. Molecular mecha- nisms of oral verrucous carcinoma are therefore increasingly being investigated. Although many different molecules associated with diagnosis, tu- mour progression and prognosis of oral verrucous carcinoma have been proposed, a reliable and ef- fective biomarker has still not been identified.35 Genetic studies have shown that several genes are differently expressed between oral verrucous carcinoma and oral SCC.52 Most investigated mark- ers in carcinogenesis of oral verrucous carcinoma are p5353,54, Ki-6753,55,56, cyclin-B156,57 and cyclin- D1.58 Except for Ki-67, their expression levels were significantly higher in conventional oral SCC than in oral verrucous carcinoma. Tumour suppression markers p21 and p27 may not be of much diagnos- tic use in distinguishing oral verrucous carcinoma from oral verrucous hyperplasia 59 and oral SCC.54,60 Components of extracellular matrix and basement membrane play an important role in tumour inva- sion and metastasis. Oral verrucous carcinoma is associated with lower expression of matrix metal- loproteinase 9 (MMP-9)53 and higher expression of laminin61 in comparison to oral SCC. Laminin and type IV collagen are good markers for base- ment membrane integrity and their discontinuity is more evident in severe oral epithelial dysplasia than in verrucous carcinoma.61 Among cell surface proteins, high level of expression of glucose trans- porter 1 (GLUT-1) in both oral SCC and oral ver- rucous carcinoma could differentiate them from oral epithelial dysplasia.62 Oral SCC could be dis- tinguished from oral verrucous carcinoma based on a higher density of CD68 (marking tumour associated macrophages) and CD31 (marking mi- crovessel density) found in immunohistochemical studies.63 Regarding cytoskeletal proteins, CK20 is highly expressed in oral verrucous carcinoma and oral SCC but not in benign squamous lesions64, and CD34 along with α-smooth muscle actin (α-SMA) seem to be helpful in the diagnosis of oral verrucous hyperplasia.65 Different expression of desmosomal proteins (up-regulation of plakophi- lin 1, desmoglein 2, desmoglein 3, desmoplakin), microRNA (miRNA) molecules (up-regulation of miRNA-203, down-regulation of miRNA-125a-5p, miRNA-125b) and proteins (down-regulation of p63) in verrucous carcinoma is useful in differen- tiation from conventional SCC and detecting foci of SCC in hybrid verrucous carcinoma as well.37,66 Treatment Due to the rarity of oral verrucous carcinoma, treatment recommendations found in the litera- ture is mostly based on case reports and small retrospective case series, and are consequently not uniform. The treatment modalities available include surgery, radiotherapy, chemotherapy, or combinations thereof. Surgery Wide surgical excision is usually considered the treatment of choice because of the wide spectrum of reconstruction possibilities of the resulting tis- sue defect in the oral cavity with good functional results, and encouraging locoregional control and survival rates.17,38,67 However, there is ongoing de- bate about the optimal width of surgical margins and the need for elective neck dissection (END). Similar to conventional oral SCC, clinical surgical margin of 10–15 mm and histological margin of at least 5 mm are still generally considered sufficient to not increase the risk of local recurrence of oral verrucous carcinoma68-70, although no worse out- comes were reported in patients with close histo- logical margins (i.e. less than 5 mm) who did not receive adjuvant radiotherapy.10 Since histological- ly pure oral verrucous carcinoma does not metas- tasize, END is usually not performed during pri- mary surgery but is indicated in hybrid oral ver- rucous carcinoma and when microvascular flap is used for reconstruction of tumour defect.10,67,68 Some authors advocate a selective supraomo- hyoid neck dissection (neck levels I–III) also in patients with advanced primary tumour stages (cT3–4) and/or clinically overt lymphadenopathy. However, in several studies, no patient with END had histologically proven nodal metastasis.10,17,38,71 Radiotherapy Oral verrucous carcinoma is thought to be less sensitive to radiotherapy than conventional oral SCC.72,73 Radiotherapy targets DNA in rapidly di- viding cells, whereas studies on cytokinetic char- acteristics of verrucous carcinoma have shown that only low proportion of tumour cells are in S-phase compartment of the cell cycle during which DNA is synthesized, corresponding to a low mitotic ac- Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma6 tivity of this tumour and reduced susceptibility to irradiation.29 Mohan et al. demonstrated that pa- tients with oral verrucous carcinoma who received postoperative radiotherapy trended toward worse disease-specific survival (DSS) than those with oral SCC, suggesting relative radioresistance of oral verrucous carcinoma.7 Studies reporting local control rate and survival rate for upfront surgery and primary radiotherapy are summarized in Table 1 and Table 2. However, a fair comparison between oncologi- cal results of surgery and radiotherapy is diffi- cult to make due to obvious lack of well-designed prospective studies or even comparisons. In most series, patients were recruited over a longer time period which resulted in suboptimal treatments in at least part of these patients. Thus, local control and survival rates of irradiated patients must be interpreted with caution and understanding that irradiation techniques and fractionation schemes used in the past changed significantly over time. Nevertheless, radiotherapy is an acceptable alter- native to surgery for patients who refuse proposed operation or are medically unfit for major surgery as well as in whom surgery would cause an im- portant functional and/or cosmetic impairment.77 In cases of radiotherapy failure, surgical salvage remains an option.78 In the past, radiotherapy was burdened with the phenomenon of anaplastic transformation which assumed the possibility of conversion of verrucous carcinoma to a less-differentiated SCC after irradiation.79 In older literature, its incidence has been reported to be as high as 30%.80 Recently, different authors questioned the concept of this phenomenon and raised the possibility of hybrid lesions containing foci of conventional SCC that had been missed at the initial biopsy and not con- trolled by irradiation, resulting in tumour recur- rence.3,79,81 This hypothesis is further justified by the reports of anaplastic transformation following primary surgery as well, probably due to the same reason as in the case of radiotherapy, i.e. an incor- rect histopathologic diagnosis.73 Postoperative radiotherapy The benefit of postoperative radiotherapy (PORT) in oral verrucous carcinoma is controversial. The decision about PORT usually follows recommen- dations for patients with conventional oral SCC (i.e. positive or close surgical margins, pT3–T4 primary tumour, perineural and lymphovascular invasion).67,82 Analysing the SEER database, how- ever, Mohan et al. demonstrated a statistically significant improvement in DSS in patients solely operated compared to those receiving surgery and PORT.7 In a recent retrospective cohort study of the National Cancer Database (NCDB), Naik et al. showed that positive surgical margins were as- sociated with significantly worse overall survival (OS) (hazard ratio [HR] 2.85, P = 0.006).82 However, TABLE 1. Primary surgery in the treatment of oral verrucous carcinoma - review of the literature series Authors and study year Number of patients Local control (%) Survival Follow-up time Kraus and Perezmesa, 196674 64 55 (85.9) N.S. N.S. Medina et al, 198430 90 74 (82.2) N.S. At least 2 years Jyothirmayi et al, 19978 11 N.S. 5-year DFS 68% Median 56 months (range 7–110) Koch et al, 20013 484 N.S. 5-year RSR 85.7% N.S. Kang et al, 200338 38 38 (100) at 3 years 3-year OSR 94.7% Median 37.5 months (range 13–76) Walvekar et al, 200917 101 80 (79.2) 5-year DFS 77.6% Median 4.61 years (range 0.5–14.3) Huang et al, 200967 39 38 (97.4) 5-year CSS 89.1% Median 90 months (range, 13–171) Candau-Alvarez et al, 201468 13 12 (92.3) OSR 92.9% for a mean follow- up of 2 years Mean 24.8 months (range 6–53) Franklyn et al, 201710 22 21 (95.5) (recurrence in a patient with hybrid OVC) N.S. Median 24 months CSS = cancer specific survival; DFS = disease free survival; N.S. = not specified; OSR = overall survival rate; OVC = oral verrucous carcinoma; RSR = relative survival rate Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma 7 in those patients the use of PORT showed no OS benefit (HR 3.12, P = 0.072). These findings suggest that the role of PORT is limited in oral verrucous carcinoma, favouring surgical re-resection, when feasible, over adjuvant radiotherapy in patients with adverse pathologic features or clinically overt residual tumour after surgery.82 Chemotherapy Experiences with chemotherapy, alone or in com- bination with other modalities, in oral verrucous carcinoma are scarce.83 Chemotherapy can be im- plemented in a neo-adjuvant setting to reduce tu- mour size before subsequent surgery, which is ex- pected to be less extensive and mutilating, result- ing in better functional and cosmetic outcome.84 It can also be used as a salvage treatment for patients with recurrent disease and to palliate symptoms in advanced tumours not suitable for aggressive radical treatment.85,86 Although the data on the use of older chemotherapy drugs are available in the literature, there is no information on the use of modern drugs (targeted agents, check-point in- hibitors) in oral verrucous carcinoma. Encouraging results were reported by Wu et al. using intra-arterial methotrexate infusion as a primary therapy in 15 patients with oral verru- cous carcinoma. Despite locally advanced T3–4 tumours in eight of these patients, a complete tu- mour remission was observed in all patients who were without disease recurrence at a mean follow- up of 42 months.83 Karkazoglou et al. reported on 12 oral verrucous carcinoma patients who had also been treated with methotrexate, given by various routes and in different doses, because of either the extent of the tumour or poor general condition of the patient. Only one patient failed to respond. Authors concluded that methotrexate reduced morbidity and improved quality of life with mini- mal and reversible toxicity.87 Preliminary observa- tions in two elderly patients with locally advanced oral verrucous carcinoma showed that single cy- cle of oral fluoropyrimidine capecitabine induced rapid and clinically significant response with near complete resolution of oral lesions within 3 weeks of initiating therapy. A durable partial response was seen at 6 months and 1 year and was associ- ated with significant improvement in life quality with acceptable toxicity profile.88 Chemoradiotherapy In addition, chemotherapy can be given simultane- ously with radiotherapy. In a group of 12 patients with previously untreated verrucous carcinoma of different head and neck mucosal sites, concurrent chemoradiotherapy with at least two cycles of in- travenous vinblastine, methotrexate and bleomy- cin and radiotherapy dose of 44–70 Gy (median 65.2 Gy) resulted in local control (median follow- up of 3.4 years) in 11 patients, nine of whom had advanced T3–4 tumours.85 Authors concluded that concomitant chemotherapy seems to successfully compensate lower effectiveness of radiotherapy in verrucous carcinoma and even allows reduction of TABLE 2. Primary radiotherapy in the treatment of oral verrucous carcinoma - review of the literature series Authors and study year Number of patients Local control rate with primary radiotherapy (%) Surgical salvage Local control rate with primary radiotherapy and salvage surgery (%) Survival Follow-up time Kraus and Perezmesa, 196674 13 0 (0) 8/13 7 (53.8) N.S. N.S. Memula et al, 198075 32 19 (59.4) 6/13 25 (78.1) 5-year DFS 31% N.S. Medina et al, 198430 12 7 (58.3) 3/5 10 (83.3) N.S. At least 2 years Nair et al, 198876 50 22 (44) at 3 years 4/28 N.S. 3-year DFS 44% At least 3 years Vidysagar et al, 199236 107 55 (51.4) (residual disease in 19 patients, recurrence in 33 patients) 20/52 N.S. 5-year DFS 49% Range 6–60 months Jyothirmayi et al, 19978 42 16 (38.1) (residual disease in 10 patients, recurrence in 16 patients) 9/26 N.S. 5-year DFS 66% Median 56 months (range 7–110) Koch et al, 20013 33 N.S. N.S. N.S. 5-year RSR 41.8% N.S. DFS = disease free survival; N.S. = not specified; OVC = oral verrucous carcinoma; RSR = relative survival rate Radiol Oncol 2023; 57(1): 1-11. Kristofelc N et al. / Review of oral verrucous carcinoma8 radiation dose bellow the standard 66–70 Gy, and, therefore, alleviates its toxicity and contributes to organ sparing.84 Promising results of chemoradio- therapy were also reported by Yoshimura et al. us- ing 5-fluorouracil and its analogues.89 Non-surgical techniques Non-surgical methods are well established treat- ment modalities in low risk nonmelanoma skin cancers and to some extent in oral benign and precancerous lesions but there are only a few case reports regarding their use in oral verrucous car- cinoma. Cryotherapy acts by freezing a lesion in situ which leads to disruption of cell membranes, dam- age of tumour vasculature, activation of cytotoxic immune mechanisms and finally cell necrosis.90 Yeh et al. reported clinically complete response to shave excision and subsequent cryotherapy with liquid nitrogen in 11 of 18 patients with oral ver- rucous hyperplasia and oral verrucous carcinoma. After a mean follow-up of 23 months, recurrence was found in three cases and all were successfully treated by the same technique.91 Photodynamic therapy (PDT) is based on topi- cal or systemic administration of an exogenous photosensitiser which increases tumour tissue sensitiveness to light of a specific wavelength.92 It mediates tumour destruction by creating oxygen free radicals, damaging tumour vasculature and activating immune response against tumour cells. Chen et al. reported a complete clinical regression and no tumour recurrence at 6 months follow-up after 22 cycles of PDT using topical 5-aminole- vulinic acid followed by multiple fractionated ir- radiations with LED red light in a 56-year-old male with oral verrucous carcinoma extending from mouth angle to buccal mucosa.93 In order to bet- ter expose deeper part of the lesion under mucosal surface to cryotherapy or PDT and make a defini- tive histopathologic diagnosis, its exophytic part may initially be removed with debulking methods such as shave or laser excision.91 CO2 laser destructs a lesion with tissue vapori- zation and is therefore proposed for treatment of tumours involving cosmetically critical areas such as lips, where wide surgical excision may lead to unacceptable aesthetic and/or functional impair- ment.94 Several authors reported good clinical re- sponse with complete tumour removal and no re- currence in a follow-up from 15 to 48 months.94-96 Described procedures are non-invasive and can be safely carried out in a local anaesthesia in the outpatient clinic. Other reported advantages are short procedure duration, ability to treat multifo- cal lesions, limited pain and scarring, fast homeo- stasis and healing process, low risk of secondary infection and little or no side effects.91,93,95 However, their effect is limited by a depth of agent penetra- tion, which therefore makes them suitable only for a treatment of superficial oral lesions.92 They also lack working precision since it is difficult to judge the final extent of tissue necrosis during a proce- dure. Moreover, tumour resolution can only be assessed clinically and not histopathologically.91 Large-scale clinical studies with longer follow-up are further necessary to evaluate their effective- ness in the management of oral verrucous carci- noma. Conclusions Oral verrucous carcinoma is a rare variant of oral SCC that must be differentiated from conventional SCC due to its locally invasive and non-metastasiz- ing behaviour with a more favourable prognosis. For making a correct diagnosis, close communica- tion between clinician and pathologist is manda- tory. Primary surgery with negative surgical mar- gins seems to be the optimal treatment for patients with oral verrucous carcinoma; whether to per- form the END remains controversial. The concern about anaplastic transformation after irradiation should not affect the decision on treatment with radiotherapy which is usually proposed to pa- tients with extensive tumours or patients in poor general condition. The role of systemic therapy, particularly immunotherapy and targeted therapy, and non-surgical treatment methods are yet to be defined. Due to rarity of the disease, pooled multi- institutional analyses are warranted to properly address opened questions. Acknowledgments This review was funded by the Slovenian Research Agency (ARRS), grant number P3-0307. References 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Verrucous hyperplasia and verrucous carcinoma in head and neck: use and benefit of methotrexate. Acta Clin Belg 2021; 76: 487-91. doi: 10.1080/17843286.2020.1752455 87. Karagozoglu KH, Buter J, Leemans CR, Rietveld DHF, Van Den Vijfeijken S, Van Der Waal I. Subset of patients with verrucous carcinoma of the oral cavity who benefit from treatment with methotrexate. Br J Oral Maxillofac Surg 2012; 50: 513-8. doi: 10.1016/j.bjoms.2011.09.011 88. Salesiotis A, Soong R, Diasio RB, Frost A, Cullen KJ. Capecitabine induces rapid, sustained response in two patients with extensive oral verrucous carcinoma. Clin Cancer Res 2003; 9: 580-5. 89. Yoshimura Y, Mishima K, Obara S, Nariai Y, Yoshimura H, Mikami T. Treatment modalities for oral verrucous carcinomas and their outcomes: contribution of radiotherapy and chemotherapy. Int J Clin Oncol 2001; 6: 192-200. doi: 10.1007/PL00012104 90. Yu CH, Lin HP, Cheng SJ, Sun A, Chen HM. Cryotherapy for oral precan- cers and cancers. J Formos Med Assoc 2014; 113: 272-7. doi: 10.1016/j. jfma.2014.01.014 91. Yeh CJ. Treatment of verrucous hyperplasia and verrucous carcinoma by shave excision and simple cryosurgery. Int J Oral Maxillofac Surg 2003; 32: 280-3. doi: 10.1054/ijom.2002.0331 92. Chen HM, Yu CH, Lin HP, Cheng SJ, Chiang CP. 5-Aminolevulinic acid-mediat- ed photodynamic therapy for oral cancers and precancers. J Dent Sci 2012; 7: 307-5. doi: 10.1016/j.jds.2012.03.023 93. Chen HM, Chen CT, Yang H, Lee MI, Kuo MYP, Kuo YS, et al. Successful treat- ment of an extensive verrucous carcinoma with topical 5-aminolevulinic acid-mediated photodynamic therapy. J Oral Pathol Med 2005; 34: 253-6. doi: 10.1111/j.1600-0714.2004.00267.x 94. Hsu CK, Lee JYY, Yu CH, Hsu MML, Wong TW. Lip verrucous carcinoma in a pregnant woman successfully treated with carbon dioxide laser surgery. Vol. 157, The British journal of dermatology. Br J Dermatol 2007; 157: 813-5. doi: 10.1111/j.1365-2133.2007.08078.x 95. 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JAAD case reports 2018; 4: 576-8. doi: 10.1016/j.jdcr.2018.02.002 Radiol Oncol 2023; 57(1): 12-19. doi: 10.2478/raon-2023-0013 12 review Molecular profiling of rare thymoma using next-generation sequencing: meta-analysis Jelena Kostic Peric1, Andja Cirkovic2, Sanja Srzentic Drazilov1, Natalija Samardzic3, Vesna Skodric Trifunovic3,4, Dragana Jovanovic5, Sonja Pavlovic1 1 Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia 2 Department for Medical Statistics and Informatics, Faculty of Medicine, University of Belgrade, Serbia 3 University Hospital of Pulmonology, Clinical Centre of Serbia, Belgrade, Serbia 4 Faculty of Medicine, University of Belgrade, Belgrade, Serbia 5 Internal Medicine Clinic “Akta Medica”, Belgrade, Serbia Radiol Oncol 2023; 57(1): 12-19. Received 21 October 2022 Accepted 31 January 2023 Correspondence to: Assist. Prof. Jelena Kostic Peric, Ph.D., Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia. E-mail: jelena.kostic@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. Thymomas belong to rare tumors giving rise to thymic epithelial tissue. There is a classification of several forms of thymoma: A, AB, B1, B2, B3, thymic carcinoma (TC) and thymic neuroendocrine thymoma. In this meta- analysis study, we have focused on thymoma using articles based on the disease’s next-generation sequencing (NGS) genomic profiling. Materials and methods. We conducted a systematic review and meta-analysis of the prevalence of studies that discovered the genes and variants occurring in the less aggressive forms of the thymic epithelial tumors. Studies pub- lished before 12th December 2022 were identified through PubMed, Web of Science (WoS), and SCOPUS databases. Two reviewers have searched for the bases and selected the articles for the final analysis, based on well-defined exclusion and inclusion criteria. Results. Finally, 12 publications were included in the qualitative as well as quantitative analysis. The three genes, GTF2I, TP53, and HRAS, emerged as disease-significant in the observed studies. The Odds Ratio for all three extracted genes GTF2I (OR = 1.58, CI [1.51, 1.66] p < 0.00001), TP53 (OR = 1.36, CI [1.12, 1.65], p < 0.002), and HRAS (OR = 1.02, CI [1.00, 1.04], p < 0.001). Conclusions. According to obtained data, we noticed that the GTF2I gene exhibits a significant prevalence in the cohort of observed thymoma patients. Moreover, analyzing published articles NGS has suggested GTF2I, TP53, and HRAS genes as the most frequently mutated genes in thymoma that have pathogenic single nucleotide variants (SNV) and Insertion/Deletion (InDel), which contribute to disease development and progression. These variants could be valuable biomarkers and target points specific to thymoma. Key words: thymoma; next-generation sequencing (NGS); SNVs/InDels; meta-analysis Introduction Thymic epithelial tumors (TETs) are localized in the anterior mediastinum and comprise several forms of thymomas with different malignant po- tentials, aggressive forms of thymic carcinoma (TC), and thymic neuroendocrine thymoma.1 Thymomas originate from thymic epithelial tis- sue. Thymoma and TC are similar and overlap- ping in many characteristics, but despite these his- topathological and cytological features, thymomas could be considered a more benign form of TETs in comparison with TC with aggressive forms. That is the reason why thymomas and TC, exhibit Radiol Oncol 2023; 57(1): 12-19. Kostic Peric J et al. / Molecular profiling of thymoma 13 main differences in therapy approach.2,3 Here we analyze thymoma including the following sub- types A, AB, B1, B2, and B3.4 A and AB subtypes belong to GTF2I, B1, and B2 are the T-cell signaling group, B2 is as well chromosomal stability group, and B3 (atypical thymoma because is the most ag- gressive, if we exclude TC) belongs to the chromo- somal instability group. Thymomas are classified as rare thoracic tumors with an overall incidence of 0.15 per 100 000 persons per year.5,6 The majority of thymomas are less aggressive forms and their treatment is based on surgery, while in the case of more aggressive TC treatment approach includes multimodal therapy. This tumor characterizes high heterogeneity which is the reason for precise molecular profiling to choose an adequate preci- sion therapy approach.7,8 Due to the rarity of the disease, there is still a lack of information about external factors that cause diseases, such as smoke or alcohol. Moreover, epidemiological studies sug- gest that it is poorly known if various environmen- tal impacts have any specific impact on disease development.8 The role of mutated genes is one of the crucial factors for thymoma formation and development. Identification of potentially patho- genic variants is obligatory to better explain the molecular milieu of the disease. Our latest study published in January 2020 related to this pathol- ogy exhibited variants that play an important role in thymoma.9 To that end, advances in high-throughput tech- nology (next-generation sequencing-NGS) have enabled assess of the mutational profiling of vari- ous types of diseases including cancer. NGS in genomics includes whole-genome sequencing (WGS), whole-exome sequencing (WES), and tar- geted sequencing (TS).10 WGS covers all genomes but provides lower sequencing coverage in com- parison to TS. Advances in NGS have contributed to a better understanding of molecular events that lead to disease origin as well as the development of various genetic tests and potential targeted therapy. TS approach targets specific regions of interest and is more cost-effective and often suit- able for diagnostic panels which include a specific set of genes characteristic of the disease. Thus, TS provides a patient-specific mutational landscape for effective targeted therapy and better patient management.11,12 Different approaches are used de- pending on the need required by the type of study. WGS represents the most comprehensive method for genome analysis and covers the entire DNA of interest. The main limitation of WGS is low se- quencing coverage (25 - 30x), high cost per sample (which is lower with the increasing role of method- ology), and complex and demanding computation analyzed genome, in comparison to WGS or TS.13 This study aimed to identify all relevant articles that evaluate the frequency of SNVs and InDels in thymomas using NGS technology through PubMed, Web of Science, and SCOPUS databases, and to perform a meta-analysis of the prevalence to get better insight into their possible involvement in thymomas. The introduction should summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study. Materials and methods This systematic review was performed by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses.14 Study selection Systematic review Publications were screened for inclusion and ex- clusion in the meta-analysis of prevalence in two phases, and all disagreements were solved by a discussion with the third reviewer. We included studies that analyze the molecular landscape of thymoma using next-generation sequencing (NGS) genomics. Studies were excluded if they: 1) investi- gated related diseases but not thymoma; 2) evalu- ated other outcomes (NGS genomics for gene ex- pression); 2) explored populations other than hu- man (animal models, cell lines); 3) were abstracts; 4) were not original articles (reviews, systematic reviews, case reports, etc.). Database search Two researchers have conducted a meta-analysis of the prevalence of the genes and variants in- volved in thymoma disease. The meta-analysis of all published peer-reviewed articles related to the study was performed by searching the PubMed, Web of Science (WoS), and SCOPUS electronic da- tabases, until the 12th of December 2022. Keywords used for article search in all databases were next- generation sequencing (NGS) and thymoma. Only publications written in English were considered. Additionally, reference lists of articles identi- fied through electronic retrieval were manually searched, as well as relevant reviews and editori- als. Experts in the field were contacted to identify other potentially relevant articles. Radiol Oncol 2023; 57(1): 12-19. Kostic Peric J et al. / Molecular profiling of thymoma14 Article screening and selection Two reviewers (JKP, AC) independently evaluated the eligibility of all titles and abstracts. Studies were included in the full-text screening if either reviewer identified the study as being potentially eligible, or if the abstract and title did not include sufficient information. Studies were eligible for full-text screening if they included NGS genomics analysis of thymoma. TETs include thymoma and TC forms, which have been distinguished. The same reviewers independently performed full-text screening to select articles for inclusion accord- ing to the criteria listed under the Inclusion and Exclusion Criteria. Disagreements were resolved by consensus (JKP, AC) or arbitration (SP). Data extraction and quality assessment Two reviewers independently abstracted the fol- lowing data: author(s), country of research, year of publication, study design, sample size, study pop- ulation, type of thymoma, inclusion and exclusion criteria used in the original articles, method of NGS variant detection, genes harboring variants, number of patients having genes with SNVs/InDel variants. Each reviewer independently evaluated the quality of selected manuscripts. Statistical analysis The primary outcome was the number of patients harboring SNVs/InDels variants per gene. The odds ratio was evaluated as the ratio between patients with variants in a specific gene per total number of observed patients with thymoma. Heterogeneity was assessed using the Chi- square Q and I2 statistics. I2 presents the inconsist- ency between the study results and quantifies the proportion of observed dispersion that is real, i.e., due to between-study differences and not due to random error. The categorization of heterogeneity was based on the Cochrane Handbook and states that I2 < 30%, 30% to 60%, or > 60%, corresponds to low, moderate, and high heterogeneity, respective- ly.15 Funnel plots were used to evaluate publication bias. Forest plots were constructed for each analy- sis showing the Odds Ratio (box), 95% confidence interval (lines), and weight (size of box) for each trial. The overall effect size was represented by a diamond. A p-value < 0.05 was considered to be statistically significant. For graph plots (Forest and Funnel) we used Cohrain’s RevMan 5.4 version.16 Results Systematic review The literature search for original articles was conducted according to the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) statement. A total of 166 potentially eligible publications were found in search of PubMed, WoS, and SCOPUS electronic databases PubMed 49 articles, WoS 44 articles, and SCOPUS 71. Moreover, we included 3 additional studies from an additional PubMed search (Additional re- cords). After duplication removal, and exclusion of review articles, case reports, abstracts, investiga- tions on animals, cell lines, and articles with un- suitable outcomes, 65 articles were excluded. After exclusion, according to previously defined criteria, 44 articles were assessed in full text. Totally, 12 full-text articles were considered for final analy- ses. The flow diagram represents the selection workflow of publications (Figure 1). Selected studies have been presented with the names of studies’ authors, year of published works, NGS approach, type of tumor, and genes that har- FIGURE 1. Flow chart to illustrate the process by which articles were selected or rejected for inclusion in the study. Radiol Oncol 2023; 57(1): 12-19. Kostic Peric J et al. / Molecular profiling of thymoma 15 bored any single nucleotide variants (SNVs) or small insertions/deletions (InDels) in Table 1. The number of patients harboring or not genes with variants, as well as a total number of patients is shown in the same table. Finally, selected studies were published from 2014 to 12th December 2022. The total number of patients analyzed for GTF2I was 433 and 193 patients harbored SNVs/InDels variants in this gene. The total number of cases with SNVs/InDels variants was 58 in TP53 in the group of 309 thymoma patients. The number of an- alyzed patients for variants in HRAS was 187, while 15 among them had SNVs/InDel variants. The max- imum sample size per group was 270. Four stud- ies were from China (4), Japan (2), the USA (1), Italy (1), Austria (1), France (1), Poland (1), and Serbia (1). Analyzed studies used different study designs, mostly cross-section, and case-control designs. Meta-analysis of prevalence The analyses of the original articles related to the next-generation sequencing genomics of thy- moma have suggested the prevalence of three genes, namely, GTF2I, TP53, and HRAS. The re- sults of analyzed genes have been represented on Forest plots (Figure 2., Figure 3., and Figure 4.) and Funnel plots (Figure 5., Figure 6., and Figure 7.) with corresponding Odds Ratio (box), Confidence Interval (CI, lines), weight (size of box), and overall size effect in diamonds. The prevalence of the GTF2I gene was the high- est (58%), a bit lower was for the TP53 gene (36%) and the lowest was the HRAS gene (2%), for the thymoma population. According to obtained data, we pointed out genes GTF2I and TP53 that exhibit the prevalence in the cohort of observed thymoma patients. Discussion The aim of this study was to explore genomic background of indolent forms of thymoma using genomic high-throughput approach. Thymoma epithelial tumors (TETs) comprise thymoma, thymic carcinoma (TC), and thymic neuroen- docrine thymoma, as we said previously. In this meta-analysis, we take into consideration several forms of thymomas, including A, AB, B1, B2, and B3 subtypes, which have diverse invasive (malig- nant) potential. Thymoma is a tumor type that be- longs to rare types of thoracic tumors, which is the reason for a lower number of studies that we have finally selected as eligible for analysis, along with the relatively recently developed method of analy- sis that we have applied. As well, keywords used in this research are limited to the NGS genomics approach to analyz- ing genes included in thymoma. The potential of high-throughput sequencing or NGS enables the detection of the molecular profile, typical for spe- cific tumors or a variety of diseases. All genomic sequencing methods, including WGS, WES, and TS have been applied in the research or in diagnos- tics. The selection of methodology depends on all advantages or disadvantages suitable for appropri- ate use. Applying previously explained inclusion/ exclusion criteria we were able to select and ana- lyze articles related to this topic, and we were able to find genes associated with thymoma. Meta-analysis of the prevalence of the gene variants that we performed using selected studies, has indicated that the majority of patients exhib- ited variants in GTF2I, TP53, and HRAS genes. The number of analyzed articles was relatively low, due to the rarity of the disease. Moreover, most of the studies have been published from 2014 to 2022. The molecular background of this pathology is still poorly understood, including all types of A, FIGURE 2. Forest plot of GTH2I gene in thymoma. FIGURE 3. Forest plot of TP53 gene in thymoma. FIGURE 4. Forest plot of HRAS gene in thymoma. Radiol Oncol 2023; 57(1): 12-19. Kostic Peric J et al. / Molecular profiling of thymoma16 AB, B1, B2, and B3 thymoma. We have analyzed only NGS genomics-based articles. Further in the discussion will also be considered other meth- odological approaches to better explain obtained results. The newest data that appeared 2022, have indicated that KIT gene is an important factor in different processes related to thymoma disease. Unfortunately, we have not had enough data to demonstrate the statistical significance of this gene to put it in a group of prevalent genes for thy- moma.17,18 GTF2I (General Transcription Factor IIi) encodes a phosphoprotein that binds to the initiator ele- ment (Inr) and Inbox element in promoters regulat- ing transcription.20 GTF2I gene is a member of a B cell receptor signaling pathway, the AKT signaling pathway (genecards.org), and is involved in crucial processes particularly, cell proliferation, cell cycle, and development.19 Finally, the advances in next- generation sequencing characterized GTF2I as a master gene in TETs pathology.20 The frequency of GTF2I variants is typical for less aggressive forms of thymoma.21 GTF2I is a gene that embryonic ab- erration could lead to lethality, which indicates its essential role in embryo development. The most pathogenic variants in this gene are Chr 7 c.1211T > A and c.1271 (COSM5095139) which could be used in targeted therapy that could lead to successful clinical application.21,22 Despite GTF2I pathogenicity in thymomas, GTF2I mutations are very rare (< 1%) in other types of cancer, accord- ing to TCGA. Moreover, many types of molecular events lead to thymoma genesis. TCGA’s studies of thymoma have shown recurrent mutations in the GTF2I gene and suggested this gene as a potential drug target for this disease.23-25 Our findings con- firm the prevalence of the SNVs/InDels variants in GTF2I in thymoma. Results have been reported in several articles which have been presented in the Forest and Funnel plots. Petrini and colleagues, using WES have discovered missense mutation Chr 7 c.1211T > A and subsequent protein change p. Leu404His in all types of thymoma A, AB, B1, B2, B3, and TC.19,26 In the publications that we se- lected for analysis, GTF2I harbored p. Leu404His and p. Leu424His (COSM5095139) pathogenic vari- ants, recognized as a disease-marker gene in thy- moma.22,26-28 TP53 is a crucial tumor suppressor gene regu- lating the most important processes in the cell in- cluding cell cycle regulation, DNA repair, senes- cence, and apoptosis.29 TP53 has been described as the gene responsible for disease pathogenesis. A recent study has demonstrated common loss-of- function in TP53 in type B thymomas and TCs.30 In a study by Peric et al., two variants with stop codon have been identified R213* and V91*, as well as four pathogenic missense changes name- ly, D281N, G244S, R158C, E221K.22,31,32 One of the newest studies by Xu S et al., included in our meta- FIGURE 7. Funnel plot of HRAS gene in thymoma. FIGURE 5. Funnel plot of GTF2I gene in thymoma. FIGURE 6. Funnel plot of TP53 gene in thymoma. Radiol Oncol 2023; 57(1): 12-19. Kostic Peric J et al. / Molecular profiling of thymoma 17 TABLE 1. Characteristics of 12 selected studies Author Year Study design Thymoma Gene (SNV) Number of cases Total number of cases NGS genomics Chen K, China 2020 Cross-sectional Type A, AB, B1, B2, B3 APC TP53 ATM AKT1 SMAD4 ALK KRAS NRAS 23 12 22 5 12 19 2 2 50 TS Enkner F, Austria 2017 Cross-sectional Type A, B HRAS SMARCB1 STK11 3 1 1 19 TS Higuchi R, Japan 2020 Cross-sectional Type A, AB, B GTF2I 14 22 TS Petrini I, Italia 2014 Cross-sectional Type A, AB GTF2I 119 270 ES Radovich M, USA 2017 Case-control Type A, AB GTF2I HRAS TP53 44 10 2 105 MOPA Sakane T, Japan 2020 Cross-sectional Type A, B2, B3, B4, B5 HRAS PIK3CA AKT1 RAS pathway EGFR pathway 1 2 1 1 3 33 SNaPshot Multiplex Song ZB, China 2016 Type B2, B3 PIK3CA EGFR 1 1 37 TS IonAmpliSeq Peric J, Serbia 2020 Case-control Type A, B1, B2, B3 SMAD4 APC ATM ERBB4 TP53 27 27 26 24 26 35 TSACP Xu S, China 2021 Cross-sectional Type A, AB B1, B2, B3 TP53 HRAS 9 2 17 ES Liang N, China 2021 Cross-sectional Type A, AB, B1, B2, B3 GTF2I 15 24 SureSelectXT TS Szpechcinski A, Poland 2022 Cross-sectional Type B2B3 KIT ERBB2 1 1 19 TS Girard N, France 2022 Cross-sectional Type A, B1, B2, B3 KIT TP53 HRAS Other genes 1 8 1 80 90 WES MOPA = multi-omics platform analysis; SNaPshot Multiplex = Snapshot multiplex assay for point mutation; TS = targeted enrichment-based sequencing: TSACP = TruSeq amplicon cancer panel; (WES) = whole exome sequencing analysis, has analyzed the genomic profile of TETs that has indicated TP53 as the most mutated gene especially in both B3 and C thymoma causing worse prognosis.33 HRAS gene belongs to the Ras signaling path- way, commonly mutated in many tumors harbor- ing pathogenic variants, for instance, G13V in thy- moma. One study suggests gain-off function mu- tations in HRAS, especially in type A and AB thy- momas.30 A recent study by Jovanovic D et al. has identified recurrent mutations in HRAS, marked as a thymoma-specific oncogene.34 TCGA’s studies of thymoma observed enrichment in the HRAS gene, as well as NRAS and TP53.24,25 Our meta-analysis of prevalence has reported the HRAS gene to be involved in the pathogenesis of thymoma.22,33,35,36 Genes that have shown the prevalence in less ag- gressive forms of thymoma, especially thymoma- specific oncogene GTF2I (transcription factor), TP53, and members of Ras family HRAS have been involved in crucial processes including cell cycle regulation, cell proliferation, cell differentiation, apoptosis, and cell survival. Therefore, pathogenic variants within these genes could be important Radiol Oncol 2023; 57(1): 12-19. Kostic Peric J et al. / Molecular profiling of thymoma18 disease markers and potential therapeutic targets for thymoma. Conclusions Our meta-analysis of articles that analyze a muta- tional portrait using NGS of thymoma has pointed out GTF2I, HRAS, and TP53 genes as thymoma- specific oncogenes. These genes harbor variants SNVs/InDels which contribute to disease devel- opment. Moreover, this study indicated the high- est prevalence of the GTF2I gene (58%). Some of the identified variants are driver mutations, for instance, GTF2I (Chr7 c.1211T > A, p. Leu404His and c.1271T > A, p. Leu424His), associated with thymoma pathology. In addition, the majority of detected molecular changes are classified as pas- senger variants, unable to cause disease and fur- ther progression, without the presence of other molecular events. However, the thymoma molecu- lar landscape is still insufficiently understood and explored. Therefore, there is a need for additional analysis and information to get a comprehensive genomic picture for better precision treatment of the patients. Acknowledgments This work was supported by the Ministry of Education, Science and Technological Development Republic of Serbia, EB: 451-03-68/2022-14/200042. References 1. Cowen D, Richaud P, Mornex F, Bachelot T, Jung GM, Mirabel X, et al. Thymoma: results of a multicentric retrospective series of 149 non- metastatic irradiated patients and review of the literature. FNCLCC trialists. Federation Nationale des Centres de Lutte Contre le Cancer. Radiother Oncol 1995; 34: 9-16. doi: 10.1016/0167-8140(94)01493-m 2. Levine GD, Rosai J. Thymic hyperplasia and neoplasia: a review of cur- rent concepts. Human Pathology 1978; 9: 495-515. doi: 10.1016/s0046- 8177(78)80131-2 3. Falkson CB, Bezjak A, Darling G, Gregg R, Malthaner R, Maziak DE, et al. The management of thymoma: a systematic review and practice guideline. J Thorac Oncol 2009; 4: 911-9. doi: 10.1097/jto.0b013e3181a4b8e0 4. Okumura M, Ohta M, Tateyama H, Nakagawa K, Matsumura A, Maeda H, et al. 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Histopathology 2019; 75: 755-66. doi: 10.1111/his.13936 Radiol Oncol 2023; 57(1): 20-34. doi: 10.2478/raon-2023-0008 20 research article Multimodality CT imaging contributes to improving the diagnostic accuracy of solitary pulmonary nodules: a multi-institutional and prospective study Gaowu Yan1, Hongwei Li2, Xiaoping Fan1, Jiantao Deng1, Jing Yan1, Fei Qiao3, Gaowen Yan4, Tao Liu1, Jiankang Chen1, Lei Wang1, Yang Yang1, Yong Li1, Linwei Zhao1, Anup Bhetuwal5, Morgan A McClure6, Na Li7, Chen Peng8 1 Department of Radiology, Suining Central Hospital, Suining, China 2 Department of Radiology, The Third Hospital of Mianyang and Sichuan Mental Health Center, Mianyang, China 3 Department of CT and MRI, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi, China 4 Department of Radiology, The First People’s Hospital of Suining, Suining, China 5 Sichuan Key Laboratory of Medical Imaging and Department of Radiology, Affiliated Hospital of North Sichuan Medical College, Nanchong, China 6 Department of Radiology and Imaging; Institute of Rehabilitation and Development of Brain Function, The Second Clinical Medical College of North Sichuan Medical College Nanchong Central Hospital, Nanchong, China 7 Department of Oncology, Suining Central Hospital, Suining, China 8 Department of Gastroenterology, The First People’s Hospital of Suining, Suining, China Radiol Oncol 2023; 57(1): 20-34. Received 15 August 2022 Accepted 5 December 2022 Correspondence to: Yong Li, Department of Radiology, Suining Central Hospital, Suining, China. E-mail: 13890893057@163.com and Na Li, Department of Oncology, Suining Central Hospital, Suining, China. E-mail: lny2008hy@163.com and Gaowu Yan, Department of Radiology, Suining Central Hospital, Suining, China. E-mail: yangaowu1989@163.com Gaowu Yan, Hongwei Li, Xiaoping Fan, Jiantao Deng, Jing Yan, and Fei Qiao all have contributed equally to this study. 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. Solitary pulmonary nodules (SPNs) are one of the most common chest computed tomography (CT) abnormalities clinically. We aimed to investigate the value of non-contrast enhanced CT (NECT), contrast enhanced CT (CECT), CT perfusion imaging (CTPI), and dual- energy CT (DECT) used for differentiating benign and malignant SPNs with a multi-institutional and prospective study. Patients and methods. Patients with 285 SPNs were scanned with NECT, CECT, CTPI and DECT. Differences between the benign and malignant SPNs on NECT, CECT, CTPI, and DECT used separately (NECT combined with CECT, DECT, and CTPI were methods of A, B, and C) or in combination (Method A + B, A + C, B + C, and A + B + C) were compared by receiver operating characteristic curve analysis. Results. Multimodality CT imaging showed higher performances (sensitivities of 92.81% to 97.60%, specificities of 74.58% to 88.14%, and accuracies of 86.32% to 93.68%) than those of single modality CT imaging (sensitivities of 83.23% to 85.63%, specificities of 63.56% to 67.80%, and accuracies of 75.09% to 78.25%, all p < 0.05). Conclusions. SPNs evaluated with multimodality CT imaging contributes to improving the diagnostic accuracy of benign and malignant SPNs. NECT helps to locate and evaluate the morphological characteristics of SPNs. CECT helps to evaluate the vascularity of SPNs. CTPI using parameter of permeability surface and DECT using parameter of normalized iodine concentration at the venous phase both are helpful for improving the diagnostic performance. Key words: solitary pulmonary nodule; non-enhanced computed tomography; contrast-enhanced computed to- mography; computed tomography perfusion imaging; dual-energy computed tomography Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 21 Introduction A solitary pulmonary nodule (SPN) is defined as a focal, round or oval area of increased opacity in the lung with its maximum diameter no larger than 3.0 cm.1-3 It is also one of the most common chest radiography (CR) or computed tomography (CT) abnormalities that are often identified incidentally in clinical practice with 150,000 SPNs incidentally detected in the United States on CR or CT every year.1 It is estimated that the prevalence of SPNs in the general population is about 2.0% to 24.0%. Among those with more risk factors of lung ma- lignancies (e.g. an older age, male, history of smok- ing or tumor, etc.), the prevalence of a SPN is up to 17.0% to 53.0%.4 With the generally increased use of CT worldwide, it can be understood that more SPNs will be detected in the coming years. The etiology of SPNs includes neoplasms (e.g., primary pulmonary carcinoma, solitary metasta- sis, or chondroma), infection (e.g., infectious gran- uloma or round pneumonia), inflammation (e.g., rheumatoid arthritis or granulomatosis with poly- angiitis), vascular (e.g., arteriovenous malforma- tion, infarct, or hematoma) and congenital abnor- mities (e.g., sequestration or bronchogenic cyst). As a result, it is important to accurately distinguish between benign and malignant SPNs before in- vasive managements; especially for solid nodules with sizes of ≥ 15 mm (≥ 1767 mm3). As the Lung- RADS guidelines have reported that their risk of malignancy is more than 15%.2 This is because for benign SPNs, conservative treatments such as drugs or observation may suffice. While for lung cancer and other malignant SPNs, we usually need surgical treatments as soon as possible. Compared with CR, magnetic resonance im- aging (MRI), and positron emission tomography (PET), a CT plays an important role in the diagno- sis and management of SPNs.1-3,5,6 For conventional CT (non-contrast enhanced CT, NECT), it is usually used to evaluate the size, margins, contour, and internal characteristics of a SPN. For contrast en- hanced CT (CECT), it helps evaluate the vascularity of a SPN. With the development of CT technologies, CT perfusion imaging (CTPI) and dual-energy CT (DECT) are increasingly used in clinical practice.7-10 For example, in a study by Wen et al.10, quantitative measures (the slope of the spectral Hounsfield Unit (HU) curve (λHU), normalized iodine concentration (NIC), CT values of 40 keV monochromatic images (CT40keV), and normalized arterial enhancement fraction (NAEF)) from DECT can help to differenti- ate benign from malignant SPNs. We hypothesized that multimodality CT imag- ing may contribute to improving the diagnostic accuracy of SPNs. However, to the authors’ knowl- edge, there are no publications in the literature in which NECT, CECT, CTPI and DECT were compre- hensively used to differentiate benign or malignant SPNs. Thus, the aim of this study was to investigate the value of NECT, CECT, CTPI, and DECT in the differentiation of benign or malignant SPNs with a multi-institutional and prospective study. Patients and methods Patients This study was conducted at our three teaching hospitals and approved by the institutional review committee of Suining Central Hospital (Suining, China; approval no. LLSNCH20200004). All pa- tients were included after providing informed consent. Patients with SPNs were admitted and treated at one of the three hospitals from January 2019 to June 2021 and were consecutively enrolled into our study. The inclusion criteria were: (1) SPNs were detected with NECT in the lung window; (2) each SPN size is 1.5 cm–3.0 cm; (3) benign or malignant SPNs were confirmed by CT-guided percutane- ous biopsy or pathology after surgery; (4) CTPI and DECT were performed within one week prior to CT-guided percutaneous biopsy or surgery; (5) SPNs were not treated by any antitumor therapies (e.g., radiotherapy, chemotherapy, or targeted drug therapy); (6) age > 18 years old; and (7) patients had acceptable liver and kidney functions and no his- tory of allergies (for example, allergic to iodine or seafood). The exclusion criteria were: (1) SPNs ap- pearing as pure ground-glass opacity (pGGO) or as subsolid nodules that could not be measured in the mediastinal window; (2) majority components of the SPNs have been calcified or liquefactive ne- crosis, leading to region of interests (ROIs) cannot be drawn; (3) body mass index (BMI) is greater than 30.0 kg/m2; (4) poor image quality (e.g., signifi- cant anomalies by heart beats or others artifacts); and (5) incomplete clinical data. Three hundred and twenty-three patients were included and 38 of them were excluded because of the exclusion crite- ria (Figure 1). CT examination All p atients were scanned with the same DECT system (Revolution CT, GE Healthcare, Milwaukee, Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules22 WI, USA), and all the scanning parameters were set at the same level. The multimodality CT imag- ing protocol included three steps, i.e., NECT, CTPI, and DECT. For NECT, the patients were asked to hold their breath at the end of inspiration, and the scan- ning ranges were taken from the thoracic inlet to the bilateral costophrenic angle. The tube voltage was 100 kVp; the tube current was modulated by a SmartmA technology, which provides reasonable tube current to decrease the radiation dose; and the preset noise index was adjusted for patient cir- cumference. For CTPI, the patients were asked to take re- laxed and slow respiration. After the position of an SPN was determined by the NECT, a GSI Chest Perfusion protocol was used with a scan- ning range of 3.0 cm above and below the central level of a SPN. A non-ionic iodine contrast agent of 400 mg/ml (Iomeron®, Shanghai Braccosine Pharmaceutical Co., Ltd.) was injected into an antecubital vein with a binocular power injector (Stellant D-CE, MEDRAD, Bayer Healthcare Co., Ltd.), at a flow rate of 5.0 ml/s (40.0 ml). After that, 20.0 ml of normal saline at a speed of 5.0 ml/s was used to flush the tube. The CTPI was performed after injection of the contrast medium with a de- lay time of 4.0 s. The tube voltage and tube current were 70 kVp and 250 mA respectively. There were 18 scanning phases with an interval of 2.5 s, and the total acquisition time was about 43.0 s. For DECT, the patients were asked to hold their breath at the end of inspiration, and the scanning ranges were the same as the NECT. Another 50.0 ml contrast agent was injected into the antecubital vein with the binocular power injector, at a flow rate of 2.5 ml/s. After that, 20.0 ml of normal saline at a speed of 2.5 ml/s was used to flush the tube. A ROI was placed in the thoracic aorta at the level of bronchial bifurcation, and the threshold was set to 120 HU. After reaching the threshold, the arte- rial phase was scanned with a delay time of 7.0 s, and the venous phase delay time was 30.0 s. The tube voltage, tube current, and noise index were the same as the NECT. The same scanning parameters for NECT, CTPI, and DECT were: detector coverage = 80 mm; pitch = 0.992: 1; coverage speed = 158.75 mm/s; rotation time = 0.5 s; preset adaptive statistical iterative reconstruction-V (ASIR-V) = 50%; postset ASIR-V = 60%; slice thickness = 5.0 mm; slice interval = 5.0 mm; reconstructed slice thickness = 1.25 mm; re- constructed slice interval = 1.25 mm. Measurement of radiation dose After the multimodality CT imaging protocol, the volume CT dose index (CTDIvol, mGy) and dose- FIGURE 1. Flow chart of patient selection. SPNs = solitary pulmonary nodules Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 23 length product (DLP, mGy.cm) were recorded. The estimated effective dose (ED, mSv) was calculated with the formula: ED = DLP × k, where the k equals 0.014 mSv/mGy.cm. Images post-processing For NECT, the lung window images were further processed with the post-processing workstation AW 4.7 (GE Healthcare, Milwaukee, WI, USA) to obtain multiplanar reconstruction (MPR) coronal and sagittal images (1.25 mm). For CTPI, the images were transferred to the post-processing workstation AW 4.7 (GE Healthcare, Milwaukee, WI, USA), CTPI parame- ters of the blood volume (BV, ml/100 g), blood flow (BF, ml/100 g/min), mean transit time (MTT, s), and permeability surface (PS, ml/100 g/min) with their perfusion artificial color maps were automati- cally generated by the CT perfusion 4D software. The ROI should be placed in the soft tissue area of a SPN at its maximum plane, and the thoracic aorta at the same level plane was selected as the reference vessel for calculating the arterial input function and a time density curve (TDC). When placing a ROI, the calcification, bleeding, liquefac- tion necrosis area, blood vessels, and anomalies by heart beats or other artifacts should be avoided as much as possible. For DECT, the arterial and venous phase me- diastinal window images were respectively pro- cessed with the GSI volume viewer software (AW 4.7, GE Healthcare, Milwaukee, WI, USA), to obtain the arterial and venous phase iodine-based imag- es. The following principles should be followed in outlining the ROI: (1) ROI should be placed in the solid area of a SPN with uniform enhancement, and the outlined area should be as large as possi- ble; (2) the calcification, bleeding, liquefaction ne- crosis area, blood vessels, and anomalies by heart beats or others artifacts should be avoided as much as possible; (3) the position and size of ROI meas- urement in each phase should be consistent. Parameter measurements The para meter measurements for CTPI, DECT, and the CT enhancement amplitude value were inde- pendently measured by two senior radiologists with more than 15 years of experience in chest CT. All indices were measured two times, and the average values were taken as the final results. Disagreements were resolved through consensus. Parameter measurements for CTPI included BV, BF, MTT, and PS. Parameter measurements for DECT included iodine overlay (OL), iodine concentration (IC), and normalized iodine concentration (NIC) at the arterial and venous phases (i.e., aOL, vOL, aIC, vIC, aNIC, and vNIC). The NIC was calculated with the formulae: NIC = ICSPN / ICThoracic aorta. The CT val- ues of a SPN at the venous CECT and NECT phas- es were also measured, and the CT enhancement amplitude value was calculated with the formula: CT enhancement amplitude value = venous CECT value - NECT value. Multimodality CT imaging for evaluating SPN First, another two senior radiologists with more than 15 years of experience in chest CT randomly and independently interpreted the conventional NECT images of each SPN. Any disagreements were solved through consensus. The SPNs were then classified into five categories, i.e., I = most likely benign, II = possibly benign, III = uncertain benign or malignant, IV = possibly malignant, and V = most likely malignant. The diagnostic meth- ods were as follows: NECT combined with CECT (Method A), and the diagnostic criteria for CT en- hancement were1: a CT enhancement amplitude value of 20–60 HU was classified as malignant, and a CT enhancement amplitude value of less than 20 HU or more than 60 HU was classified as benign; NECT combined with DECT (Method B); NECT combined with CTPI (Method C). If meth- ods A, B, or C were consistent when classifying a benign or malignant SPN, the SPN would be judged as benign or malignant. However, if meth- ods A, B, or C were not consistent when classifying a benign or malignant SPN, the following princi- ples would be followed: (1) if a benign or malig- nant SPN was definitely diagnosed as category I or V by NECT, the diagnostic results of CECT, DECT and CTPI would not be considered, and the SPN would be directly characterized as benign or ma- lignant; (2) if the nature of a benign or malignant SPN could not be confirmed by NECT (i.e. a cate- gory of II, III or IV SPN), the final diagnosis would be made according to the diagnostic thresholds of CECT, DECT and CTPI, respectively. There were four combined diagnostic methods: method A + B, method A + C, method B + C, and method A + B + C (Figure 2). In combined diagnostic methods, the results of benign or malignant SPNs may be inconsistent according to the diagnostic thresholds of CECT, CTPI or DECT. Our method is that if a SPN is diagnosed as malignant according to one of Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules24 the thresholds (CECT, CTPI, or DECT), it would be preliminarily categorized as malignant; then, the result would be compared with the pathological result to calculate the sensitivity, specificity and accuracy of the combined diagnostic method. Statistical analysis Measurement data were expressed as the mean ± standard deviation or median and interquartile range (P25, P75). The comparisons were performed with the two independent samples t-test or Mann Whitney rank sum test, as appropriate. The count data were expressed as the percentage (%) or com- position, and the comparisons were performed with the Fisher’s exact test. Receiver operating characteristic (ROC) curve analysis was performed on statistically significant CTPI and DECT param- FIGURE 2. Technology roadmap of multimodality computed tomography (CT) imaging for evaluating solitary pulmonary nodules (SPNs). BF = blood flow; BV = blood volume; CECT = contrast enhanced CT; CTPI = CT perfusion imaging; DECT = dual-energy CT; IC = iodine concentration; MPR = multiplanar reconstruction; NECT = non-contrast enhanced CT; MTT = mean transit time; NIC = normalized iodine concentration; OL = iodine overlay; PS = permeability surface eters to obtain the area under the curves (AUC) and diagnostic thresholds, and the Delong-test was performed to compare the differences.11 In using the pathological diagnosis as the gold standard, the sensitivities, specificities, accuracies, positive predictive values (PPVs) and negative predictive values (NPVs) of the three diagnostic methods separately used (Method A, B, and C) and in combination (Method A + B, Method A + C, Method B + C, and Method A + B + C) for the diagnosis of benign and malignant SPNs were cal- culated, and the McNemar’s test was used to com- pare the sensitivity, specificity, and accuracy of the various diagnostic methods. All statistical analyses were performed by using the software of GraphPad Prism 8.0.0 and MedCalc 19.5.3. Significant differences were set at a p value < 0.05. Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 25 < 0.05). However, no significant differences were noted between the malignant and benign SPNs in cavity sign, air bronchogram, calcification, fat (defined as CT attenuation of -40 HU to -120 HU), adjacent bronchial changes, location, or size on NECT (all p values were > 0.05). TABLE 1. Patient characteristics in 285 patients with solitary pulmonary nodules Characteristics Pathology P valueBenign SPNs (n = 118) Malignant SPNs (n = 167) Gender* Male 56 (47.46%) 96 (57.49%) 0.1170 Female 62 (52.54%) 71 (42.51%) Age (years)# 50.84 ± 19.60 52.93 ± 20.30 0.3952 Smoking status* Yes 52 (44.07%) 81 (48.50%) 0.4721 No 66 (55.93%) 86 (51.50%) Tumor history* Yes 17 (14.41%) 28 (16.77%) 0.6245 No 101 (85.59%) 139 (83.23%) Tumor biomarkers* Normal 103 (87.29%) 139 (83.23%) 0.4026 Abnormal 15 (12.71%) 28 (16.77%) SPNs = solitary pulmonary nodules; *compared with the Fisher’s exact test; #compared with the two independent samples t-test TABLE 2. Pathological results of the 285 solitary pulmonary nodules (SPNs) included in this study SPNs Pathology Datum (%) Benign SPNs (n = 118) Tuberculosis 46 (29.0%) Acute and chronic inflammation 32 (27.1%) Inflammatory pseudotumor 14 (11.9%) Hamartoma 9 (7.6%) Pulmonary sclerosing hemangioma 6 (5.1%) Sequestration 4 (3.4%) Bronchogenic cyst 3 (2.5%) Rheumatoid arthritis 2 (1.7%) Granulomatosis with polyangiitis 2 (1.7%) Malignant SPNs (n = 167) Primary pulmonary carcinoma 116 (69.5%) Solitary metastasis 23 (13.8%) Primary lung neuroendocrine tumor 21 (12.6%) Primary pulmonary lymphoma 7 (4.2%) Results Patient characteristics Ultimately, 285 SPNs were included in this study. Of these, 178 (62.46%, 178/285) SPNs were con- firmed by CT-guided percutaneous biopsy, and 107 (37.54%, 107/285) SPNs were confirmed by pa- thology after surgery. The patients’ characteristics are shown in Table 1. There were no significant differences between the malignant and benign SPNs relative to gender, age, smoking status, his- tory of tumors, or tumor biomarkers (all p values > 0.05). Pathological results Of the 285 SPNs, 118 were benign SPNs (41.4%, 118/285) and 167 were malignant SPNs (58.6%, 167/285). Of the 118 benign SPNs, 46 were tuber- culosis (29.0%, 46/118), 32 were acute and chronic inflammation (27.1%, 32/118), and 14 were inflam- matory pseudotumors (11.9%, 14/118). Of the 167 malignant SPNs, 116 (69.5%, 116/167) were primary pulmonary carcinomas (including adenocarcino- mas in 52, squamous cell carcinomas in 35, and small cell lung cancer in 17, etc.), 23 (13.8% , 23/167) were solitary metastasis (this included five from the liver, five from the breast, etc). Pathological results of the 285 SPNs included in this study are shown in Table 2. Radiation dose After the multimodality CT imaging protocol, the CTDIvol, DLP, and ED in the SPNs were 66.88 ± 4.36 mGy, 768.29 ± 91.65 mGy.cm, and 10.76 ± 1.28 mSv, respectively. NECT and CECT in evaluating SPNs In the malignant SPNs (n = 167), 49 (29.34%, 49/167), 115 (68.86%, 115/167), 108 (78.81%, 108/167), 38 (22.75%, 38/167), 89 (53.29%, 89/167), and 84 (50.30%, 84/167) cases were seen with smooth margins, lob- ulated sign, spiculated sign, vacuole sign, pleural indentation, and vessel convergence, respectively. In the benign SPNs (n = 118), the same CT findings were seen in 84 (71.19%, 84/118), 29 (24.58%, 29/167), 25 (21.19%, 25/118), 13 (11.02%, 13/118), 25 (21.19%, 25/118), and 33 (27.97%, 33/118) cases (Table 3), re- spectively. There were significant differences be- tween the malignant and benign SPNs in above mentioned findings on NECT (all p values were Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules26 For the classification of SPNs (Table 4), among the 118 benign SPNs, there were 39, 9, 48, 22, and 0 SPNs that were classified as the Category I, II, III, IV, and V nodules, respectively. Among the 167 malignant SPNs, there were 0, 5, 54, 19, and 89 SPNs that were classified as the Category I, II, III, IV, and V nodules, respectively. For CECT, among the 167 malignant SPNs, 118 cases (70.66%, 118/167) were noted with a CT en- hancement amplitude value of 20–60 HU; and among the 118 benign SPNs, 75 cases (63.56%, 75/118) were noted with a CT enhancement ampli- tude value of less than 20 HU or more than 60 HU (Figure 3). CTPI in evaluating SPNs The parameters of BF, BV, MTT, and PS of CTPI in malignant SPNs were higher than those of benign SPNs (Figure 4, Table 5). However, there was on- ly significant difference in the PS parameter (p < 0.05). A ROC curve analysis was performed on the PS parameter for differentiating benign and ma- lignant SPNs (Figure 5). It showed an AUC of 0.739 (95% confidence interval of 0.684–0.789, p < 0.0001) with the sensitivity, specificity, and diagnostic threshold of 85.03 %, 66.10%, and 9.88 ml/100g/ min, respectively. DECT in evaluating SPNs For DECT in evaluating SPNs (Figure 6), parame- ters of aOL, vOL, aIC, vIC, aNIC, and vNIC in ma- lignant SPNs were significantly higher than those of benign SPNs (all p values were < 0.05, Table 6). ROC curve analyses were performed on these parameters for differentiating benign and malig- nant SPNs (Figure 7). The AUCs, sensitivities, and specificities were 0.636 to 0.790, 59.88% to 75.45%, and 61.02% to 80.51%, respectively. The diagnostic thresholds were 13.89 HU, 12.79 HU, 0.65 mg/ml, 0.85 mg/ml, 0.12, and 0.35, respectively (Table 7). Compared with the AUC of aOL, vOL, aIC, vIC, and aNIC by the Delong test, vNIC had the largest AUC (all p values < 0.05, Table 8). Multimodality CT imaging in evaluating SPNs For methods A, B, C, A+B, A+C, B+C, and A+B+C, the sensitivities, specificities, accuracies, PPVs, and NPVs were 83.23% to 97.60%, 63.56% to 88.14%, 75.09% to 93.68%, 76.37% to 92.09%, and 72.82% to 96.30%, respectively (Table 9). TABLE 3. Solitary pulmonary nodules evaluated with non-contrast enhanced CT CT findings* Benign SPNs(n = 118) Malignant SPNs (n = 167) P Values Smooth margin Yes 84 (71.19%) 49 (29.34%) < 0.0001 No 34 (28.81%) 118 (70.66%) Lobulated sign Yes 29 (24.58%) 115 (68.86%) < 0.0001 No 89 (75.42%) 52 (31.14%) Spiculated sign Yes 25 (21.19%) 108 (78.81%) < 0.0001 No 93 (64.67%) 59 (35.33%) Vacuole sign Yes 13 (11.02%) 38 (22.75%) 0.0120 No 105 (88.98%) 129 (77.25%) Cavity sign Yes 9 (7.63%) 16 (9.58%) 0.6727 No 109 (92.37%) 151 (90.42%) Air bronchogram Yes 33 (27.97%) 56 (33.53%) 0.3643 No 85 (72.03%) 111 (66.47%) Calcification Yes 10 (8.47%) 6 (3.59%) 0.1149 No 108 (91.53%) 161 (96.41%) Fat Yes 6 (5.08%) 4 (2.40%) 0.3277 No 112 (94.92%) 163 (97.60%) Pleural indentation Yes 25 (21.19%) 89 (53.29%) < 0.0001 No 93 (78.81%) 78 (46.71%) Vessel convergence Yes 33 (27.97%) 84 (50.30%) 0.0002 No 85 (72.03%) 83 (49.70%) Adjacent bronchial changes Yes 26 (22.03%) 43 (25.75%) 0.4867 No 92 (77.97%) 124 (74.25%) Location Upper and middle lobes 53 (44.92%) 79 (47.31%) 0.7186 Inferior lobe 65 (55.08%) 88 (52.69%) Size (mm) 15–20 29 (24.58%) 46 (27.54%) 0.5883 20–30 89 (75.42%) 121 (72.46%) SPNs = solitary pulmonary nodules; *compared with the Fisher’s exact test Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 27 There were no significant differences between the methods A, B, and C in sensitivities, specifici- ties, and accuracies, and so did the methods A+B, A+C, B+C, and A+B+C (all p values > 0.05). However, the methods A+B, A+C, B+C, and A+B+C all had higher sensitivities, specificities, and accuracies than the methods A, B, and C for distinguishing benign from malignant SPNs (all p values < 0.05). Discussion NECT and CECT in evaluating SPNs As low-dose CT scanning is more and more wide- ly used in the early screening of lung cancers, to some extent, NECT scanning has become one of the most commonly used examination modalities for SPNs.12 In addition to confirming the location, NECT is also used to perform a morphologic eval- uation of SPNs. Generally speaking, the smaller the SPN, the more likely it is to be a benign lesion. In terms of the margins and contours of an SPN, it can be classified as smooth, lobulated, irregular, or spiculated.1,5,6 Studies have reported that about 80% of benign SPNs are not larger than 2.0 cm in diameter.1,5,6,13 However, some studies showed that about 15% of malignant SPNs are not larger than 1.0 cm in diameter, and 42% of malignant SPNs are not larger than 2.0 cm in diameter.1,14 Most benign SPNs have smooth and well-defined mar- gins. However, about 21% of malignant SPNs have well-defined margins.15 A lobulated, irregular, or spiculated contour is usually associated with a malignant SPN. However, lobulation also occurs in up to 25% of benign SPNs.16 Therefore, there are considerable overlaps in the size, margins, and contours of benign and malignant SPNs. Internal characteristics (e.g. homogeneous attenuation, cav- itation, intranodular fat, presence and pattern of intranodular calcification, etc.) and abnormalities surrounding nodules (e.g. pleural indentation, ves- sel convergence, adjacent bronchial changes, etc.) are also helpful for distinguishing benign from malignant SPNs.1 For example, intranodular fat and a popcorn like calcification are typical find- ings seen in hamartomas. However, intranodular fat or calcification alone cannot be used to differ- entiate benign from malignant SPNs confidently. In our study, there were significant differences between the malignant and benign SPNs only in smooth margins (29.34% vs 71.19%), lobulated sign (68.86% vs 24.58%), spiculated sign (78.81% vs 21.19%), vacuole sign (22.75% vs 11.02%), pleural indentation (53.29% vs 21.19%), and vessel con- FIGURE 3. A solitary pulmonary nodule (SPN) with the size of 17.0 × 19.0 mm located in the middle lobe of right lung of a 59 years old male. Non-contrast enhanced CT (NECT), both upper (A) and (B) images, showed that there was sign of smooth margin, but without signs of lobulation, spiculation, vacuole, cavitation, air bronchogram, calcification, fat, pleural indentation, vessel convergence, or adjacent bronchial changes. Contrast enhanced CT (CECT) showed that there were mild and obvious enhancements in the arterial (C) and venous (D) phases, respectively. The patient was scanned with a CT-guided percutaneous lung biopsy procedure, and the pathological result showed chronic inflammatory disease. This SPN disappeared after a week of antibiotic therapy. FIGURE 4. A solitary pulmonary nodule (SPN) with the size of 25.0 × 27.0 mm located in the superior lobe of left lung of a 61 years old female evaluated by CT perfusion imaging (CTPI). Blood volume (BV) (A), blood flow (BF) (B), mean transit time (MTT) (C), and permeability surface (PS) (D) for the SPN were 6.16 ml/100 g, 34.16 ml/100 g/min, 10.94 s, and 25.97 ml/100 g/min, respectively. Pathology of the SPN after the surgery confirmed the diagnosis of an adenocarcinoma. Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules28 vergence (50.30% vs 27.97%) on NECT (all p values < 0.05). Our results are consistent with some pre- vious studies.17-19 Some previous studies also re- ported that these findings on NECT are risk factors for malignant SPNs. However, we did not identify cavity sign, air bronchogram, calcification, fat, ad- jacent bronchial changes, location, or size as risk factors for malignant SPNs (all p values > 0.05). This may be explained by the number of patients with SPNs included in our and previous studies which varied largely. Our results and previous studies also showed considerable overlaps in the findings of benign and malignant SPNs on NECT. Therefore, when we performed the classification of the 285 SPNs, there were 157 cases that were classi- fied as the Category II, III, and IV nodules. For CECT, it is helpful to improve the accuracy of differential diagnosis between benign and ma- lignant SPNs. The degree of enhancement is di- rectly associated with the possibility of malignan- cy and the vascularity of the SPNs. Swensen et al.20 reported that nodular enhancement of more than 20 HU on CECT highly implies a malignant SPN. Whereas, nodular enhancement of less than 15 HU on CECT typically indicates a benign SPN, with a sensitivity, specificity, accuracy, PPV, and NPV of 98%, 58%, 77%, 68%, and 96%, respectively. In our study, the diagnostic criteria for CT enhancement were: a CT enhancement amplitude value of 20– 60 HU was classified as malignant, and a CT en- hancement amplitude value of less than 20 HU or more than 60 HU was classified as benign. We also want to highlight that CT postprocessing technol- ogy of thin-section and multiplanar reconstruction (MPR) are indeed beneficial when differentiating benign and malignant SPNs. This is because more details would be identified with thin-section CT and MPR. On the other hand, comprehensive mor- phological features evaluation is far better than a single morphological features assessment because more negative (or positive) morphological features will increase the likelihood of a malignant (or be- nign) SPN. CTPI in evaluating SPNs CTPI allows the derivation of several physiologic parameters, including BV, BF, MTT, and PS. BV is defined as the integral under a corrected attenua- tion curve for enhancement values from a contrast material bolus. It is a relative measure of the blood volume within small vessels in a region of specific tissue. BV is related to the number and diameter of open vessels, etc. MTT is a measure of the time it takes for blood to pass through small vessels. BF refers to the blood flow passing through a section of the blood vessel in unit time which is related TABLE 4. Non-enhanced computed tomography (NECT) in evaluating solitary pulmonary nodules (SPNs) with various categories in 285 patients Items Category I Category II Category III Category IV Category V Benign SPNs (n = 118) 39 9 48 22 0 Malignant SPNs (n = 167) 0 5 54 19 89 Category I = most likely benign; Category II = possibly benign; Category III = uncertain benign or malignant; Category IV = possibly malignant; Category IV = most likely malignant TABLE 5. Solitary pulmonary nodules evaluated with CT perfusion imaging Parameters* Benign SPNs (n = 118) Malignant SPNs (n = 167) P values BF (ml/100 g/min) 49.34 (27.78, 72.81) 58.44 (24.91, 80.47) 0.1022 BV (ml/100 g) 4.79 (2.87, 7.66) 4.84 (2.90, 7.74) 0.1829 MTT (s) 6.71 (3.05, 9.58) 7.66 (3.83, 10.54) 0.2034 PS (ml/100 g/min) 8.89 (4.94, 12.45) 14.37 (11.50, 16.29) < 0.0001 BF = blood flow; BV = blood volume; CT = computed tomography; MTT = mean transit time; PS = permeability surface; SPNs = solitary pulmonary nodules; *compared with the Mann Whitney rank sum test TABLE 6. Solitary pulmonary nodules (SPNs) evaluated with dual-energy CT Parameters* Benign SPNs (n = 118) Malignant SPNs (n = 167) P Values aOL (HU) 13.24 (10.97, 21.58) 19.58 (13.29, 26.07) < 0.0001 vOL (HU) 11.09 (10.09, 14.86) 14.99 (10.59, 23.98) < 0.0001 aIC (mg/ml) 0.69 (0.47, 0.985) 1.13 (0.70, 1.56) < 0.0001 vIC (mg/ml) 0.55 (0.44, 1.00) 0.97 (0.50, 1.46) < 0.0001 aNIC 0.10 (0.06, 0.13) 0.18 (0.11, 0.25) < 0.0001 vNIC 0.23 (0.13, 0.32) 0.54 (0.43, 0.65) < 0.0001 aIC = Iodine concentration at the arterial phase; aNIC = normalized iodine concentration at the arterial phase; aOL = Iodine overlay at the arterial phase; CT = computed tomography; HU = Hounsfield unit; SPNs = solitary pulmonary nodules; vIC = Iodine concentration at the venous phase; vNIC = normalized iodine concentration at the venous phase; vOL = Iodine overlay at the venous phase; *compared with the Mann Whitney rank sum test Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 29 to the patency of drainage vein, lymphatic reflux, blood volume level, etc. BF is calculated with the equation: BF = BV/MTT, where BV is blood volume and MTT is mean transit time. PS refers to the dif- fusion coefficient of the unidirectional transmis- sion velocity of the contrast agent through capil- lary endothelial cells. CTPI can be used to evaluate the perfusion and vascularity of lesions in the lung and many other organs.21-28 Wang et al.21 reported that parameters of CTPI (including BF, BV and PS but not MTT) of SPNs were significantly correlated with SPNs’ mi- crovessel density (MVD) and luminal vascular pa- rameters such as luminal vascular number (LVN), luminal vascular area (LVA), and luminal vascular perimeter (LVP). Huang et al.22 analyzed the CT perfusion parameters (BF, BV, MTT, and PS) and the microvessel parameters (MVD, LVN, LVA, and LVP) in the non-small cell lung cancer (NSCLC) patients with and without lymph node metastasis. In our study, only the PS parameter of CTPI was found to be significant difference between the malignant and benign SPNs (p < 0.05), and a ROC curve analysis performed on the PS param- eter showed that the AUC, sensitivity, specific- TABLE 7. Solitary pulmonary nodules evaluated with dual-energy CT Parameters AUC Threshold Sensitivity Specificity 95% CI P values aOL (HU) 0.636 13.89 70.66 61.02 0.577–0.692 < 0.001 vOL (HU) 0.638 12.79 59.88 72.03 0.580–0.694 < 0.001 aIC (mg/ml) 0.657 0.65 67.66 69.49 0.599–0.712 < 0.001 vIC (mg/ml) 0.703 0.85 68.86 71.19 0.646–0.755 < 0.001 aNIC 0.728 0.12 67.66 74.58 0.672–0.778 < 0.001 vNIC 0.790 0.35 75.45 80.51 0.738–0.836 0.0001 aIC = Iodine concentration at the arterial phase; aNIC = normalized iodine concentration at the arterial phase; aOL = Iodine overlay at the arterial phase; AUC = area under the curve; CT = computed tomography; HU = Hounsfield unit; vIC = Iodine concentration at the venous phase; vOL = Iodine overlay at the venous phase; vNIC = normalized iodine concentration at the venous phase 95 % CI = 95 % confidence interval TABLE 8. Pairwise comparison of AUC of dual energy CT parameters in 285 patients with solitary pulmonary nodules Parameters* Z statistic P value aOL vs vOL 0.0995 0.9207 aOL vs aIC 0.813 0.4162 aOL vs vIC 2.485 0.0129 aOL vs aNIC 3.171 0.0015 aOL vs vNIC 5.170 < 0.0001 vOL vs aIC 0.702 0.4829 vOL vs vIC 2.567 0.0103 vOL vs aNIC 3.280 0.0010 vOL vs vNIC 5.345 < 0.0001 aIC vs vIC 2.034 0.0420 aIC vs aNIC 2.755 0.0059 aIC vs vNIC 4.728 < 0.0001 vIC vs aNIC 1.036 0.3001 vIC vs vNIC 3.227 0.0013 aNIC vs vNIC 2.708 0.0068 aIC = Iodine concentration at the arterial phase; aOL = Iodine overlay at the arterial phase; aNIC = normalized iodine concentration at the arterial phase; AUC = area under the curve; CT = computed tomography; vIC = Iodine concentration at the venous phase; vNIC = normalized iodine concentration at the venous phase; vOL = Iodine overlay at the venous phase; *compared with the DeLong test FIGURE 5. Receiver operating characteristic curve for distinguishing benign from malignant nodules using CT perfusion imaging parameter of permeability surface (PS). Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules30 ity, and diagnostic threshold were 0.739, 85.03%, 66.10%, and 9.88 ml/100g/min, respectively. Our results are not completely consistent with previous reports.7,8,29 For example, in the systematic review and meta-analysis performed by Huang et al.8, lung cancer was found having higher BV, BF, MTT, and PS values than benign lesions; and AUC values of BV and PS were 0.92 (0.90, 0.94) and 0.83 (0.80, 0.86), respectively. The explanations may be as follow. PS mainly reflects the structural integrity of the capillary wall and the osmotic pressure between plasma and tissue fluid. There are a large number of microvessels and rich blood supply in malig- nant tumors. However, the capillary wall is imma- ture, which is mainly reflected in the incomplete structure of vascular wall, including the basement membrane and epithelial cells. As a result, the vas- cular permeability will increase, promoting a large number of contrast agents to enter the tissue space. By contrast, the capillary wall of benign nodules is mature with a complete basement membrane and continuous endothelial cells which decreases the contrast agents to enter into the tissue space. On the other hand, although there are abundant microves- sels in inflammatory lesions in benign nodules, in addition to the mature development of vascular wall, the edema and congestion of surrounding tis- sues will lead to the increase of tissue hydrostatic pressure which will also slow down the penetra- tion rate of contrast medium. Therefore, the overall result is that the PS value in malignant SPNs was higher than that of benign SPNs. FIGURE 6. A solitary pulmonary nodule (SPN) with the size of 24.0 × 26.0 mm located in the superior lobe of left lung of a 57 years old female evaluated by arterial (A) and venous phases (B) of dual-energy CT (DECT). Iodine concentration at the arterial phase (aIC), Iodine concentration at the venous phase (vIC), normalized iodine concentration at the arterial phase (aNIC), and normalized iodine concentration at the venous phase (vNIC) were 2.409 mg/mL, 10.23 mg/mL, 0.17 (2.409/14.18), 0.53 (10.23/19.18) respectively. Pathology of the SPN after the surgery confirmed the diagnosis of an adenocarcinoma. TABLE 9. Solitary pulmonary nodules evaluated with multimodality CT imaging Methods* Sensitivity (%) Specificity (%) Accuracy (%) PPV (%) NPV (%) Method A 83.23 63.56 75.09 76.37 72.82 % Method B 85.63 67.80 78.25 79.01 76.92 % Method C 84.43 66.10 76.84 77.90 75.00 % Method A+B 94.61 74.58 86.32 84.04 90.72 % Method A+C 92.81 77.97 86.67 85.64 88.46 % Method B+C 95.81 81.36 89.82 87.91 93.20 % Method A+B+C 97.60 88.14 93.68 92.09 96.30 % CT = computed tomography; Method A = non-contrast enhanced CT combined with contrast enhanced CT; Method B = non-contrast enhanced CT combined with dual energy CT; Method C = non-contrast enhanced CT combined with CT perfusion imaging; Method A + B = non-contrast enhanced CT combined with contrast enhanced CT and dual energy CT; Method A + C = non-contrast enhanced CT combined with contrast enhanced CT and CT perfusion imaging; Method B + C = non-contrast enhanced CT combined with dual energy CT and CT perfusion imaging; Method A + B + C = non-contrast enhanced CT combined with contrast enhanced CT, dual-energy CT, and CT perfusion imaging; NPV = Negative predictive value; PPV = Positive predictive value; *compared with the McNemar’s test Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 31 DECT in evaluating SPNs There are mainly three techniques commercially available for DECT scanning. These techniques include: (a) fast voltage switching DECT, (b) layer detector DECT, and (c) dual-source DECT.30 In this study, we used a fast voltage switching DECT system. It has the ability to obtain virtual nonen- hanced images, arterial phase and venous phase contrast enhanced images, and iodine maps in one examination.30 An iodine map is the decomposition of mate- rial components realized by dual DECT accord- ing to the different attenuation characteristics of substances under high and low energy. It is the imaging of iodine material density extracted from enhanced iodine components. Iodine maps can effectively inhibit the background CT value and match with artificial color maps which can direct- ly reflect the difference in iodine concentration in the lesion. The iodine concentration in the lesions can be measured quantitatively, and the lesions with slight enhancement can be displayed more sensitively. The CT value of the lesions measured on the iodine maps is the net enhancement value of the lesions. By measuring the enhancement val- ue of lesions in iodine maps, we can evaluate the hemodynamic changes of tumors and the curative effects after tumor treatments. In our results, parameters of DECT, which in- cluded aOL, vOL, aIC, vIC, aNIC, and vNIC, in ma- lignant SPNs, were significantly higher than those of benign SPNs (all p values < 0.05). Compared with the AUC of aOL, vOL, aIC, vIC, and aNIC, the vNIC had the largest AUC (all p values < 0.05). Our results are consistent with many previous publications.9,10,31-34 For example, Ha et al.31 differ- entiated pulmonary metastasis from benign lung nodules in thyroid cancer patients by using DECT parameters finding that the DECT parameters (IC, NIC, NIC in using pulmonary artery, λHU, and Z-effective values) of the metastatic nodules were significantly higher than those of the benign nod- ules (all p values < 0.05). Multimodality CT imaging in evaluating SPNs Zhou et al.35 analyzed the relationship between clinical data, tumor markers, chest high-resolution CT (HRCT), and pathology in patients with SPNs finding that a joint evaluation model had bet- ter diagnostic efficiency for the diagnosis of SPN with diameter ≤ 2.0 cm. In another study, Zhu et al.9 reported that IC from DECT was significantly correlated with low-dose volume perfusion CT (VPCT) parameters (BF, BV, MTT, flow extraction product (FED), pulmonary nodule enhancement peak (PPnod), and VPCT parameters (BV, FED, and PPnod) had better diagnostic performance for SPN than DECT parameters (IC). Both of the above studies showed that multimodal evaluation is helpful in improving the diagnostic efficiency of SPNs. In this study, the diagnostic performances of the three diagnostic methods were separately used or in combination for differentiating benign and malignant SPNs were calculated, and the re- sults showed that multimodality CT imaging had higher performances than single modality CT im- aging in differentiating between benign and ma- lignant SPNs in sensitivity, specificity, and accu- racy (all p values < 0.05). To the best of the authors’ knowledge, this is the first study in the literature in which NECT, CECT, CTPI and DECT were com- prehensibly used to differentiate benign from ma- lignant SPNs. FIGURE 7. Receiver operating characteristic curves for distinguishing benign from malignant nodules using dual-energy CT parameters. aOL = Iodine overlay at the arterial phase (AUC = 0.636); vOL = Iodine overlay at the venous phase (AUC = 0.638); aIC = Iodine concentration at the arterial phase (AUC = 0.657); vIC = Iodine concentration at the venous phase (area under the curve [AUC] = 0.703); aNIC = normalized iodine concentration at the arterial phase (AUC = 0.728); vNIC = normalized iodine concentration at the venous phase (AUC = 0.790); all p values < 0.05 Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules32 Radiation in multimodality CT imaging Radiation is an important consideration when using a multimodality CT imaging protocol. Previous studies have reported that the radiation dose of CTPI is the key factor hindering its appli- cation.9,36 In our study, the CTDIvol, DLP, and ED in the SPNs were 66.88 ± 4.36 mGy, 768.29 ± 91.65 mGy.cm, and 10.76 ± 1.18 mSv, respectively. We have tried to make the radiation dose in our study at an acceptable level by using low tube voltage, SmartmA, and iterative reconstruction.37-39 Tube voltage and tube current are positively correlated with the radiation dose.40-42 In addition, it is recom- mended that postset ASIR-V% (60% in this study) should be higher than or equal to preset ASIR-V% (50% in this study).43-45 All these radiation reduc- tion techniques have been shown to decrease the radiation dose while maintaining the image qual- ity.40-45 Some limitations of this study are: (a) only a fast voltage switching DECT was used in our study, and we did not compare our results with param- eters from a layer detector DECT or dual-source DECT; (b) we did not compare our results with parameters from a magnetic resonance imaging (MRI) or positron emission tomography computed tomography (PET/CT); (c) some parameters of CTPI and DECT were not included into our study for evaluating; (d) radiation dose of our multimodal- ity CT imaging protocol is still at a relatively high level; and (e) we did not include some advanced medical image analysis methods, such as artificial intelligence (AI), deep learning, or radiomics in our study. As a result, further investigations are needed to strengthen our findings in the future. Conclusions In conclusion, SPNs evaluated with multimodal- ity CT imaging contributes to improving the di- agnostic accuracy of benign and malignant SPNs (Table 10). NECT helps to locate and evaluate the morphological characteristics of SPNs. CECT helps to evaluate the vascularity of SPNs. CTPI using pa- rameter of PS and DECT using parameter of vNIC both are helpful for improving the diagnostic per- formance. Acknowledgments The authors would like to thank co-authors AB and MM for their help in editing and proofread- ing the article. This study was supported by grants TABLE 10. Step-wise approach of multimodality CT imaging for evaluating solitary pulmonary nodules1,46,47 Non-contrast enhanced CT Density 1. Solid 2. Subsolid Shape 3. Round or oval 4. Triangular or polygonal Margins 5. Smooth 6. Lobulated 7. Spiculated Internal characteristics 8. Fat 9. Calcification 10. Cavitation Some complex findings 11. Pleural retraction 12. Air bronchogram 13. Bubble like lucencies (pseudocavitation) 14. Cystic airspace 15. Vascular convergence Contrast enhanced CT Parameter (s) 16. Degree of enhancement CT perfusion imaging Parameter (s) 17. Permeability surface Dual-energy CT Parameter (s) 18. Normalized iodine concentration at the venous phase Radiol Oncol 2023; 57(1): 20-34. Yan G et al. / Multimodality CT imaging for solitary pulmonary nodules 33 from the Sichuan Provincial Commission of Health (Nos. 18PJ138, 19PJ283, 19PJ284, and 20PJ284), the Sichuan Provincial Department of Science and Technology (No. 2019YFQ0028), the Science and Technology Association of Suining City (Nos. 6 and 10), and the Science and Technology Bureau of Mianyang City (No. 2020YJKY004). References 1. Erasmus JJ, Connolly JE, McAdams HP, Roggli VL. Solitary pulmonary nodules: Part I. Morphologic evaluation for differentiation of benign and malignant lesions. 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This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Peripheral nerve tumours (PNTs) are rare, but important cause of peripheral nerve dysfunction. The aim of the study was to present a series of consecutive patients with PNTs evaluated in authors’ ultrasonography (US) practice. Patients and methods. The electronic medical records of patients with PNTs examined at our US laboratory from February 2013 to May 2020 were retrospectively reviewed. Data on gender, age, clinical features, PNT location, elec- trodiagnostic (EDx) features and US findings were collected. Results. In the analyzed period 2845 patients were examined in our US laboratory. From these 15 patients (0.5%) with PNTs were identified. Four of them (3 with confirmed neurofibromatosis) had multiple PNTs. Half of patients (53%) presented with features of peripheral nerve damage, and others with palpable mass or pain. The most often involved nerve was ulnar (36%). PNT cross sectional areas varied from 24 mm2 to 1250 mm2 (median, 61 mm2). Based in 5 pa- tients on histological and in remaining patients on US features, schwannoma was diagnosed in 40%, neurofibroma in 27%, and perineurioma in 27% of patients. Conclusions. As in previous reports, PNTs in our series presented with neurological symptoms, palpable mass or pain. In contrast to other focal neuropathies, particularly nerves with schwannomas, in spite of their large thickening, often demonstrated well preserved function. Adding US to our clinical practice, enabled us to diagnose these rare periph- eral nerve lesions that we missed before. Key words: electrodiagnosis; nerve cross-sectional area; peripheral nerves; peripheral nerve tumors; ultrasonography Introduction Peripheral nerve tumours (PNTs) are rare, but important cause of peripheral nerve dysfunction. Usually they present with neurological symptoms (muscle atrophy and weakness, paresthesia or sen- sory loss), palpable mass or pain.1,2 On examina- tion neurological deficits can be found distally to PNTs in the affected nerve innervation area. Mass movable perpendicular, but not along the periph- eral nerve axis, and sensations along the affected nerve elicited on mass percussion (i.e., Tinel’s sign) are also pointing to possible PNT.1,2 Even before PNT diagnosis is known, electrodiagnostic (EDx) testing is often performed to evaluate severity of peripheral nerve damage. However, EDx is not useful for PNT diagnosis1, but imaging studies are much more relevant. Magnetic resonance (MR) is regarded as the most useful method1,3, although ul- trasonography (US) is also gaining support among clinicians.3,4 Particularly when diagnosis of PNT is not known, US is most useful, because it is cheap and widely accessible. Imaging delineates the le- sion, identifies its relation to peripheral nerve, and helps to differentiate various types of PNTs (Table 1).5 There are several nice reviews describ- ing US characteristics of PNT.3,4 However, only few publications written mainly by radiologists Radiol Oncol 2023; 57(1): 35-41. Podnar S / Ultrasonography of peripheral nerve tumours36 describe actual clinical experiences with US diag- nosis of PNTs.5-7 In the present study we report a series of con- secutive patients referred to our US unit mainly from EDx laboratories in whom we have diag- nosed PNTs. Patients and methods We retrospectively reviewed the electronic medical records of all patients referred from February 2013 to May 2020 to the US laboratory at the Institute of Clinical Neurophysiology, University Medical Centre Ljubljana, Slovenia. Our unit is the only one dedicated to peripheral nerve US in Slovenia, a country with a population of two million. The National Ethics Committee of Slovenia approved the study (approval code: 63/07/17), and at the time of analysis all patients signed written informed consent. During the whole review process, all pa- tients’ personal information was carefully protect- ed. We were not blinded to the findings of the clinical neurologic examination and EDx testing. Before US examinations we performed a focused neurological examination of patients by ourselves. We used standard US equipment (ProSound Alpha 7, Hitachi Aloka Medical, Ltd., Tokyo, Japan), with a 4–13 MHz linear array transducer. We meas- ured tumor cross sectional areas (CSAs) by a trace method that excluded the hyperechoic rim.8 During review of US images we observed PNT features typical for neurofibromas and schwan- nomas (Table 1).5 In patients without histological diagnosis presumptive PNT diagnoses were based on clinical, EDx and particularly US features. In all included patients, we collected data on gender, age, symptoms (including duration), neurological, EDx features, US findings, and PNT location. Results In the analyzed period, in our US unit we exam- ined 2845 patients, and we found PNTs in 15 (0.5%) of them. Demographic features of individual pa- tients, PNT anatomic, clinical, EDx and US features are shown in Table 2. Our patients’ median age was 33 years (range: 16–69 years). Slightly less than half of them were male (47%). Four had multiple PNTs; in 2 neurofibromatosis type 1 (NF1), and in 1 neu- rofibromatosis type 2 (NF2) were previously di- agnosed. In another (patient #13, Tables 2–3) gene sequencing results are pending. Of 11 patients with single PNTs, 7 (64%) had lesions on the right side. Ulnar nerve was involved in 4 (36%), median, sci- atic and tibial in 2 (18%) patients each, and fibu- lar in 1 (9%) patient with single PNT. Elbow and forearm were each involved in 3 (27%), thigh and ankle in 2 (18%) patients each, and knee in 1 (9%) patient with single PNT. The most common clinical findings in our patients were weakness and senso- ry abnormalities, each found in 7 (47%) patients, followed by muscle atrophy in 6 (40%), pain in 3 (20%), sensitivity on mass percussion in 2 (13%) and only palpable mass in 1 patient (7%). At the time of US study, EDx report was available for 12 (80%) patients. Affected nerve compound muscle action potential (CMAP) amplitude was markedly FIGURE 1. (A) Transverse ultrasonographic (US) view of the median (M) and ulnar (U) nerve in the axillary region showing numerous globular hypoechoic peripheral nerve tumours (PNTs) causing largely increased nerve cross sectional areas (CSAs, 148 mm2 and 101 mm2, respectively). (B) Longitudinal view of a single partially encapsulated, slightly lobulated and rather homogenous oval PNT (length 24 mm, thickness 9 mm) with central, but poorly defined nerve-tumour transition.5 Most probably these numerous PNTs are neurofibromas, although diagnosis in this 52-year-old woman presenting with peripheral neuropathy, primary lymphedema, and history of mitral and aortic valve surgery (patient #13, Tables 2–3), is not known yet. A B Radiol Oncol 2023; 57(1): 35-41. Podnar S / Ultrasonography of peripheral nerve tumours 37 reduced or absent in 5 (42%) patients, and senso- ry nerve action potential (SNAP) amplitude in 9 (75%) patients. In our series CSAs of PNT varied from 24 mm2 to 1250 mm2 (median, 61 mm2). Ratio of PNT/unaffected segment of the same nerve CSA varied from 2.9 to 156 (median, 6.0). Morphological features of PNTs are presented in Table 3. Ratio of maximum/minimum PNT diameter varied from 1.5 to > 10 (median, 4.5). Majority (64%) of PNTs were of fusiform shape. PNT contour was smooth in 8 (53%), and lobulated in remaining 7 (47%). PNT encapsulation was complete in 7 (47%), partial in 6 (40%), and absent in 2 (13%). PNTs echotexture was heterogenous in 13 (87%). Nerve entrance into PNT was central in 10 (67%), eccentric in 3 (20%), and not possible to asses in 2 (13%). In our series infiltrative nerve-tumor transition was observed in 7 PNTs (58%), poorly defined in 5 (42%), and could not be observed in 3 (20%). Histological diagnosis was available in 5 patients (Tables 2 and 3) with schwannoma. In another patient with NF1 histol- ogy of multiple PNTs could be also established – TABLE 1. Morphological features useful for differentiation between neurofibromas and schwannomas5 Peripheral nerve tumor (PNT) feature Comment Maximum to minimum diameter Ratio > 3 → neurofibroma Shape: round, oval, fusiform Fusiform → neurofibroma Contour: smooth, lobulated Lobulated → neurofibroma Encapsulation: absent, partial, complete Complete → schwannoma Echogenicity: hypo-, iso-, hyper- Hypoechoic → PNT Echo texture: homogenous, heterogenous Heterogenous → schwannoma Cystic changes: absent, focal, partial, large Cystic changes → schwannoma Calcifications: absent, present Present → schwannoma Target sign: absent, present Nerve entrance: not identified, identified Nerve-tumor position: central, eccentric Central → neurofibroma Nerve-tumor transition: clear, poorly defined, infiltrative Infiltrative → neurofibroma Vascularity: increased, normal, decreased Hypovascular → neurofibroma TABLE 2. Demographic, anatomical, clinical, electrodiagnostic (EDx) and ultrasonographic (US) features of patients with peripheral nerve tumors (PNTs) # Gender Age Side Nerve Location Symptoms& Signs CMAP amp. (mV) SNAP amp. (μV) Tumor CSA (mm2) Tumor diagnosis Other 1 Male 69 R Ulnar Elbow AWS 43 Neurofibroma 2 Male 24 L #Radial Upper arm W 0.2 4 24 Schwannoma* NF2 3 Male 66 R Median Forearm Æ 6.9 5 49 Schwannoma 4 Male 16 L #Median Upper arm WS 61 Neurofibroma NF1 5 Female 26 R Ulnar Forearm AWS 0.2 0 30 Perineurioma 6 Female 18 L Sciatic Thigh AWS 0.4 0 109 Perineurioma 7 Female 18 R Fibular Knee AWS 0 0 47 Perineurioma 8 Male 47 L Ulnar Elbow M 7.6 3 348 Schwannoma* 9 Female 58 R Median Forearm P 7.6 16 45 Neurofibroma 10 Female 22 R Sciatic Thigh AWS 0 0 97 Perineurioma 11 Female 34 R Tibial Ankle PAWS 10.6 7 1250 Schwannoma* 12 Male 63 L Ulnar Elbow L 8.2 5 368 Schwannoma* 13 Female 52 R #Ulnar Forearm 6.2 12 212 Neurofibroma NF? 14 Male 24 R #Median Upper arm P 6.3 33 26 Neurofibroma* NF1 15 Female 33 L Tibial Ankle L 92 Schwannoma A = muscle atrophy; amp. = amplitude; CMAP = compound muscle action potential; CSA = cross sectional area; L = left; L = local sensitivity; M = palpable mass; NF1 = neurofibromatosis type 1; NF2 = neurofibromatosis type 2; P = pain; R = right; S = sensory loss; SNAP = sensory nerve action potential; W = weakness; # = patients had multiple tumors; * = histological diagnosis of PNT available Radiol Oncol 2023; 57(1): 35-41. Podnar S / Ultrasonography of peripheral nerve tumours38 neurofibroma. Based on histological definite, and based on clinical and US features probable diag- noses of schwannoma were made in 6 (40%), neu- rofibroma in 5 (27%), and perineurioma in 4 (27%) patients.5 Discussion PNTs are known to be rare, which was also con- firmed by our US experience, demonstrating PNTs in only every 200th examined patient. As in our US laboratory we see referrals from a number of Slovenian EDx laboratories, and examine primarily patients with unclear etiology of peripheral nerve lesions, in reality PNTs are probably even rarer. It was reported that PNTs constitute only 5% of adult, and 2% of pediatric upper extremity tumors.9 With median age of 33 years our patients were much younger compared to typical oncological patients, which is similar to previously reported series of patients with PNTs reporting mean age of 36 years10, and 37 years.7 As reported by others7,10, we also found equal distribution of PNTs between both genders. TABLE 3. Morphological features of peripheral nerve tumors (PNTs) found on ultrasonographic (US) examination5 of individual patients # Ratio Shape Contour Encapsulation Echo texture Nerve position Nerve transition Number Tumor diagnosis 1 5 Fusiform Lobulated Partial Heterogeneous Central Infiltrative Single Neurofibroma 2 ? Lobulated Partial Heterogeneous ? ? Several Schwannoma* 3 6 Fusiform Smooth Whole Heterogeneous Central Poorly defined Single Schwannoma 4 6 Fusiform Fusiform None Heterogeneous Central Infiltrative Several Neurofibroma 5 8 Fusiform Lobulated Partial Heterogeneous Central Infiltrative Single Perineurioma 6 > 10 Fusiform Lobulated Partial Heterogeneous ? ? Single Perineurioma 7 6 Fusiform Smooth None Homogenous Central Infiltrative Single Perineurioma 8 3 Oval Smooth Whole Heterogeneous Central Poorly defined Single Schwannoma* 9 > 10 Fusiform Lobulated Partial Heterogeneous Eccentric Infiltrative Single Neurofibroma 10 5 Fusiform Smooth Whole Heterogeneous Central Infiltrative Several Perineurioma 11 1,5 Oval Smooth Whole Homogenous Eccentric ? Single Schwannoma* 12 2,5 Oval Smooth Whole Heterogeneous Central Poorly defined Single Schwannoma* 13 2,5 Oval Smooth Whole Heterogeneous Central Poorly defined Several Neurofibroma 14 4 Fusiform Lobulated Partial Heterogeneous Central Infiltrative Several Neurofibroma* 15 1,7 Oval Smooth Whole Heterogeneous Eccentric Poorly defined Single Schwannoma Ratio = Maximum/minimum PNT diameter; * = histological diagnosis of PNT available FIGURE 2. Comparison of transverse image of the sciatic peripheral nerve tumour (PNT) on (A) magnetic resonance (MR) T1 (arrow) and (B) ultrasonography (US). (C) Longitudinal US view of PNT in the middle thigh (A – small picture on the left), affecting the tibial portion of sciatic nerve. In this 22-year-old girl MR revealed at the exit of sciatic nerve from the pelvis another PNT affecting its fibular portion, and causing right foot drop of 14 years duration (patient #10, Tables 2–3). Based on clinical and imaging features in this patient diagnosis of probable perineurioma was made. A B C Radiol Oncol 2023; 57(1): 35-41. Podnar S / Ultrasonography of peripheral nerve tumours 39 We could divide our patient cohort into several groups. The first group consisted of 4 patients with numerous PNTs in several peripheral nerves. Two of patients from this group with numerous neurofi- bromas had known NF1 and a single patient with schwannomas had NF2. Diagnosis in a remaining patient (#13), with numerous neurofibromas in all US examined peripheral nerves (Figure 1), has not been established yet. The second group consisted of three girls (pa- tients #6, 7, 10) presenting in the first decade of life with unilateral foot drop. Each of them had sev- eral lumbo-sacral spine MRs that all proved nega- tive, and their PNTs were not diagnosed until they presented to our US laboratory 8–15 years after symptom onset. At that time their clinical features were unchanged for several years, so none of them decided to have a nerve biopsy to establish a his- tological PNT diagnosis. Nevertheless, we believe these 3 patients most probably have sciatic nerve perineurioma.11 One of them (patient #10) had two PNTs, separated by 20–30 cm segment of US rela- tively normal sciatic nerve. Using US we demon- strated the more distal PNT of the tibial portion of sciatic nerve (Figure 2). Additional more proximal PNT affecting fibular portion of sciatic nerve, and causing foot drop was found on MR. The third group consisted of four patients with PNTs on the ulnar nerve in the elbow seg- ment (Figure 3). The main challenge in this group of patients is differentiation of PNTs from ulnar neuropathy at the elbow (UNE) due to entrapment under the humeroulnar aponeurosis (i.e., cubital tunnel syndrome). The main distinctive feature of PNTs is their large CSA (43 mm2, 348 mm2, 45 mm2, and 368 mm2). We found CSA larger than 40 mm2 in only 1 of 202 (0.5%) UNE patients,12 and is there- fore extremely rare. Another characteristic feature of PNTs is rather well preserved nerve function in spite of large nerve thickening. By contrast, large CSA in UNE is as a rule accompanied by severe nerve dysfunction.12 What is the reason for such high number of PNTs on ulnar nerve in the elbow area is not clear. One possibility would be expo- sure of the nerve in this segment to mechanical stress. Alternative explanation would be a sam- pling artefact, as we see a plenty of patients with suspected UNE. If the latter case that would mean that a large number of PNTs on other nerves and locations are still missed. A B FIGURE 3. (A) Transverse and (B) longitudinal ultrasonographic (US) view of a large peripheral nerve tumour (PNT) on the left ulnar nerve just above the elbow. Three years before this 47-year-old man noted a palpable mass that in the last 6 months on touching started to elicit electrisation spreading into the last two fingers (patient #8, Tables 2–3). Well encapsulated, slightly lobulated, predominantly cystic and highly heterogenous hypoechoic oval lesion with central and poorly defined nerve-tumour transition can be seen. Histological examination confirmed a diagnosis of schwannoma. Radiol Oncol 2023; 57(1): 35-41. Podnar S / Ultrasonography of peripheral nerve tumours40 Of remaining 4 patients, 1 had definite and another probable tibial nerve schwannoma at the ankle, which is again a region of considerable me- chanical stress. Another young woman had a fusi- form thickening of the ulnar nerve in the forearm (patient #5, Figure 4). She had surgical release of the ulnar nerve exit from the flexor carpi ulnaris muscle, with no apparent benefit. She might also have perineurioma, or less likely neurofibroma. In the fourth man probable median nerve schwan- noma in the forearm was a coincidental finding during US evaluation due to Lewis-Sumner syn- drome, and caused no additional symptoms. As described previously1 according to clinical presentation, our patients could be divided into two groups. Eight (53%) patients presented with features of peripheral nerve lesion (e.g., muscle atrophy, weakness, sensory loss). Remaining 7 pa- tient presented by other clinical features (i.e., pain, local sensitivity, palpable mass), or as coincidental FIGURE 4. (A) Hands of a 26-year-old woman with 4-year history of muscle atrophy, weakness and numbness in the distal ulnar nerve territory (patient #5, Tables 2–3). Note intrinsic right palm muscle atrophy and clawing of the last two fingers. (B) On transverse ultrasonographic (US) view ulnar nerve cross sectional area (CSA) increased from 7 mm2 both proximally and distally to 20 mm2 in the middle of the lesion. (C) On longitudinal view a partially encapsulated, lobulated, fusiform hypoechoic right ulnar peripheral nerve tumour (PNT) of the forearm can be seen. Based on clinical and US features, we made a diagnosis of probable perineurioma. A B C Radiol Oncol 2023; 57(1): 35-41. Podnar S / Ultrasonography of peripheral nerve tumours 41 PNT finding without symptoms. In the first group CMAP and SNAP amplitudes of affected nerves were severely reduced or absent (42% and 75%, respectively), and in the second group they were mainly preserved (58% and 25%, respectively). The first group consisted of all 4 young women with probable perineurioma, and additional 3 patients with probable neurofibroma. By contrast, majority of the second group consisted of 4 patients with probable or definite schwannoma (Table 2). Before introduction of US into our institution, we diagnosed PNTs only very rarely. This changed after we started to perform US studies. Some PNTs that we finally diagnosed using US, were causing patients’ unexplained severe nerve dysfunction for more than a decade. Without US we would prob- ably not be able to diagnose PNTs in majority of patients from this series, and some of them would probably remain without diagnosis to this day. The main limitation of the present study was that in majority of patients histological diagno- sis of PNTs was not available. Therefore, in these patients we based our PNT diagnoses on clinical and particularly imaging features of the lesions. For differentiation between schwannomas and neurofibromas we applied US criteria of Ryu et al. that demonstrated high diagnostic accuracy.5 Unfortunately, no similar criteria are available for US diagnosis of perineurioma. Another limitation of the present study was its retrospective design; at the time of image analysis all projections needed for optimal differentiation between schwannomas and neurofibromas were therefore not available (Table 3). In conclusion, the present study confirmed that PNTs are rare, but important cause of peripheral nerve dysfunction. We found US critically impor- tant for demonstration of PNTs, and published US criteria as useful to differentiate schwanno- mas from neurofibromas. Unfortunately, no such criteria are available for perineuriomas. PNTs are most likely when their continuity with peripheral nerves is demonstrated, and discrepancy between lesions’ large size and well preserved nerve func- tion is found. Acknowledgments Author thanks Dr. Gregor Omejec for collabora- tion in performance of US examinations, and Mr. Boštjan Kastelic for help with preparing of fig- ures. The author is supported by the Republic of Slovenia Research Agency, Grant No. P3-0338. References 1. July J, Guha A. Peripheral nerve tumors. Handb Clin Neurol 2012; 105: 665- 74. doi: 10.1016/b978-0-444-53502-3.00016-1 2. Kubiena H, Entner T, Schmidt M, Frey M. Peripheral neural sheath tumors (PNST) – what a radiologist should know. Eur J Radiol 2013; 82: 51-5. doi: 10.1016/j.ejrad.2011.04.037 3. Abreu E, Aubert S, Wavreille G, Gheno R, Canella C, Cotten A. Peripheral tumor and tumor-like neurogenic lesions. Eur J Radiol 2013; 82: 38-50. doi: 10.1016/j.ejrad.2011.04.036 4. Gruber H, Glodny B, Bendix N, Tzankov A, Peer S. High-resolution ultrasound of peripheral neurogenic tumors. Eur Radiol 2007; 17: 2880-8. doi: 10.1007/ s00330-007-0645-7 5. Ryu JA, Lee SH, Cha EY, Kim TY, Kim SM, Shin MJ. Sonographic differentiation between Schwannomas and neurofibromas in the musculoskeletal system. J Ultrasound Med 2015; 34: 2253-60. doi: 10.7863/ultra.15.01067 6. Telleman JA, Stellingwerff MD, Brekelmans GJ, Visser LH. Nerve ultrasound in neurofibromatosis type 1: a follow-up study. Clin Neurophysiol 2018; 129: 354-9. doi: 10.1016/j.clinph.2017.11.014 7. Reynolds DL, Jr., Jacobson JA, Inampudi P, Jamadar DA, Ebrahim FS, Hayes CW. Sonographic characteristics of peripheral nerve sheath tumors. AJR Am J Roentgenol 2004; 182: 741-4. doi: 10.2214/ajr.182.3.1820741 8. Omejec G, Zgur T, Podnar S. Diagnostic accuracy of ultrasonographic and nerve conduction studies in ulnar neuropathy at the elbow. Clin Neurophysiol 2015; 126: 1797-804. doi: 9. Forthman CL, Blazar PE. Nerve tumors of the hand and upper extremity. Hand Clin 2004; 20: 233-42, v. doi: 10.1016/j.hcl.2004.03.003 10. Pedro MT, Antoniadis G, Scheuerle A, Pham M, Wirtz CR, Koenig RW. Intraoperative high-resolution ultrasound and contrast-enhanced ultra- sound of peripheral nerve tumors and tumorlike lesions. Neurosurg Focus 2015; 39: E5. doi: 10.3171/2015.6.focus15218 11. Salvalaggio A, Cacciavillani M, Coraci D, et al. Nerve ultrasound and 3D-MR neurography suggestive of intraneural perineurioma. Neurology 2016; 86: 1169-70. doi: 10.1212/wnl.0000000000002488 12. Omejec G, Podnar S. Utility of nerve conduction studies and ultrasonography in ulnar neuropathies at the elbow of different severity. Clin Neurophysiol 2020; 131: 1672-7. doi: 10.1016/j.clinph.2020.02.019 Radiol Oncol 2023; 57(1): 42-50. doi: 10.2478/raon-2023-0007 42 research article Effects of dynamic contrast enhancement on transition zone prostate cancer in Prostate Imaging Reporting and Data System Version 2.1 Jiahui Zhang1, Lili Xu1, Gumuyang Zhang1, Xiaoxiao Zhang1, Xin Bai1, Hao Sun1,2, Zhengyu Jin1,2 1 Department of Radiology, State Key Laboratory of Complex Severe and Rare Disease, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China 2 National Center for Quality Control of Radiology, Beijing, China Radiol Oncol 2023; 57(1): 42-50. Received 9 October 2022 Accepted 18 November 2022 Correspondence to: Hao Sun, Hao Sun, M.D., M.P.H., Department of Radiology, State Key Laboratory of Complex Severe and Rare Disease, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Shuaifuyuan No.1, Wangfujing Street, Dongcheng District, Beijing 100730, China. E-mail: sunhao_robert@126.com; Zhengyu Jin, Department of Radiology, State Key Laboratory of Complex Severe and Rare Disease, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Shuaifuyuan No.1, Wangfujing Street, Dongcheng District, Beijing 100730, China. E-mail: jinzy@pumch.cn 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 analyse the effects of dynamic contrast enhanced (DCE)-MRI on transi- tional-zone prostate cancer (tzPCa) and clinically significant transitional-zone prostate cancer (cs-tzPCa) in Prostate Imaging Reporting and Data System (PI-RADS) Version 2.1. Patients and methods. The diagnostic efficiencies of T2-weighted imaging (T2WI) + diffusion-weighted imaging (DWI), T2WI + dynamic contrast-enhancement (DCE), and T2WI + DWI + DCE in tzPCa and cs-tzPCa were compared using the score of ≥ 4 as the positive threshold and prostate biopsy as the reference standard. Results. A total of 425 prostate cases were included in the study: 203 cases in the tzPCa group, and 146 in the cs- tzPCa group. The three sequence combinations had the similar areas under the curves in diagnosing tzPCa and cs- tzPCa (all P > 0.05). The sensitivity of T2WI + DCE and T2WI + DWI + DCE (84.7% and 85.7% for tzPCa; 88.4% and 89.7% for cs-tzPCa, respectively) in diagnosing tzPCa and cs-tzPCa was significantly greater than that of T2WI + DWI (79.3% for tzPCa; 82.9% for cs-tzPCa). The specificity of T2WI + DWI (86.5% for tzPCa; 74.9% for cs-tzPCa) were significantly greater than those of T2WI + DCE and T2WI + DWI + DCE (68.0% and 68.5% for tzPCa; 59.1% and 59.5% for cs-tzPCa, respectively) (all P < 0.05). The diagnostic efficacies of T2WI + DCE and T2WI + DWI + DCE had no significant differ- ences (all P > 0.05). Conclusions. DCE can improve the sensitivity of diagnosis for tzPCa and cs-tzPCa, and it is useful for small PCa lesion diagnosis. Key words: prostate cancer; transitional zone; magnetic resonance imaging; dynamic contrast enhancement Introduction Prostate cancer (PCa) is a common malignant tumour and has the second highest incidence of all cancers worldwide in males1; it is the sec- ond deadliest among male cancer patients in the United States.2 Prostate-specific antigen (PSA) as- says and magnetic resonance imaging (MRI) are widespread in the diagnosis and follow-up ex- amination of PCa, and thus the PCa detection rate continues to increase yearly worldwide.3 MRI is a non-invasive examination and can clearly show the anatomy of the prostate. It is one of the most valuable imaging methods for the diagnosis of PCa.4,5 PCa is primarily located in the peripheral zone, but approximately 25% of PCa cases are lo- Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 43 cated in the transitional zone. The MRI features of PCa in the transitional zone are similar to central glandular benign prostatic hyperplasia (BPH) in signal intensity and morphological characteristics, thus making it difficult to differentiate between BPH and PCa.6 The early diagnosis and timely in- tervention of PCa is key to improve the prognosis of PCa patients.7 Thus, it is of great significance to improve the diagnostic accuracy of transitional- zone prostate cancer (tzPCa). The Prostate Imaging Reporting and Data System (PI-RADS) is currently recognized as an important guideline for prostate examination and diagnosis via MRI. The first and second editions of PI-RADS guidelines (v1, v2) were proposed in 2012 and 2015, respectively.8,9 In 2019, PI-RADS Version 2.1 (v2.1) reformulated the research speci- fications for MRI acquisition technology in pros- tate scanning and analysed the diagnostic values of multiparametric MRI (mpMRI), which includes T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), and dynamic contrast-enhanced (DCE)-MRI sequencing.10 A prostate transitional- zone lesion score criterion of PI-RADS v2.1 only includes T2WI and DWI sequences; DCE-MRI has no effect on the transitional-zone lesion score. The benefits and drawbacks of the DCE-MRI se- quence for PCa diagnosis are increasingly being recognized. The advantages of omitting the DCE sequence are that it shortens the scan time and reduces examination costs. However, the DCE se- quence with a higher spatial resolution could be less prone to motion artefacts, susceptibility arte- facts from metallic implants, or rectal gas. It can be used informally as a “safety-net” or “back-up” sequence to detect lesions that might otherwise be missed on T2WI or DWI, e.g., when the readers have less experience or when there is insufficient image quality.10-12 Therefore, the value of DCE-MRI as a routine sequence has been debated.10,13-17 Prompt intervention is needed for clinically sig- nificant prostate cancer (Gleason score ≥ 3 + 4), which usually has poor differentiation, a damaged gland structure, a high degree of malignancy, and strong invasiveness.9 This study used three se- quence combinations (T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE) to analyse the diagnostic value of DCE-MRI for detecting tzPCa and clini- cally significant transitional-zone prostate cancer (cs-tzPCa) in PI-RADS Version 2.1, using trans- perineal ultrasound-guided prostate biopsy as the reference standard. Patients and methods Patients The Institutional Review Board (IRB) of our hospi- tal approved this retrospective study (IRB number JS-2114), and the requirement for informed consent was waived. The clinical and MRI data of 894 pa- tients with prostate diseases admitted to our hos- pital from January 2015 to December 2019 were continuously collected. The inclusion criteria were FIGURE 1. Flowchart showing the patient recruitment process. Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 44 as follows: (1) a complete MRI examination includ- ing T1WI, T2WI, and DWI with the correspond- ing apparent diffusion coefficient (ADC) map and DCE sequences; (2) diagnosis confirmed by trans- perineal ultrasound-guided prostate biopsy after MRI; and (3) no biopsy, radiation therapy, chemo- therapy, hormonal therapy, or other therapies prior to MRI examination. The exclusion criteria were as follows: (1) after MRI examination, the lesion was suspected to be located in the peripheral zone (n = 438); (2) poor MRI image quality affected scoring (n = 5); and (3) biopsy or other therapies prior to MRI (n = 26). Figure 1 shows the flowchart of the pa- tient recruitment process. In total, 425 cases were included. Non-tzPCa indicates transitional-zone lesions other than cancer, such as BPH or normal prostate. Free cs-tzPCa indicates transitional-zone lesions other than clinically significant prostate cancer, including insignificant prostate cancer and non-tzPCa. MRI protocol This work used a 3.0-T, eight-channel, surface- phased array coil abdominal MRI scanning sys- tem (GE750; GE Health care, Milwaukee, WI, USA). The centre of the coil was placed at the pubic sym- physis during positioning and fixed with a band to reduce artefacts from breathing movements. The scan sequence included axial T1WI and T2WI, coronal and sagittal T2WI, DWI, and DCE. When the b-value was 100 and 1000 sec/mm2, we used the corresponding ADC graph for evaluation. Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) offered enhanced contrast at a dose of 0.1 mmol/kg and a flow rate of 3 ml/s. The MRI im- age-acquisition parameters are shown in Table 1. Pathological diagnosis The Gleason grading system for histopathologi- cal grading of PCa was adopted.18 Here, cs-tzPCa was confirmed if the tumour’s Gleason score was ≥ 3 + 4 with or without ≥ 0.5-cm3 tumour volume and/or extraprostatic extension.9,19 According to the 2014 International Society of Urological Pathology (ISUP) criteria, cs-tzPCa was defined as ISUP grade≥2.20 Patients biopsied all underwent a 12-core systematic trans-perineal ultrasound- guided prostate biopsy by one urinary specialist. The prostate gland was divided into 11 regions on ultrasound scans. At least one additional targeted biopsy was performed; the cognitive targeted bi- opsy using cognitive registration was based on zonal anatomy or imaging landmarks such as re- markable nodules. The details of the cognitive tar- geted biopsy are as follows: first, the urologist re- viewed the MRI results; and second, the urologist used the MRI information to perform the targeted biopsy for the most remarkable nodules guided by ultrasound images. A pathologist reported the pathological score of prostate biopsy specimens. The radiologist matched the lesions with the high- est PI-RADS v2.1 scores on the MRI images with the pathology report. Image analysis Two radiologists with 10 and 5 years of experi- ence in prostate imaging/diagnosis independently TABLE 1. Sequence parameters for prostate MRI Parameters T2WI DWI DCE Sequence FRFSE SE-EPI 3D-GRE TR/TE (ms) 4137/86 4200/90 4.3/1.3 Flip angle 110° 90° 12° Echo train length 32 1 N/A FOV (mm × mm) 270 × 270 360 × 360 400 × 400 Matrix size 288 × 192 128 × 96 320 × 192 Slice thickness (mm) 3.0 3.0 3.0 Other b values = 0, 50, 100, 150, 200, 500, 800, 1000, 1500, 2000 sec/mm2 Temporal resolution < 10s, total scan time of 5 min DCE = dynamic contrast-enhancement; DWI = diffusion-weighted imaging; FOV = field of view; FRFSE = fast relaxation fast spin echo; SE-EPI = spin echo echo planar imaging; T2WI = T2-weighted imaging; TE = time echo; TR = repetition time; 3D-GRE = 3D-gradient echo Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 45 analysed the images under double-blinded con- ditions. The radiologists scored the images of in- dex lesions in each sequence using PI-RADS v2.1 and then calculated scores for T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE. When multiple lesions were present, the lesion with the highest PI-RADS score was used for statistical analysis. In the case of disagreement between the two radiolo- gists, the final score was discussed with a third ra- diologist to reach a consensus. On PI-RADS v2.1, both T2WI and DWI scored 1–5 points whereas the DCE score was binary [negative (−) or positive (+)]. The final total score of T2WI + DCE and T2WI + DWI + DCE was increased by one point when the image showed early and focal enhancement (positive) of DCE; the final total score of T2WI + DCE and T2WI + DWI + DCE remained un- changed when the image was negative regarding DCE. The final score was 5 when the image scored 5 on T2WI regardless of the DWI and DCE scores. Detailed scoring criteria are shown in Table 2. Figure 2 shows a case of cs-tzPCa located in the right lobe of the prostate. Statistical analysis SPSS version 22.0 (IBM Corp., Armonk, NY, USA) and MedCalc version 15.0 (MedCalc Software, Ostend, Belgium) were used for statistical analy- sis. Agreement of readings between the two radi- ologists was evaluated by a kappa (κ) coefficient with κ < 0.20 indicating no agreement, κ = 0.21–0.40 indicating fair agreement, κ = 0.41–0.60 indicat- ing moderate agreement, κ = 0.61–0.80 indicating good agreement, and κ = 0.81–1.00 indicating ex- cellent agreement. Using PI-RADS ≥ 4 as the posi- tive threshold and prostate biopsy as the reference standard, receiver operating characteristic (ROC) curves for T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE in the diagnosis of tzPCa and cs- tzPCa were separately drawn to calculate areas under the curve (AUCs), sensitivities, specificities, positive predictive values (PPVs), negative pre- dictive values (PNVs), and accuracies to evaluate the corresponding diagnostic efficacies. We also compared differences among the detection rates of tzPCa and cs-tzPCa via three sequence combina- tions. The DeLong test was used to evaluate differ- ences among AUCs, and McNemar’s test was used to compare the sensitivities, specificities, and ac- curacies of different sequence combinations. The chi-square test was used to compare differences among the detection rates. P < 0.05 was considered statistically significant for all statistical tests. TABLE 2. Scoring criteria of transition zone prostate for three sequence combinations Scoring Criteria T2WI DWI DCE T2WI + DWI T2WI + DCE T2WI + DWI + DCE 1 1~5 − 1 1 1 1 1~5 + 1 2 2 2 1~3 − 2 2 2 2 1~3 + 2 3 3 2 4~5 − 3 2 3 2 4~5 + 3 3 4 3 1~4 − 3 3 3 3 1~4 + 3 4 4 3 5 − 4 3 4 3 5 + 4 4 5 4 1~5 − 4 4 4 4 1~5 + 4 5 5 5 1~5 − 5 5 5 5 1~5 + 5 5 5 DCE = dynamic contrast-enhancement; DWI = diffusion-weighted imaging; T2WI = T2-weighted imaging TABLE 3. Clinicopathological data of patients included in this study Clinicopathological data Patients Age (year), Mean ± SD 66 ± 9.0 T-PSA (ng/ml), Median (Upper and lower quartiles) 9.4 (6.3 + 15.4) tzPCa, n (%) 203 (48%) Non-tzPCa, n (%) 222 (52%) cs-tzPCa, n (%) 146 (34%) Free cs-tzPCa, n (%) 279 (66%) Tumor size (mm), Median (Upper and lower quartiles) 12.7 (9.1 + 22.5) GS, n (%) 3 + 3 57 (28%) 3 + 4 56 (28%) 4 + 3 24 (12%) 3 + 5 3 (1.5%) 4 + 4 18 (8.9%) 4 + 5 28 (13.8%) 5 + 3 1 (0.5%) 5 + 4 13 (6.4%) 5 + 5 3 (1.5%) cs-tzPCa = clinically significant transitional-zone prostate cancer; PSA = prostate-specific antigen; SD = standard deviation; tzPCa = transitional-zone prostate cancer Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 46 Results Clinicopathological data This study included 425 prostate cases per the afore- mentioned inclusion criteria. There were 203 cases in the tzPCa group (48%) and 222 in the non-tzPCa group (52%). There were 146 cases in the cs-tzPCa group (34%) and 279 in the free cs-tzPCa group (66%). The patient age range was 34–86 years (mean age, 66 years), and the median PSA value was 9.4 ng/ mL. The main clinical manifestations were elevated PSA, dysuria, frequent urination, and urinary ur- gency. Abnormalities may be found in the patients during digital rectal examination, including a stiff prostate or palpation of nodules. Table 3 shows the patients’ clinicopathological information. Agreement between the two radiologists The PI-RADS v2.1 scores provided by the two radi- ologists were analysed for agreement. The reading agreement between PI-RADS v2.1 scores for T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE was κ = 0.869, 0.855, and 0.868, respectively. Comparative analysis of the diagnostic efficacies of three sequence combinations in tzPCa and cs-tzPCa The diagnostic efficacies of three sequence com- binations in tzPCa and cs-tzPCa were shown in Tables 4 and 5. The AUCs were 0.863, 0.868, and 0.863 for tzPCa and 0.840, 0.833, and 0.824 for cs- tzPCa in T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE, respectively. The AUCs of three se- quence combinations in diagnosing tzPCa and cs- tzPCa had no significant differences (all P > 0.05). The sensitivity of T2WI + DCE and T2WI + DWI + DCE in diagnosing tzPCa and cs-tzPCa was sig- nificantly greater than that of T2WI + DWI (all P < 0.05). The specificity and accuracy of T2WI + DWI in diagnosing tzPCa and cs-tzPCa were signifi- cantly greater than those of T2WI + DCE and T2WI + DWI + DCE (all P < 0.05). The sensitivity, specific- ity, and accuracy of T2WI + DCE and T2WI + DWI + DCE in diagnosing tzPCa and cs-tzPCa had no significant differences (all P > 0.05). Comparative analysis of the cancer detection rates of three sequence combinations in tzPCa and cs-tzPCa Figures 3 and 4 show the cancer detection rates of three sequence combinations in tzPCa and cs-tzP- Ca. When the PI-RADS score was 4, T2WI + DWI had the highest cancer detection rate. The cancer detection rates of T2WI + DWI were significantly greater than those of T2WI + DCE and T2WI + DWI + DCE in tzPCa (69.4%, 29.8%, and 33.7%, respec- tively, all P < 0.05) and cs-tzPCa (43.9%, 19.2%, and 25.0%, respectively, all P < 0.05). The cancer detec- tion rates of the three sequence combinations had no significant differences in tzPCa or cs-tzPCa when the PI-RADS score was 1, 2, 3, and 5 (all P > 0.05). Discussion Some studies questioned whether DCE is a neces- sary sequence for routine MRI dynamic scanning FIGURE 2. Images from a 62-year-old male patient with a total prostate-specific antigen (PSA) of 7.66 ng/mL. (A) Axial T2-weighted imaging (T2WI) showed focal hypointensity on the right lobe of the prostate in the transitional zone with a blurred margin and a T2WI score of 3. (B) Diffusion-weighted imaging (DWI) showed a slight increase in lesion signal. (C) Reduced corresponding apparent diffusion coefficient (ADC) value with a score of 3. (D) dynamic contrast- enhancement (DCE) showed obvious enhancement in the early stage of the lesion, thus indicating that the DCE score was positive. Scores were 3, 4, and 4 based on T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE, respectively. The lesion was confirmed to be clinically significant transitional-zone prostate cancer (cs- tzPCa) based on biopsy (Gleason score of 5 + 4). A B C D Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 47 of the prostate.13-17 To the best of our knowledge, no prior studies have yet analysed the value of DCE- MRI in diagnosing tzPCa and cs-tzPCa. To inves- tigate whether DCE-MRI can improve the diag- nostic accuracy of tzPCa, this study innovatively compared and analysed the diagnostic efficiencies of T2WI + DWI, T2WI + DCE, and T2WI + DWI + DCE for detecting tzPCa and cs-tzPCa. This study had a large sample size and included 425 prostate cases—the results were reliable. The results of this study showed T2WI + DWI had higher specificity and accuracy, and DCE-MRI had higher sensitiv- ity in diagnosing tzPCa and cs-tzPCa. DCE-MRI could have a potential impact on the detection of tzPCa and cs-tzPCa. The AUCs of three sequence combinations in diagnosing tzPCa and cs-tzPCa had no significant differences. However, AUC values only reflect the global performance of the test; sensitivity and specificity trade-offs must also be compared be- tween the three sequence combinations. The speci- ficity and accuracy of T2WI + DWI was significant- ly greater than those of T2WI + DCE and T2WI + DWI + DCE in diagnosing tzPCa and cs-tzPCa. The prostate patients with an elevated PSA value and a positive digital rectal examination were more con- TABLE 4. Comparison of diagnostic efficacy of three sequence combinations in tzPCa tzPCa (n =203) Non-tzPCa (n=222) Sensitivity (%) Specificity (%) PPV (%) NPV(%) Accuracy (%) AUC (95% CI) T2WI + DWI ≥ 4 (n=191) 161 30 79.3 86.5 84.3 82.1 83.1 0.863(0.827–0.894)< 4 (n=234) 42 192 T2WI + DCE ≥ 4 (n=243) 172 71 84.7 68.0 70.8 83.0 76.0 0.868(0.832 + 0.899)< 4 (n=182) 31 151 T2WI + DWI + DCE ≥ 4 (n=244) 174 70 85.7 68.5 71.3 84.0 76.7 0.863(0.827 + 0.895)< 4 (n=181) 29 152 aP 0.001 < 0.001 NA NA 0.002 0.424 bP < 0.001 < 0.001 NA NA < 0.001 0.968 cP 0.500 1.000 NA NA 0.250 0.369 AUC = area under curve; CI = confidence interval; DCE = dynamic contrast-enhancement; DWI = diffusion-weighted imaging; NPV = negative predictive value; PPV = positive predictive value; tzPCa = transitional-zone prostate cancer; T2WI = T2-weighted imaging aP value between T2WI + DWI and T2WI + DCE; bP value between T2WI + DWI and T2WI + DWI + DCE; cP value between T2WI + DCE and T2WI + DWI + DCE TABLE 5. Comparison of diagnostic efficacy of three sequence combinations in cs-tzPCa cs-tzPCa (n=146) Free cs-tzPCa (n=279) Sensitivity (%) Specificity (%) PPV (%) NPV (%) Accuracy (%) AUC (95%CI) T2WI + DWI ≥ 4 (n=191) 121 70 82.9 74.9 63.4 89.3 77.6 0.840(0.802–0.874)< 4 (n=234) 25 209 T2WI + DCE ≥ 4 (n=243) 129 114 88.4 59.1 53.1 90.7 69.2 0.833(0.795–0.868)< 4 (n=182) 17 165 T2WI + DWI + DCE ≥ 4 (n=244) 131 113 89.7 59.5 53.7 91.7 69.9 0.824(0.785–0.869)< 4 (n=181) 15 166 aP 0.008 < 0.001 NA NA < 0.001 0.430 bP 0.002 < 0.001 NA NA < 0.001 0.101 cP 0.500 1.000 NA NA 0.250 0.193 AUC = area under curve; CI = confidence interval; DCE = dynamic contrast-enhancement; DWI = diffusion-weighted imaging; NPV = negative predictive value; PPV = positive predictive value; tzPCa = transitional-zone prostate cancer; T2WI = T2-weighted imaging aP value between T2WI + DWI and T2WI + DCE; bP value between T2WI + DWI and T2WI + DWI + DCE; cP value between T2WI + DCE and T2WI + DWI + DCE Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 48 fident in the diagnosis of cancer when DWI results were positive. T2WI + DCE and T2WI + DWI + DCE showed significantly higher sensitivity in both tzPCa and cs-tzPCa. Tamada et al.21 compared and analysed the diagnostic efficiencies of PI-RADS v2.1 scoring between T2WI + DWI + DCE and T2WI + DWI in clinically significant prostate cancer of 165 cases. Their results were similar to ours, but their study did not distinguish between peripheral zone cancers and transitional zone cancers. Eleven tzPCa cases that were not diagnosed by T2WI + DWI were diagnosed by T2WI + DCE and T2WI + DWI + DCE. The reason is that DCE can clearly show small PCa lesions and improve the sensitivity of diagnosis22, while the T2WI and DWI sequences are more likely to miss small lesions. A previous study demonstrated that DCE also im- proves the differential diagnosis of lesions located on the front edge of the peripheral zone with an unclear transition zone from the normal transition zone.19 T2WI + DCE and T2WI + DWI + DCE had higher diagnostic sensitivity, which could poten- tially help reduce repeat biopsies when the results were negative. However, active surveillance is still necessary. PCa is characterized by more angio- genic factors and numerous tumour blood vessels FIGURE 3. Histograms showing cancer detection rates of tzPCa based on the T2-weighted imaging (T2WI) + diffusion-weighted imaging (DWI), T2WI + dynamic contrast-enhancement (DCE), and T2WI + DWI + DCE. FIGURE 4. Histograms showing cancer detection rates of cs-tzPCa based on the T2-weighted imaging (T2WI) + diffusion-weighted imaging (DWI), T2WI + dynamic contrast-enhancement (DCE), and T2WI + DWI + DCE. Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 49 that exhibit increased permeability versus healthy blood vessels. PCa lesions typically enhance earlier and more obviously on DCE than adjacent prostate tissues.23,24 Vascular permeability also increases in benign diseases such as BPH.25 These factors might explain the low specificity and high false-positive rate of T2WI + DCE and T2WI + DWI + DCE in the diagnosis of tzPCa and cs-tzPCa. When the PI-RADS score was 4, the cancer detec- tion rates of T2WI + DWI were significantly greater than those of T2WI + DCE and T2WI + DWI + DCE in tzPCa and cs-tzPCa, which showed T2WI + DWI had lower false-positive rate than T2WI + DCE and T2WI + DWI + DCE. This may be because when BPH lesions strengthened earlier and were more obvious on DCE than adjacent prostate tissues, and when the score of T2WI or T2WI + DWI was 3, the scores of T2WI + DCE and T2WI + DWI + DCE would be increased by one point, thus becoming 4. This study does have some limitations: (1) This was a single-centre clinical retrospective analysis, which may have selection bias. In future work, we will use a multi-centre, prospective clinical study. (2) This work used prostate biopsy as the reference standard. Prostate biopsy specimens sometimes did not reflect the final pathological findings, which might have yielded false-negative results. (3) In addition to providing qualitative param- eters, DCE-MRI can also provide quantitative and semi-quantitative parameters26, which were not discussed further in this work. It remains unclear whether these parameters are useful in the early diagnosis of tzPCa and cs-tzPCa. In conclusion, DCE-MRI can improve the sen- sitivity of diagnosis and has a potential impact on the detection and active surveillance of tzPCa and cs-tzPCa. Meanwhile, DCE-MRI can clearly show small cancers and is useful for small PCa lesion diagnosis. Acknowledgments National High Level Hospital Clinical Research Funding (grant no.2022-PUMCH-A-033), the CAMS Innovation Fund for Medical Sciences (grant no.2022-I2M-C&T-B-019), National High Level Hospital Clinical Research Funding (grant no.2022-PUMCH-B-069), National High Level Hospital Clinical Research Funding (grant no.2022- PUMCH-A-035), the Natural Science Foundation of China (grant no.81901742), 2021 Key clinical Specialty Program of Beijing, Beijing Municipal Key Clinical 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. 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Gatti M, Faletti R, Calleris G, Giglio J, Berzovini C, Gentile F, et al. Prostate cancer detection with biparametric magnetic resonance imaging (bpMRI) by readers with different experience: performance and comparison with multiparametric (mpMRI). Abdom Radiol 2019; 44: 1883-93. doi: 10.1007/ s00261-019-01934-3 12. Wassberg C, Akin O, Vargas HA, Dave AS, Zhang Jb, Hricak H. The incre- mental value of contrast-enhanced MRI in the detection of biopsy-proven local recurrence of prostate cancer after radical prostatectomy: effect of reader experience. AJR Am J Roentgenol 2012; 199: 360-6. doi: 10.2214/ AJR.11.6923 13. Tamada T, Sone T, Jo Y, Hiratsuka J, Higaki A, Higashi H, et al. Locally recurrent prostate cancer after high- dose-rate brachytherapy: the value of diffusion- weighted imaging, dynamic contrast-enhanced MRI, and T2- weighted imaging in localizing tumors. AJR Am J Roentgenol 2011; 197: 408-14. doi: 10.2214/AJR.10.5772 14. Kuhl CK, Bruhn R, Krämer N, Nebelung S, Heidenreich A, Schrading S. Abbreviated biparametric prostate MR imaging in men with elevated prostate-specific antigen. Radiology 2017; 285: 493-505. doi: 10.1148/ radiol.2017170129 15. Junker D, Steinkohl F, Fritz V, Bektic J, Tokas T, Aigner F, et al. Comparison of multiparametric and biparametric MRI of the prostate: are gadolinium- based contrast agents needed for routine examinations? World J Urol 2019; 37: 691-9. doi: 10.1007/s00345-018-2428-y 16. Di Campli E, Delli P A, Seccia B, Cianci R, d’Annibale M, Colasante A, et al. Diagnostic accuracy of biparametric vs multiparametric MRI in clinically significant prostate cancer: comparison between readers with different ex- perience. Eur J Radiol 2018; 101: 17-23. doi: 10.1016/j.ejrad.2018.01.028 17. Huebner NA, Korn S, Resch I, Grubmüller B, Gross T, Gale R, et al. Visibility of significant prostate cancer on multiparametric magnetic resonance imaging (MRI)-do we still need contrast media? Eur Radiol 2021; 31: 3754-64. doi: 10.1007/s00330-020-07494-1 18. Epstein JI, Amin MB, Reuter VE, Humphrey PA. Contemporary Gleason grad- ing of prostatic carcinoma: an update with discussion on practical issues to implement the 2014 International Society of Urological Pathology (ISUP) consensus conference on Gleason Grading of Prostatic Carcinoma. Am J Surg Pathol 2017; 41: e1-7. doi: 10.1097/PAS.0000000000000820 Radiol Oncol 2023; 57(1): 42-50. Zhang J et al. / Effects of dynamic contrast enhanced on transitional-zone prostate cancer in PI-RADS V2.1 50 19. Weinreb JC, Barentsz JO, Choyke PL, Cornud F, Haider MA, Macura KJ, et al. PI-RADS Prostate Imaging - Reporting and Data System: 2015, Version 2. Eur Urol 2016; 69: 16-40. doi: 10.1016/j.eururo.2015.08.05 20. Oishi M, Shin T, Ohe C, Nassiri N, Palmer SL, Aron M, et al. Which patients with negative magnetic resonance imaging can safely avoid biopsy for prostate cancer? J Urol 2019; 201: 268-76. doi: 10.1016/j.juro.2018.08.046 21. Tamada T, Kido A, Yamamoto A, Takeuchi M, Miyaji Y, Moriya T, et al. Comparison of biparametric and multiparametric MRI for clinically sig- nificant prostate cancer detection with PI-RADS version 2.1. J Magn Reson Imaging 2021; 53: 283-91. doi: 10.1002/jmri.27283 22. Taghipour M, Ziaei A, Alessandrino F, Hassanzadeh E, Harisinghani M, Vangel M, et al. Investigating the role of DCE-MRI, over T2 and DWI, in accu- rate PI-RADS v2 assessment of clinically significant peripheral zone prostate lesions as defined at radical prostatectomy. Abdom Radiol 2019; 44: 1520-7. doi: 10.1007/s00261-018-1807-6 23. Engelbrecht MR, Huisman HJ, Laheij RJ, Jager GJ, van Leenders GJ, Hulsbergen-Van De Kaa CA, et al. Discrimination of prostate cancer from normal peripheral zone and central gland tissue by using dynamic con- trast-enhanced MR imaging. Radiology 2003; 229: 248-54. doi: 10.1148/ radiol.2291020200 24. Nicholson B, Theodorescu D. Angiogenesis and prostate cancer tumor growth. 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Eur J Radiol 2016; 85: 2119-26. doi: 10.1016/j.ejrad.2016.09.022 Radiol Oncol 2023; 57(1): 51-58. doi: 10.2478/raon-2023-0006 51 research article Pancreatic islets implanted in an irreversible electroporation generated extracellular matrix in the liver Yanfang Zhang1,2, Yanpeng Lv2,3, Yunlong Wang4, Tammy T Chang5, Boris Rubinsky2 1 Department of Endocrinology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, China 2 Department of Mechanical Engineering and Department of Bioengineering, University of California, Berkeley, USA 3 School of Electrical Engineering, Zhengzhou University, Zhengzhou, China 4 Henan Bioengineering Research Center, Zhengzhou, China 5 Department of Surgery, University of California, San Francisco, San Francisco, USA Radiol Oncol 2023; 57(1): 51-58. Received 5 November 2022 Accepted 24 November 2022 Correspondence to: Prof. Boris Rubinsky, Department of Mechanical Engineering and Department of Bioengineering, University of California Berkeley, Berkeley CA 94720, USA. E-mail: rubinsky@berkeley.edu 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. Pancreatic islet transplantation via infusion through the portal vein, has become an established clinical treatment for patients with type 1 diabetes. Because the engraftment efficiency is low, new approaches for pan- creatic islets implantation are sought. The goal of this study is to explore the possibility that a non-thermal irreversible electroporation (NTIRE) decellularized matrix in the liver could be used as an engraftment site for pancreatic islets. Materials and methods. Pancreatic islets or saline controls were injected at sites pre-treated with NTIRE in the liv- ers of 7 rats, 16 hours after NTIRE treatment. Seven days after the NTIRE treatment, islet graft function was assessed by detecting insulin and glucagon in the liver with immunohistochemistry. Results. Pancreatic islets implanted into a NTIRE-treated volume of liver became incorporated into the liver paren- chyma and produced insulin and glucagon in 2 of the 7 rat livers. Potential reasons for the failure to observe pancre- atic islets in the remaining 5/7 rats may include local inflammatory reaction, graft rejection, low numbers of starting islets, timing of implantation. Conclusions. This study shows that pancreatic islets can become incorporated and function in an NTIRE-generated extracellular matrix niche, albeit the success rate is low. Advances in the field could be achieved by developing a better understanding of the mechanisms of failure and ways to combat these mechanisms. Key words: liver cancer; pancreatic islet transplantation; non-thermal irreversible electroporation; tissue engineering; diabetes Introduction The experimental work of Ballinger and Lacy was the first to demonstrate the feasibility of using pan- creatic islets transplants in the treatment of diabe- tes.1 Intraperitoneal and intramuscular injections of pancreatic islets reduced hyperglycemia, polyu- ria and glycosuria in rats, but consistently normal values were rarely achieved.1 Kemp et al. suggested that since insulin from the pancreatic cells is nor- mally secreted into the portal venous system, the liver may provide a more physiological environ- ment for transplanted islets in intraperitoneal or subcutaneous locations.2 Indeed, injection of islets into the rat portal vein resulted in normal urine volume, normal glycemia and abolition of glyco- Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation52 suria for 2 months after transplantation, suggest- ing that the liver was an effective site for pancreatic islets transplantation.2 Pancreatic islet transplantation via infusion through percutaneous transhepatic access of the portal vein, has since become a clinical treatment for patients with type 1 diabetes or undergoing to- tal pancreatectomy.3,4,5 Despite major advances in pancreatic islet transplantation via the portal vein, the engraftment efficiency of islets remains low and only about 10–20% of transplanted islets are estimated to survive.5 Approximately 60% of the transplanted islets are lost in the very early stages of the post-transplantation period.6 The causes of low islet engraftment and functional durability are multi-factorial. Major contributing factors include early islet death due to failed engraftment and the thrombotic/inflammatory reaction induced within the portal vein.5 Instant blood-mediated inflam- matory reaction triggered by the direct exposure of the islets to blood contributes to the loss of trans- planted islets.7 As the technology to develop stem cell-derived beta-cells and islets mature, demand for islet transplantation is expected to increase and the procedure become more widespread.8 Identification of new and improved methods for pancreatic islets implantation has become an im- portant area of research.9,10 In an attempt to over- come deficiencies related to the injection of pancre- atic islets through the portal vein, while retaining the advantages of islet implantation into the liver, Fujita et al. reported on a method of direct trans- plant of pancreatic islets, in the form of an islet- cell-sheet, onto the liver surface.11 They found that the implanted islet tissue consisted predominantly of insulin-positive beta cells. Glucagon-positive alpha cells were also present, but their numbers were small and they were sparsely distributed within the islet tissue.11 Here, we investigate a different approach to transplant pancreatic islets within the liver utiliz- ing the special attributes of non-thermal irrevers- ible electroporation (NTIRE). NTIRE has become a clinically useful modality for cancer treatment in the liver.12 NTIRE is a minimally-invasive tissue ablation technique in which electric field pulses are delivered across a treatment volume to ir- reversibly permeabilize the cell membrane. Cell death is induced in the NTIRE-treated volume13,14 likely through necroptosis and/or pyroptosis pathways.15,16 The extracellular matrix within the treated volume remains intact and blood vessels remain patent.14,17 Accordingly, NTIRE has been used to ablate solid tumors near sensitive body structures, such as large blood vessels within the liver18 and pancreas.19 Large blood vessels in the NTIRE-treated region remain intact; the blood ves- sels do not leak and the endothelial layer regen- erates.20 Our earlier research has shown that the extracellular matrix that remains after NTIRE can serve as a scaffold for native cell regeneration in the liver14, blood vessels21 intestines.17 These stud- ies also suggested the use of NTIRE to generate an extracellular matrix for regenerative medicine.22-24 This study was motivated by findings from our earlier studies.25,26 In25 it was shown that trans- planted hepatocytes engraft into host liver paren- chyma when directly implanted into an NTIRE pre-treated area. NTIRE improved exogeneous cell engraftment likely by killing host hepatocytes while preserving the extracellular matrix, thereby creating space and a supportive niche for new cell engraftment. Importantly, NTIRE induces a pro- reparative innate immunity milieu, which may further promote integration of transplanted cells.26 In contrast, an earlier study has shown that in the absence of NTIRE pre-treatment, direct implan- tation of three-dimensional cell clusters, such as hepatocyte organoids, into the liver, has limited durability and tissue incorporation.27 In that study, hepatic organoids were directly implanted within a hepatotomy site in the liver of immune compe- tent mice.27 By day 3 after the implantation, most hepatocytes within organoids were apoptotic or necrotic, and by day 7, chronic inflammatory reac- tions developed around the implanted organoids.27 The goal of this study is to evaluate if a NTIRE decellularized matrix in the liver could serve as a graft site for pancreatic islets directly implanted in the liver and if grafted pancreatic islets could sur- vive and become functional in this type of niche. Materials and methods Approvals Experiments were performed on Sprague Dawley male rats weighing 250–350 g. Seven rats received NTIRE and islet transplants, and 7 rats were used as islet donors. All animals received humane care from properly trained professionals in compliance with both the Principals of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Institute of Health (NIH publication no. 85-23, revised 1985), and treated according to an animal protocol ap- proved by the Animal Care and Use Committee of the University of California, Berkeley. The Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation 53 University of California Berkeley Animal Care and Use Committee approved the animal protocol AUP-2017-12-10605. NTIRE ablation protocol After partial mobilization of the liver from adja- cent tissue, the liver lobe was gently clamped be- tween two 10-mm-diameter electrodes (Harvard Apparatus, Holliston, MA, USA), as shown in Figure 1. The measured distance between the two electrodes is 3.0 ± 0.2 mm. A sequence of 10 square pulse with an electric field of 1000 V/cm, 100 μs pulse width, separated by 100 ms was applied between the electrodes, across the liver, using an electroporator (ECM 830, Wave Electroporation System (BTX Harvard Apparatus, Holliston, MA, USA). Previous studies with the experimental configuration in this study have shown that this electroporation protocol produces minimal ther- mal damage, and the tissue is affected primarily by irreversible electroporation.23 Because the elec- trodes are flat parallel surfaces, the voltage on the electrodes can be set to achieve a uniform electric field through most of the liver volume bound be- tween the surface electrodes, except at the edges. This voltage was adjusted to yield an electric field of 1000 V/cm in the central area of the parallel elec- trodes. Two different lobes were electroporated in each animal, one lobe for the injection of pancreatic islets and the second lobe for injection of a control of a phosphate buffered solution (PBS). Seven rats were used as recipients in the pancreatic islet trans- plant experiment. Procurement of pancreatic islets Pancreatic islets were prepared as described.28 Rats were initially anesthetized with 3–5% isoflurane by placing them in an induction chamber. No more than 8 mg/kg bupivacaine was injected once subcu- taneously prior to skin incision. Once anesthetized, the rat was then transferred to a nose cone respira- tor connected to a precision vaporizer that deliv- ered 1–3% isoflurane for maintenance. The perito- neal cavity was entered via a midline incision of the abdomen. The common bile duct was cannulat- ed and injected with digestion solution-containing collagenase P (Roche, #11249002001). The pancreas was carefully excised and pulled away from the in- testines, stomach, mesentery and spleen, put into a 50 ml centrifuge tube, and immediately incubated in a 37°C water bath for 20 min. The rats were eu- thanized by a combination of an overdose of va- porized isoflurane and a bilateral chest dissection. Pancreas digestion was then terminated by adding cold washing buffer containing Hanks’ Buffered Salt Solution (HBSS, Hyclone SH3058802), 1% Penicillin-Streptomycin (Pen-Strep, Invitrogen, 15070063), 1% 1M N-2-hydroxyethylpiperazine- N-2-ethane sulfonic acid (HEPES, Invitrogen, 15630080), 0.1% Deoxyribonuclease I(DNase I), 1.7 ml 1 M CaCl2/1000 ml and 1.2 ml 1 M MgCl2/1000 ml. After being washed in washing buffer several times and filtered by a strainer, the digested islets were separated on a Histopaque (Sigma-Aldrich, 11191) density gradient. The islets were then hand- picked and the Histopaque was washed out by a washing buffer. The islets were resuspended in 5 ml culture media and poured into a Petri dish for hand-picking. A few sample fresh islets were stained with dithizone (DTZ) to confirm their puri- ty. Stock solution of DTZ (0.5 mg/ml) was prepared in dimethyl sulfoxide (DMSO). The stock solution was diluted five times in Dulbecco’s Phosphate FIGURE 1. Photograph of a clamp electroporation electrode on a liver lobe. Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation54 Buffered Solutions, (DPBS) solution. The morphol- ogy of purified islets is shown in Figure 2. The islet cells were scarlet with DTZ staining, while exo- crine cells were devoid of staining. Arrows point to the few exocrine cells left after digestion by col- lagenase. The pancreatic islets were generated in seven rats, with one rat providing the pancreatic islets per transplant recipient. Implantation of pancreatic islets Rats pre-treated with NTIRE were anesthetized and the previously made midline incision was re-opened 16 hours later. Approximately 200 pan- creatic islets were transplanted into a recipient. Islets were washed and re-suspended in 0.2 ml PBS. Using a 1 ml syringe and 26-gauge needle, islets were injected into the center of one of the IRE-treated liver lobes. The second, control lobe was injected with the same volume of PBS into the IRE- treated region in that lobe. Figure 3 illustrates the site of the injections at the center of the ablated region. At the completion of the pancreatic islet in- jection procedure, the abdominal wall was closed and sutured. The animals were kept two in a cage, given food and water freely and continuously monitored for well-being by a veterinarian affili- ated with the animal care facility. No controls with injection of pancreatic islets in the untreated liver parenchyma was performed. A previous study has shown that in the absence of NTIRE pre-treatment, direct implantation of three-dimensional hepato- cytes cell clusters into the liver had limited dura- bility and tissue incorporation.27 In contrast, trans- planted hepatocytes engraft into host liver paren- chyma when directly implanted into an NTIRE pre-treated area.25 Islet graft analysis We collected and examined the livers 7 days after the NITRE procedure. Islet graft function was as- sessed by detecting insulin and glucagon in the liver by immunohistochemistry. The treated liver tissue samples were cut in a plane normal to the liver lobe surface, through the center of the treated lesion. The liver was fixed in formalin, embedded in paraffin, and sectioned 5 μm thick (Histo-Tec Laboratory, Hayward, CA, USA). The samples were sent to UT Health San Antonio STRL Histology/ Immunohistochemistry Laboratory Department of Pathology and Laboratory Medicine where they were stained with guinea pig anti-insulin antibod- ies and rabbit anti-glucagon antibodies. Results and discussion The livers of recipient rats were treated with 1000 V/cm NTIRE in 2 locations. Sixteen hours later, one site was injected with PBS and the other site was injected with approximately 200 donor rat pancre- atic islets. We chose to analyze the livers 7 days af- FIGURE 2. Photograph of the pancreatic islets used in this study stained by dithizone (DTZ). Arrows point to the few unstained exocrine cells left after digestion by collagenase. FIGURE 3. An illustration showing the implantation site in the liver. The dark area is the non-thermal irreversible electroporation (NTIRE) treated area. Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation 55 ter the NTIRE treatment because, in rats, the liver completely regenerates 7 days after injury.29 Figure 4 shows the appearance of the NTIRE- treated lobe that was injected with PBS. To show that staining by insulin and glucagon antibodies was specific to the sites injected with pancreatic is- lets, the sites injected with PBS were stained with insulin or glucagon antibodies, Figure 4 A and B, respectively. The control NTIRE-treated liver re- generated and was not stained by the insulin or glucagon antibodies. Panel 4C is a higher magni- fication of the previously NTIRE treated region. It shows that 7 days after IRE ablation, the hepato- cytes were intact and well-developed. Nuclei were clear, and sinusoids were seen. There was no evi- dence of scar tissue or fibrosis. This illustrates the regenerative capability of NTIRE-treated livers. Within the NTIRE-treated liver parenchyma that received islet implantation, we found evi- dence of engrafted insulin- and glucagon-pro- ducing cells in 2 of the 7 animals. Figures 5A and 5B show the presence of engrafted pancreatic is- lets within the liver as demonstrated by positive insulin and glucagon staining. Insulin-stained cells (beta cells) and glucagon-stained cells (alpha cells) are marked with full arrows in the higher magnification images Fig 5C and 5D, respectively. Fully developed and normal hepatocytes were ob- served (dotted arrows) around the glucagon- and insulin-positive cells. These findings show that it is possible for pancreatic islets, injected in tissue treated with NTIRE, to become integrated in the liver parenchyma and become functional produc- ing glucagon and insulin. The fact that 7 days after the implantation the pancreatic islets have become incorporated in the liver parenchyma and generate insulin and gly- cogen in 2 of the 7 animals, is encouraging. By comparison, in previous experiments with direct injection of hepatocyte organoids in the liver pa- renchyma, most hepatocytes within implanted or- ganoids were apoptotic or necrotic by day 3 after transplant.27 The goal of this study is limited to showing that pancreatic islets can become incorporated in a niche formed by NTIRE treatment of the liver. In that sense, the results are promising. Nevertheless, the incorporation was observed in only 2 of the 7 repeats, which is a low rate of success. It should be emphasized, however, that engraftment efficiency of pancreatic islet transplantation through the por- tal vein is not optimal, which is why alternative approaches for transplantation are explored.5 We speculate that there are several reasons for our low rate of success. An important aspect of the procedure is choos- ing the optimal time for the exogenous pancre- atic islet implantation in the NTIRE treated liver. In view of the limited success in this study, this must be investigated further. In our previous study of implantation of hepatocytes in the liver, the implantation was done 3 days after the NTIRE procedure.25 In this study, the choice for the time of implantation is based on the results of a previ- ous study on the mechanisms of cell death in the NTIRE-treated liver.16 That study followed the tem- poral events in a liver treated with the same NTIRE protocol as in this study. It was observed that the hepatocytes appear morphologically intact one hour after the NTIRE procedure. This suggested that the implantation cannot be done at the same time as the NTIRE ablation, because the hepato- cytes still occupied the treated volume. At 6 hours after the procedure, dead hepatocytes are seen FIGURE 4. Liver tissue treated with non-thermal irreversible electroporation (NTIRE) and injected with PBS served as the control. Seven days after treatment with NTIRE and injection of PBS, immunohistochemical staining was performed on the controls using insulin and glucagon antibodies (University of Texas at Houston, USA). In the control slides, no cells were stained with insulin or glucagon antibodies, shown in (A) and (B), respectively. The higher magnification panel 4C shows normal hepatocytes with no evidence of scar tissue. A B C Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation56 throughout the treated volume. By 24 hours after NTIRE, hepatocyte regenerative proliferation may be initiated.29 On the basis of these considerations, we implanted the pancreatic islets at 16 hours after the NTIRE treatment. Ideally, one would choose a time in which the NTIRE niche is free from dying hepatocytes and from regenerating hepatocytes, so that pancreatic islets have optimized conditions to become incorporated into the extracellular matrix. However, the inflammation and repair responses within of the NTIRE-treated region are also impor- tant factors. Our group showed that pro-reparative macrophages start to replace pro-inflammatory macrophages at day 3 after NTIRE treatment and become the dominant macrophage subtype by day 7.26 Therefore, it is quite possible that 16 hours af- ter the NTIRE treatment is not the optimal time. A later timepoint in which pro-reparative mac- rophages predominant within the NTIRE-treated volume may be more conducive to islet engraft- ment. Determining the optimal time for implan- tation, in context of an immunological response coupled with hepatocyte regeneration, is a critical aspect of this procedure and requires much more research. Finally, islet graft rejection may have had an effect on the limited rate of success. No anti- rejection or immunosuppressive medications were used in this study. Technical reasons may have also played a role in the results. The rate and mode of injection of the pancreatic islets into the liver parenchyma is important. The injection should be slow, con- trolled and gradual.27 In this study, injection with a hand-held syringe may not have been the same in all the repeats. Another technical reason may be the injection of the pancreatic islets along the tract of the 26-gauge needle. As shown in Figure 5, the injection site is relatively narrow, on the order of 200 μm, and therefore, the histology section through the center of the lesion may have missed FIGURE 5. Liver tissue treated with non-thermal irreversible electroporation (NTIRE) and injected with 200 rat donor pancreatic islets. Seven days later, immunohistochemical staining was performed with anti-insulin and anti-glucagon antibodies (University of Texas at Houston, USA). In the pancreatic islets injected tissue, insulin-stained cell clusters are shown in (A) and (C) (full arrows). Normal hepatocytes were observed (dotted arrows) around the insulin-stained cells in the liver lobe. Glucagon- stained cells cluster are shown in (B) and (D) (full arrows). Normal hepatocytes were observed (dotted arrows) around the glucagon-stained cells in the liver lobe. A B C D Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation 57 the injected islets in some of the repeats. Another technical reason may be the small number of the injected pancreatic islets. In this study, we used 200 pancreatic islets per injection site, and this may have been too low a number. Obviously, the low rate of success is of concern and should be further investigated. Conclusions This study shows that pancreatic islets injected into a niche formed in the liver by NTIRE abla- tion became incorporated and functional in 2 of 7 experimental animals 7 days after NTIRE treat- ment. While the rate of success is low, the study demonstrates that pancreatic islets can become in- corporated in a tissue niche generated by NTIRE ablation. More research is needed to improve the success rate of this technique before it can have clinical value. Author contributions YZ designed and performed the animal experiment and aided in the analysis of the data. YL designed and performed the electroporation. YW advised on the animal experiment and edited the animal ex- periment section. TC, contributed to the analysis of the results, writing of the paper and with advice. BR supervised the research, designed the experi- ment, analyzed the data, and wrote the paper. Acknowledgments This work was supported by the Department of Mechanical Engineering, University of California, Berkeley, the National Natural Science Foundation of China (No. 81702768), Science and technology project of Henan province (No. 212102310193), Innovative leading talents of science and tech- nology project of Health Commission of Henan Province (No. YXKC2021024), NIH R21-EB024135, and Open Philanthropy Project. We thank the associate chief pathologist Junhong Li who generously aided in the pathologi- cal analysis of slides. References 1. Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972; 72: 175-86. PMID: 4262169 2. Kemp CB, Knight MJ, Scharp DW, Ballinger WF, Lacy PE. Effect of trans- plantation site on the results of pancreatic islet isografts in diabetic rats. Diabetologia 1973; 9: 486-91. doi: 10.1007/BF00461694 3. Shapiro AJ, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson RP, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 355: 1318-30. doi: 10.1056/NEJMoa061267 4. McCall M, Shapiro AJ. Update on islet transplantation. Cold Spring Harb Perspect Med 2012; 2: a007823. doi: 10.1101/cshperspect.a007823 5. Korsgren O, Nilsson B, Berne C, Felldin M, Foss A, Kallen R, et al. Current status of clinical islet transplantation. Transplantation 2005; 79: 1289-93. doi: 10.1097/01.tp.0000157273.60147.7c 6. Biarnés M, Montolio M, Nacher V, Raurell M, Soler J, Montanya E. β-cell death and mass in syngeneically transplanted islets exposed to short-and long-term hyperglycemia. Diabetes 2002; 51: 66-72. doi: 10.2337/diabe- tes.51.1.66 7. Bennet W, Groth CG, Larsson R, Nilsson B, Korsgren O. Isolated human islets trigger an instant blood mediated inflammatory reaction: implications for in- traportal islet transplantation as a treatment for patients with type 1 diabe- tes. Upsala J Med Sci 2000; 105: 125-33. doi: 10.1517/03009734000000059 8. Silva IB, Kimura CH, Colantoni VP, Sogayar MC. Stem cells differentiation into insulin-producing cells (IPCs): recent advances and current challenges. Stem Cell Res Ther 2022; 13: 1-24. doi: 10.1186/s13287-022-02977-y 9. Cayabyab F, Nih LR, Yoshihara E. Advances in pancreatic islet transplantation sites for the treatment of diabetes. Front Endocrinol 2021: 12: 732431. doi: 10.3389/fendo.2021.732431 10. Rickels MR, Robertson RP. Pancreatic islet transplantation in humans: recent progress and future directions. Endocr Rev 2019; 40: 631-68. doi: 10.1210/ er.2018-00154 11. Fujita I, Utoh R, Yamamoto M, Okano T, Yamato M. The liver surface as a favorable site for islet cell sheet transplantation in type 1 diabetes model mice. Regen Ther 2018; 8: 65-72. doi: 10.1016/j.reth.2018.04.002 12. Koethe Y, Wilson N, Narayanan G. Irreversible electroporation for colorectal cancer liver metastasis: a review. Int J Hyperthermia 2022; 39: 682-7. doi: 10.1080/02656736.2021.2008025 13. Davalos RV, Mir LM, Rubinsky B. Tissue ablation with irreversible electropo- ration. Ann Biomed Eng. 2005; 33: 223-31. doi: 10.1007/s10439-005-8981-8 14. Rubinsky B, Onik G, Mikus P. Irreversible electroporation: a new ablation modality—clinical implications. Technol Cancer Res Treat 2007; 6: 37-48. doi: 10.1177/153303460700600106 15. Al-Sakere B, André F, Bernat C, Connault E, Opolon P, Davalos RV, et al. Tumor ablation with irreversible electroporation. PloS ONE 2007; 2: e1135. doi: 10.1371/journal.pone.0001135 16. Zhang Y, Lyu C, Liu Y, Lv Y, Chang TT, Rubinsky B. Molecular and histological study on the effects of non-thermal irreversible electroporation on the liver. Biochem Biophys Res Commun 2018; 500: 665-70. doi: 10.1016/j. bbrc.2018.04.132 17. Phillips MA, Narayan R, Padath T, Rubinsky B. Irreversible electroporation on the small intestine. Br J Cancer 2012; 106: 490-5. doi: 10.1038/bjc.2011.582 18. Hitpass L, Distelmaier M, Neumann UP, Schöning W, Isfort P, Keil S, et al. Recurrent colorectal liver metastases in the liver remnant after major liver surgery – IRE as a salvage local treatment when resection and thermal ablation are unsuitable. Cardiovasc Intervent Radiol 2022; 45: 182-9. doi: 10.1007/s00270-021-02981-4 19. Lee EW, Shahrouki P, Peterson S, Tafti BA, Ding PX, Kee ST. Safety of irrevers- ible electroporation ablation of the pancreas. Pancreas 2021; 50: 1281-6. doi: 10.1097/MPA.0000000000001916 Radiol Oncol 2023; 57(1): 51-58. Zhang Y et al. / Irreversible electroporation-enabled islet transplantation58 20. Maor E, Ivorra A, Mitchell JJ, Rubinsky B. Vascular smooth muscle cells ablation with endovascular nonthermal irreversible electroporation. J Vasc Interv Radiol 2010; 21: 1708-15. doi: 10.1016/j.jvir.2010.06.024 21. Maor E, Ivorra A, Leor J, Rubinsky B. Irreversible electroporation attenuates neointimal formation after angioplasty. IEEE Trans Biomed Eng 2008; 55: 2268-74. doi: 10.1109/TBME.2008.923909 22. Phillips M, Maor E, Rubinsky B. Principles of tissue engineering with non- thermal irreversible electroporation. J Heat Transfer 2011; 133: 011004. doi: 10.1115/1.4002301. 23. Phillips M, Maor E, Rubinsky B. Nonthermal irreversible electropora- tion for tissue decellularization. J Biomech Eng 2010; 132: 091003. doi: 10.1115/1.4001882 24. Phillips M, Maor E, Rubinsky B. Lavee J. Extracellular matrix material cre- ated using non-thermal irreversible electroporation. US Patent 08835166, Sep 16 2014. 25. Chang TT, Zhou VX, Rubinsky B. Using non-thermal irreversible elec- troporation to create an in vivo niche for exogenous cell engraftment. Biotechniques 2017; 62: 229-31. doi: 10.2144/000114547 26. Lopez-Ichikawa M, Vu NK, Nijagal A, Rubinsky B, Chang TT. Neutrophils are important for the development of pro-reparative macrophages after irreversible electroporation of the liver in mice. Sci Rep 2021; 11: 1-3. doi: 10.1038/s41598-021-94016-8 27. Zhou VX, Lolas M, Chang TT. Direct orthotopic implantation of hepatic organoids. J. Surg. Res 2017; 211: 251-60. doi: 10.1016/j.jss.2016.12.028 28. Szot GL, Koudria P, Bluestone JA. Murine pancreatic islet isolation. J Vis Exp 2007; 22: e255. doi: 10.3791/255 29. Michalopoulos GK. Liver regeneration. J. Cell. Physiol 2007; 213: 286-300. doi: 10.1002/jcp.21172 Radiol Oncol 2023; 57(1): 59-69. doi: 10.2478/raon-2023-0002 59 research article Estimating exposure to extremely low frequency magnetic fields near high-voltage power lines and assessment of possible increased cancer risk among Slovenian children and adolescents Tina Zagar1, Blaz Valic2.3, Tadej Kotnik3, Sara Korat1, Sonja Tomsic1, Vesna Zadnik1, Peter Gajsek2,3 1 Slovenian Cancer Registry, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 INIS - Institute for Non-Ionizing Radiation, Ljubljana, Slovenia 3 Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(1): 59-69. Received 25 October 2022 Accepted 10 November 2022 Correspondence to: Peter Gajšek, Ph.D., Department of Dosimetry–Institute of Nonionizing Radiation, Pohorskega bataljona 215, SI-1000 Ljubljana, Slovenia. E-mail: peter.gajsek@inis.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. Some previous research showed that average daily exposure to extremely low frequency (ELF) mag- netic fields (MF) of more than 0.3 or 0.4 μT could potentially increase risk of childhood leukaemia. Materials and methods. To allow calculations of ELF MF around high voltage (HV) power lines (PL) for the whole Slovenia, a new three-dimensional method including precision terrain elevation data was developed to calculate the long-term average ELF MF. Data on population of Slovenian children and adolescents and on cancer patients with leukaemia’s aged 0–19 years, brain tumours at age 0–29, and cancer in general at age 0–14 for a 12-year period 2005–2016 was obtained from the Slovenian Cancer Registry. Results. According to the large-scale calculation for the whole country, only 0.5% of children and adolescents under the age of 19 in Slovenia lived in an area near HV PL with ELF MF density greater than 0.1 μT. The risk of cancer for children and adolescents living in areas with higher ELF MF was not significantly different from the risk of their peers. Conclusions. The new method enables relatively fast calculation of the value of low-frequency magnetic fields for arbitrary loads of the power distribution network, as the value of each source for arbitrary load is calculated by scaling the value for nominal load, which also enables significantly faster adjustment of calculated estimates in the power distribution network. Key words: exposure assessment; childhood cancer; extremely low frequency magnetic fields; modelling; cancer; high voltage power lines Introduction Ionizing radiation is a known risk factor for the de- velopment of leukaemia and thyroid cancer, while the carcinogenicity of non-ionizing radiation has so far not been scientifically proven. Non-ionizing radiation includes the extremely low-frequency (ELF) magnetic fields (MF) that originate from electric current flowing through artificial sources, among which the most widely present in the living and working environments are the high-voltage (HV) power lines (PL) and transformer substations. Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 60 The exposure to ELF MP is the highest in the im- mediate vicinity of the source and decreases very rapidly with distance. For sources in the vicinity of residences, the Slovenian legislation prescribes a ten times lower permissible ELF MF values (10 μT) than the European Recommendation (100 μT). During the last few decades, several pooled analyses have been published that combined all available data with various exposure indices.1-4 These pooled analyses consistently found statisti- cally significant increased relative risk estimates for childhood leukaemia in the case of prolonged daily high exposure to ELF MF (above 0.3 or 0.4 μT) compared with low exposure (below 0.1 μT).5-8 In 2001, the International Agency for Research on Cancer (IARC) examined the available body of sci- entific literature on ELF MF and from the subset of studies concerning childhood leukaemia con- cluded that ELF MF should be classified in group 2B as possibly carcinogenic to humans based on “limited evidence of carcinogenicity in humans” and “inadequate evidence of carcinogenicity in ex- perimental animals”.9 Associations between other cancers and ELF MF were not observed or were statistically insignificant, while it is emphasized the biological mechanisms on how would ELF MF induce cancer growth remain unknown despite broad research also in this area.9-14 The fear of an increased risk of cancer that could occur after long-term exposure to ELF MF is also expressed by general public15, which additionally drives the research in this area. ELF MF that originate mainly from the use of electricity have been studied as a risk factor for childhood leukaemia since 1979.16 The available studies ever since used different approaches on estimating the long-term exposure to ELF MF. The simplest possible way of assessing exposure is to calculate distance to a facility which is likely to be a source of the field.17 One of the common methods to estimate the ELF MF exposure is socalled wire code which uses the distance to and the configu- ration of HV PL wiring to estimate the long-term exposure to ELF MF.16 Wire code method does not require any consent of the participants (compared to measurements at homes) and therefore the se- lection of the case and control groups is not biased by their willingness to participate.18-21 However, because of the overlap in field levels between cat- egories, wire code was not a good predictor of ELF MF levels, accounting for less than 21% of the vari- ance in magnetic-field measurements.22 Another method has used estimated historical fields where the ELF MF exposure was defined by a combina- tion of measurements and calculations at homes.23 The inclusion criteria for participants was the dis- tance of the dwelling from the HV PL. The calcu- lations took into account all important technical parameters of HV PL. The aim of the study was to determine the ELF MF due to all HV PL in Slovenia. We developed a new method for more reliable real-life exposure assessment, since previous research revealed this as a major source of bias. Further, we investigated whether children and adolescents exposed to ELF MF due to living near HV PL in Slovenia have a higher risk of leukaemia. Additionally, to address concerns of the Slovenian public, we analysed the possible association of ELF MF exposure and the incidence of brain tumours and of all childhood cancers together. Materials and methods High voltage power lines registry in Slovenia There are about 670 km of 400 kV PL, 330 km of 220 kV PL, 2600 km of 110 kV PL and 25 km of 110 kV underground cables in Slovenia. The technical data necessary to calculate and determine the spatial distribution of the ELF MF in the vicinity of HV PL were obtained from national power grid company ELES and from 5 power distribution companies covering Slovenia. The following data about each PL tower were collected: the coordinates, altitude, height and type. The tower type defines the type of the PL geometry (Barrel, Danube...) as well as the exact geometry of the tower cross arms. To take the sag of each span into account, the data about the exact catenary of each span was also obtained. For most of the HV PL the catenary for each span was available in the form of 3D lines, generated by national power grid company from the results of the measurements with LIDAR system. For a few HV PL where the 3D lines were not available, the values of the sag of each span or cable tension in each span were used to calculate the 3D lines. For 110 kV underground cables their exact course was available in a form of points along the cable course, but detailed information about the geometry and depth of the cables was not available. Based on these technical data, each PL was fur- ther split into sections with identical tower types and identical or very similar geometries (position and length of tower cross arm). If the PL was dou- ble circuit (in Slovenia only single or double cir- cuit PL are in use), the load data were checked for Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 61 each system of the PL. If the loads were mostly the same, then both systems were deemed as one sec- tion, otherwise each system was deemed as sepa- rate segment. In total the HV distribution system was split into 17 segments for 400 kV, 13 segments for 220 kV and 411 segments for 110 kV. The load data provided by national power grid company ELES consisted of the data about the working and reactive energy transferred by each HV PL in 15-minute intervals in the period from 1. 1. 2006 to 31. 12. 2017. From these data the val- ues of electric currents were calculated for every 15 minutes, as well as several geometric mean values: daily, weekly (7 day), monthly (30 day) and total mean value. There is no unified approach among the stud- ies regarding which mean value to use as the sur- rogate for long term exposure. Some studies have estimated the variations over time from available data, for example, on electricity consumption.24,25 Several studies used the yearly mean load value for the year of the diagnosis with some adding also the year of birth.23,26-29 In this study, the total mean value covers the two-year period from 1. 1. 2006 to 31. 12. 2017. Calculating ELF MF The ELF MF level at one location is the result of the contributions of all nearby HV PL. As the ELF MF is a vector quantity, the total value depends not only on the amplitude of each contribution, but al- so on their direction and the angles between them. Typically, when calculating the ELF MF, a numeri- cal model is created that contains all sources and thus the calculation determines the total ELF MF level for the selected loads of HV PV. With the available programs and computational capacity, such a calculation is not feasible for large areas. In addition, usual approach requires recalculation of the whole model for each load conditions (for example at nominal loads, actual maximum loads, average day or annual loads, conditions in case of one HV PL failure...) or if there are changes in technical characteristics like construction of new or reconstruction of existing HV PL. To allow largescale ELF MF calculations, a new method was developed and validated, which ena- bles the calculation of the total ELF MF based on the values of the ELF MF of individual sources. The methodology was divided into two steps: the first step was to calculate the ELF MF of one HV PL and the second was to calculate the total ELF MF due to all HV PL for desired loads. Methodology to calculate the ELF MF of single HV PL New method to calculate the ELF MF of one HV PL was based on the principle that the distribution of the ELF MF in the vicinity of the PL is not affected by the nearby objects or terrain, and on the fact that as long as the geometry of the PL and its load are the same, the value of the ELF MF in a selected point depends only on the distance between the se- lected point and the HV PL and on the difference of the altitude of the selected point and the HV PL. Each HV PL was split into sections with identical or quite similar geometries. From the data in the registry, a 3D line was generated for each segment, representing the course of the lowest cable of the HV PL. Additionally, for each HV PL segment the ELF MF distribution in a vertical cross section was calculated with a Narda EFC-400 EP program package at the nominal load. The program package calculated the ELF MF by splitting all conductors into short parts and summing their contributions. The value of the ELF MF of one short part of con- ductor was calculated by the Biot-Savart law: where is magnetic flux density contribution from a short part of the conductor at the point given by positon vector and I is electric current in the cable. The calculation took into account exact geom- etry of the towers in this segment and extended to the distance where the value of the ELF MF fell below 0.05 μT. The result was stored in a GRID ma- trix with the step of 1 m and positioned so that the center of the matrix aligned with the 3D line of the segment. The 3D line representing the course of the PL and the matrix with the values of the ELF MF in a vertical cross section were used as the input to a customdeveloped program that calculated the val- ue of the ELF MF along the whole segment length. The program first split the segment into spans. For each span, the program generated a 1m grid of points that covered the area of the whole length of span in one dimension and the same distance as covered by the matrix of the values of ELF MF in a vertical cross section. At both ends of the span (at location of the HV PL towers), additional points were added by rotating the last line of points as presented by blue and black dots in Figure 1. We can see that blue and black dots do overlap in a certain area around the tower. For each point in the 1m grid, first the height difference was calcu- Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 62 lated between the absolute height of the 3D line representing the course of the PL and the point 1 m above the ground. The absolute height of the point 1 m above the ground was determined from the digital elevation model of Slovenia with the resolution of 1 m constructed based on the LIDAR data. Second, the distance of each point in the 1 m grid from the HV PL was calculated. Both values were rounded to the nearest integer and used to pick the corresponding value from the matrix with the calculated values of the ELF MF in a vertical cross section. By this process the proper value of the ELF MF was assigned to each point in the 1 m grid. Finally, a 10 m orthogonal grid was generated (red circles in Figure 1) to which the highest value of nearby points in a 2 m radius was assigned. The output of the program for each segment was a 10m grid of points with the values of the ELF MF. FIGURE 1. Points in 1m grid for two spans of a power line are shown with blue and black dots in which the program determined the values of the extremely low- frequency (ELF) magnetic fields (MF). Red circles represent the final 10 mgrid for which the final results were generated. FIGURE 2. Comparison of results obtained by all four algorithms to determine total value of extremely low-frequency (ELF) magnetic fields (MF) for a case of crossing of two 2×110 kV power lines of type barrel under the nominal load of 650 A. The color scale shows the deviation of the estimated value of ELF MF values determined by all four new algorithms from the actual value of ELF MF determined by the usual numerical modeling procedure. The black contours represent the limits of 0.1, 0.4 and 1.0 μT for the actual values, and the red contours for the estimated values. Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 63 Finally, if HV PL was split in more than one seg- ment, all 10 m grids of one HV PL were merged together. For those coordinates where values were available in two (or theoretically more) grids, the highest value was used for the final result. Methodology to calculate the total ELF MF of multiple HV PL When multiple HV PL either cross or run in paral- lel, the total value of the ELF MF is a result of the contributions of all nearby HV PL. As ELF MF is a vector quantity, the resulting value is typically significantly lower than a sum of all contributions. Therefore, four different algorithms to determine the total value of the ELF MF in each considered point were analysed and compared: • the MAX algorithm took the higher value of the two contributions as the total value; • the MAX + MIN algorithm took the sum of the higher and the lower values as the total value; • the MAX + 0.3 MIN and MAX + 0.6 MIN cal- culated the total value by adding to the higher value the lower value multiplied by a specific weight factor of 0.3 and 0.6, respectively. As, the actual total value of the ELF MF can nev- er exceed the sum of the two values considered, the MAX + MIN algorithm is the most conserva- tive, the MAX algorithm is the least conservative, and the other two are inbetween. In order to evaluate all four algorithms, various realworld scenarios were analysed where cumula- tive effects of several HV PL occur, e.g. crossing of two HV PL, or parallel courses of two and three HV PL. Figure 2 presents the analysis of one such case –the intersection of two 110 kV double-circuit HV PL. The colour scale shows the deviation of the estimated ELF MF determined by all four new algorithms from the actual ELF MF determined by usual numerical modelling procedure. Green colour means that there is practically no devia- tion, red colour marks areas where the algorithm overestimated the values, and blue colour shows areas where the algorithm underestimated the val- ues. Among four proposed algorithms, the MAX algorithm underestimated the values of the ELF MF the most (as the blue areas are the largest) and overestimated them the least (as the red areas are the smallest). The MAX + MIN algorithm never un- derestimated the values of the ELF MF, and over- estimated them the most among the four proposed algorithms. Information regarding the deviation of the estimated value from the actual value is also provided in Figure 2 by black and red contours. The black contours represent the field lines of 0.1, 0.4 and 1.0 μT for the actual value obtained by usual modelling procedure, and the red contours for the estimated value using a novel method. For the MAX + MIN algorithm red contours never lie inside black contours, which means that the MAX + MIN algorithm overestimated the values of the ELF MF. Using the MAX algorithm, red contours often or even predominantly lie within black con- tours. The analysis showed that the most realistic results for the conditions considered was given by the MAX + 0.6 MIN algorithm, which was thus chosen as the optimal algorithm. Subsequent fea- sibility analysis showed that calculations could be performed according to all four algorithms, which allowed later sensitivity analysis, i.e. whether the choice of algorithm affects the result of analyses. A novel program with all four analysed algo- rithms was developed to determine total value of the ELF MF. The program also featured the func- tionality to scale the values of each HV PL with a certain factor before calculating the total value of the ELF MF. This functionality enables relatively easy and fast calculation of the total ELF MF not only for the nominal load of HV PL, but for any load conditions or operating conditions of the whole power distribution network. For the later analysis, the total geometric mean value of the load was used that covers the whole period from 1. 1. 2006 to 31. 12. 2017. Cancer data We conducted retrospective geographical epide- miological study in population of Slovenian chil- dren and adolescents aged 0–19 years diagnosed in a 12-year period 2005–2016. Address of residence served as a proxy indica- tor of ELF MF exposure. From Central Population Registry data on permanent address, which was georeferenced to X and Y coordinates, was ob- tained for all Slovenian children and adolescents in the studied population. Modelled values for ELF MF density near HV PL were used to classify all population of children and adolescents into five categories of ELF MF exposure: B < 0.1 μT; 0.1 μT ≤ B < 0.2 μT; 0.2 μT ≤ B < 0.3 μT; 0.3 μT ≤ B < 0.4 μT and 0.4 μT ≤ B. Georeferenced addresses were allocated to the closest point on the 10m grid by using the k-nearest neighbours (kNN) method on the Quad Tree to avoid calculation of Euclidian distances be- tween each grid point and each address location (building Quad Tree algorithm has running time O(n·log(n)) ).30 Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 64 From the Slovenian Cancer Registry incidence (number of all newly diagnosed cancer cases in one calendar year) data on cancer patients were obtained and linked to population data based on Slovenian personal identification number. Patients with leuk emias were identified based on codes C91–C95 according to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10). In addition, we analysed all cancer cases occurring in child- hood (age at diagnosis 0–14 years) and cases with malignant tumour of the central and autonomic nervous system (or brain tumour in short) with codes C70–C72 diagnosed up to 29 years of age. Relative risk of cancer was assessed using a standardized incidence ratio (SIR), which is a method for indirect age standardization and is cal- culated as a ratio between observed and expected incidence for each studied group, i.e. cancer in- cidence in each of the five categories of ELF MF exposure. Expected incidence is calculated from age distribution in studied group and age-specific incidence rates in the overall population.31 A SIR value of 1 indicates that observed incidence in the studied group is the same as expected incidence, less than 1 indicates a lower than expected inci- dence, and over 1 indicates a higher than expected incidence. Data management and analysis of population and cancer data was performed in R software (ver- sion 4.0.2) and R packages dplyr (version 1.0.2) and SearchTrees (version 0.5.2). Results ELF MF around HV PL To measure the ELF MF at the participants homes it is necessary to obtain their consent. This can be potential source of selection bias.29 It is suggested 32 that although it is an imperfect measure of magnet- ic field exposure, wire code is the only method ap- plicable to nonparticipating subjects. But if techni- cal data about the HV PL near the residence of par- ticipants are available, it is possible to determine the exposure at the desired time interval based on the numerical calculations. Such combined estima- tion of ELF MF exposure has been used in several studies.8,27-29 As numerical calculation of ELF MF is very demanding for larger areas, the studies usu- ally limit the area of interest to a pre-selected dis- tance to the HV PL, usually in the range of a few hundreds of meters. The value of the ELF MF in a selected point near PL depends on the geometry of the PL, the load of the PL, the distance from the HV PL, and the el- evation difference between the PL and the selected point. It is not affected by the terrain and nearby objects. This principle was used by Miravet- Garret33, where the distance and difference of the altitude of the HV PL and of the selected point was determined from the detailed technical data of the HV PL and the ground profile, and then the value of the ELF MF at the selected point was calculated by a simplified analytical formula. The mean values of the load of HV PL in Slovenia have an increasing tendency, meaning that on average the energy transfer through HV PL is increasing. For 110 kV PL the average value of geometric means of the loads of all PL was 84 A in 2006 and 87 A in 2017, for 220 kV PL it was 185 A in 2006 and 213 A in 2017, and for 400 kV PL it was 282 A in 2006 and 360 A in 2017. The increase is sig- nificantly higher for 400 KV PL compared to either 220 or 110 kV PL. However, the mean loads are not high. Among all 400 kV PL, the highest total mean value of the load for one PL for the whole period from 1. 1. 2006 to 31. 12. 2017 was 742 A and it in- creased to 842 A for the highest yearly mean value (in year 2016). In Figure 3 the daily, monthly and yearly mean values for this HV PL are presented. Yearly mean loads do not differ significantly from the total mean loads (1. 1. 2006 ‒ 31. 12. 2017). The highest yearly value was achieved in 2016 (842 A), whereas the lowest was achieved in 2007 (374 A). The fluctuations in monthly mean loads are larger, the highest monthly load was achieved in November 2010 (1588 A) and the lowest in August FIGURE 3. The daily, monthly, yearly and total (1. 1. 2006 – 31. 12. 2017) mean values of the load of a 400 kV power line with the highest mean values in Slovenia. Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 65 2010 (1 A). For weekly mean loads, the highest val- ues were achieved at the end of November 2010 (1987 A). The ELF MF around HV PL was calculated for the entire territory of Slovenia on the 10m grid. On Figure 4 the value of the ELF MF of several HV PL around one transformer stations are presented for nominal load (top) and the total mean geometric load covering the period from 1. 1. 2006 to 31. 12. 2017. The value of the ELF MF was determined by the new algorithm (from chapter Methodology to calculate the total ELF MF of multiple sources). For the total mean geometric load, the value of the ELF MF was higher than 0.1 μT on almost 2 mil- lion points. This represents the area of about 191 km2, which means that on about 1% of the terri- tory of Slovenia the value of the ELF MF is 0.1 μT or higher, while on more than 99% of the territory of Slovenia the value of the ELF MF is below 0.1 μT. For 0.4 μT the areas are significantly smaller, as this value is exceeded only on 0.36% of Slovenian territory. The results were validated by comparing calcu- lated values of ELF MF with measurements in the vicinity of two 400 kV PL and two 220 kV PL. The same load conditions (nominal load) were used for comparison. For the 400 kV PL with the highest mean load, the comparison is given in Table 2. Childhood cancers and ELF MF exposure Cancer in children and adolescents is a rare dis- ease, as it represents less than 1% of all cancers in Slovenia as well as in Europe.34,35 In recent years, around 70 cancers are diagnosed annually in Slovenia during childhood and adolescence (from birth to the age of 19), 20% more among boys than among girls.34 Age-standardized incidence rate has been increasing by an average of slightly more than 1% annually since 1961 in Slovenia and also increased by about 1% per annum in Europe in the past three decades.34,36 The causes for the increas- ing incidence are unknown; although it is largely attributed to improved diagnostic and registration procedures, the possibility of increased exposure to different harmful factors, especially intrauterine, cannot be excluded.36 The most common groups of malignant can- cers in the age group of 0–19 years are leukaemias, lymphomas and tumours of the central nervous system (in short brain tumours). According to the average for the period 1961–2019, leukaemias represented a quarter of all newly diagnosed can- cers, followed by brain tumours with around 15%, Hodgkin’s lymphoma with 10% and non-Hodg- kin’s lymphoma with 8%.34 Apart from exposure to benzene and ionizing radiation, the risk factors for leukaemias are not well known.37,38 The so- called hygiene hypothesis is also possible, where, due to the improvement of infection prevention in the modern era, leukaemia may develop in geneti- FIGURE 4. Example of the total value of the extremely low-frequency (ELF) magnetic fields (MF) of several high voltage power lines around distribution transformer stations for nominal load in the area of the capital city of Ljubljana (approximately 5 x 2.7 km). TABLE 1. The distances from the 400 kV power line with the highest mean load, at which the value of the extremely low frequency magnetic field falls below 0.1, 0.2, 0.3 and 0.4 μT, respectively Distance B = 0.1 μT B = 0.2 μT B = 0.3 μT B = 0.4 μT Nominal load 340 m 238 m 193 m 167 m Highest daily mean load (2164 A, 23. 11. 2010) 303 m 212 m 172 m 149 m Highest weekly mean load (1987A, 22. – 28.11.2010) 290 m 203 m 165 m 143 m Highest monthly mean load (1588 A, 11. 2010) 258 m 181 m 147 m 127 m Highest yearly mean load (842A, 2016) 187 m 131 m 107 m 92 m Total mean load (742 A, 1. 1. 2006 – 31. 12. 2017) 175 m 123 m 99 m 87 m Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 66 cally predisposed individuals due to an incorrect response to infection by the immune system of a child who did not come into contact with non- dangerous infectious agents in early childhood at the right time (early enough).39 Some (but not all) studies have shown that the risk may be increased (only) for childhood leukaemias with longterm daily exposure to ELF MF densities greater than 0.3 or 0.4 μT, depending on the study. The key advantage of our study was that with in- clusion of the population data from the Slovenian Cancer Registry, we avoided performing a case- control study and, at the same time, selection and participation bias.8 Research indicates that the fraction of children exposed to higher values of ELF MF (>0.4 μT) is likely to be less than 2% of the population.40,41 For Slovenia, it was estimated in the past that no more than 1% of children were exposed to long term ELF MF greater than 0.4 μT.42 In presented study, using an innovative model for evaluating ELF MF on a small spatial network and georeferencing residences and sources of the electricity network in Slovenia, we precisely deter- mined the exposure to ELF MF in children aged up to 14 years in the period 2005–2016 in Slovenia, who live nearby HV PL. We found that as many as 99.5% of all children did not live in the areas with the mean value of ELF MF 0.1 μT or higher. And only 0.09% of all children were exposed for at least one year to the area of potentially carcino- genic ELF MF density, that is greater than 0.4 μT, in the vicinity of HV PL. This population results and results for age groups 0–19 and 0–29 are presented in Table 3 (the results are equal to the first decimal point). These results emphasize the importance of performing exact calculations on real-life popula- tion-based data in order to obtain realistic picture of the situation in a specific country. In Table 3 we also present results according to five categories of ELF MF exposure for observed and expected number of cancer cases, and stand- ardized incidence ratio for all cancers combined, leukaemia and brain tumours. All of the 516 can- cer cases in children aged 0–14 years in 2005–2016 were classified in the lowest category of exposure (ELF MF below 0.1 μT) near HV PL. Relative risk cannot be calculated for other categories of ELF MF exposure, since there were no cases. Among 195 diagnosed cases of leukaemia in children and adolescents aged 0–19 years in 2005– 2016 only one adolescent was classified in the cat- egory of ELF MF density between 0.1 and 0.2 μT – a person lived in the vicinity of 110 kV PL. This single case of leukaemia represents big variability and thus “shifted” all residents from this group from a relative risk of zero to a relative risk of more than one. Although the value for relative risk in this group appears high (2.4), the confidence inter- val is far too wide for statistical significance (since it is based on only one case). No leukaemia case was classified in categories with higher ELF MF exposures (i.e., above 0.2 μT). Among 196 patients aged 0–29 years diagnosed with brain tumour only one adolescent was classi- fied in the category of ELF MF exposure between 0.2 and 0.3 μT, giving standardized incidence ratio 4.4 with wide 95% confidence interval (0.1-24.6). Also, this person lived in the vicinity of 110 kV PL at the time of diagnosis. No brain tumour case was classified in other categories of ELF MF exposure. We conclude that none of the cases of leukae- mia in the studied population can be attributed to exposure to ELF MF – the relative risk of leu- kaemia in children and adolescents living near HV PL does not differ from the average risk in the general population. Additionally, we could not as- sess a possible risk trend over exposure categories, because there were also no cases of leukaemias in other categories. Our findings are in line with other similar stud- ies, but we cannot directly compare the results with other studies due to the different (in our opin- ion better, more accurate assessment methodology based on actual exposure and availability of exact coordinates for all dwellings on Slovenian popu- lation.41,43 Using place of residence as a surrogate indicator of ELF MF exposure is more reliable in children than in adults, as they spend most of the day in the vicinity of home and the surrounding area. In addition, they migrate less. Discussion For the ELF MF exposure assessment, we devel- oped an innovative, accurate model on a fine spa- tial grid, which is more reliable than exposure approximations used by previous studies (e.g. Euclidean distance from guides, questionnaires, TABLE 2. Comparison of the calculated and measured value of the extremely low frequency magnetic field for 400 kV power line with the highest mean load Location 1 2 3 4 5 Calculated value [μT] 7.4 3.8 3.0 9.1 3.4 Measured value [μT] 7.3 3.4 4.1 12.3 2.7 Radiol Oncol 2023; 57(1): 59-69. Zagar T et al. / Extremely low frequency magnetic fields and cancer risk 67 etc.). With this new method the calculations of ELF MF values generated by all HV PL were performed for the entire territory of Slovenia with the grid of 10 m for average loads of HV PL in the period from 2006 to 2017. The value of ELF MF was higher than 0.1 μT in a little less than 2 million points, which corresponds to an area of about 200 km2, or one percent of the whole territory of Slovenia. The new method enables relatively fast calculation of the value of ELF MF for arbitrary loads of the power distribution network, as the value of each source for arbitrary load is calculated by scaling the value for nominal load, and at the end the total value is determined by new methodology. The advantage of this approach is also significantly faster adjust- ment of calculated estimates in the power distri- bution network, such as a new power line or the reconstruction of an existing power line, as new calculation is required only for a new or modified source. The new methodology was validated on smaller areas by comparing the total values of ELF MF, determined according to the new methodol- ogy, with the values obtained with the traditional approach of numerical modelling and with the re- sults of measurements. Both comparisons showed the expected agreement of the results. Among Slovenian children and adolescents, only 0.5% live in the areas with the mean value of ELF MF 0.1 μT or higher. And only 0.09% of all children were exposed for at least one year in the area of potentially carcinogenic ELF MF density in the vicinity of power lines, which is greater than 0.4 μT. Although the incidence of childhood can- cer, which is a rare disease, is gradually increas- ing in Slovenia, based on our population study, we cannot attribute any case of childhood cancer up to 14 years of age, any leukaemia diagnosed up to 19 years, or any tumour of the central nervous system up to 29 years in the twelve years under study (2005–2016) to the exposure to ELF MF near HV PL. Finally, although here the new method for es- timation of exposures to ELF MF was used for as- sessment of possible increased cancer risk in chil- dren and adolescents, it is also applicable for the broader purpose of exposure assessment, includ- ing the planning of proper placement of HV PL and transformer substations into the environment, and of new housing near the existing HV PL. Acknowledgments This work was funded by the Slovenian Research Agency (ARRS) and the Slovenian Ministry of Health through the targeted research project “Health risk assessment for exposures of chil- dren to lowfrequency electric and magnetic fields TABLE 3. The proportion of Slovenian population of children and adolescents in 2005–2016, observed and expected number of cancer cases, and standardized incidence ratio with 95% confidence interval for all cancers combined (age 0–14 years), leukemia (age 0–19 years) and brain tumors (age 0–29 years) are presented according to five categories of ELF MF exposure < 0.1 mT 0.1–0.2 mT 0.2–0.3 mT 0.3–0.4 mT ≥ 0.4 mT Population* (proportion) 99.5 % 0.2 % 0.1 % 0.1 % 0.1 % All cancers (age 0–14 years) Observed number of cases 516 0 0 0 0 Expected number of cases 513.6 1.1 0.6 0.3 0.5 Standardized incidence ratio (95% confidence interval) 1.0 (0.9–1.1) no cases no cases no cases no cases Leukemia (age 0–19 years) Observed number of cases 194 1 0 0 0 Expected number of cases 194.1 0.4 0.2 0.1 0.2 Standardized incidence ratio (95% confidence interval) 1.0 (0.9–1.2) 2.4 (0.1–13.3) no cases no cases no cases Brain tumors (age 0–29 years) Observed number of cases 195 0 1 0 0 Expected number of cases 195.1 0.4 0.2 0.1 0.2 Standardized incidence ratio (95% confidence interval) 1.0 (0.9–1.2) no cases 4.4 (0.1-24.6) no cases no cases * Population’s proportions for age groups 0–14, 0–19 and 0–29 are equal to the first decimal point. 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Br J Cancer 2016; 115: 122-8. doi: 10.1038/bjc.2016.142 Radiol Oncol 2023; 57(1): 70-79. doi: 10.2478/raon-2023-0001 70 research article Comparison of CalliSpheres® microspheres drug-eluting beads and conventional transarterial chemoembolization in hepatocellular carcinoma patients: a randomized controlled trial Zhongxing Shi1, Dongqing Wang1, Tanrong Kang1, Ru Yi2, Liming Cui1, Huijie Jiang2 1 Department of Interventional Radiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China 2 D epartment of Radiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China Radiol Oncol 2023; 57(1): 70-79. Received 3 October 2022 Accepted 8 November 2022 Correspondence to: Huijie Jiang, Ph.D., Department of Radiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, China. E-mail: ji anghuijie@hrbmu.edu.cn 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/). Ba ckground. This trial aimed to compare the outcomes of drug-eluting beads transarterial chemoembolization (DEB-TACE) with CalliSpheres® microspheres (CSM) and conventional transarterial chemoembolization cTACE in the treatment of patients with unresectable hepatocellular carcinoma (HCC). Patients and methods. A total of 90 patients were divided into DEB-TACE group (n = 45) and cTACE group (n = 45). The treatment response, overall survival (OS), progression-free survival (PFS), and the safety were compared between the two groups. Results. The objective response rate (ORR) in the DEB-TACE group was significantly higher than that in cTACE group at 1, 3, and 6 months of follow-up (P = 0.031, P = 0.003, P = 0.002). The complete response (CR) in DEB-TACE group was significantly higher than that in cTACE group at 3 months (P = 0.036). Sur vival analysis revealed that, DEB-TACE group had better survival benefits than cTACE group (median OS: 534 days vs. 367 days, P = 0.027; median PFS: 352 days vs. 278 days P = 0.004). The degree of liver function injury was more serious in DEB-TACE group at 1 week, but was similar between the two groups at 1 month. DEB-TACE with CSM caused a high incidence of fever and a severe abdominal pain (P = 0.031, P = 0.037). Conclusions. DEB-TACE with CSM showed better treatment response and survival benefits than cTACE group. Although a transient more severe liver damage, high incidence of fever and a s evere abdominal pain occurred in the DEB-TACE group, it could be resolved through symptomatic treatment. Key words: cTACE; CalliSpheres® microspheres; DEB-TACE; hepatocellular carcinoma; tumo r response Introduction Hepatocellular carcinoma (HCC) is the most com- mon type of primary liver cancer and has been re- ported to be the sixth most common cancer and the third most frequent cause of cancer-related deaths worldwide, and the leading cause of cancer-related deaths in men in China.1 HCC occurs in the setting of chronic liver inflammation, which is closely re- lated to chronic viral hepatitis infection (hepatitis B or C) or exposure to toxins, including alcohol or aflatoxin. Despite improvements in the early diagnosis and various treatment methods, most patients with HCC lose the chance of surgical re- section, liver transplantation, and radiofrequency ablation.2,3 Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma 71 According to Barcelona Clinic Liver Cancer (BCLC) staging system guidelines, transarterial chemoembolization (TACE) is widely applied in HCC patients not suitable for surgical treatment in intermediate and advanced stages.4 In addi- tion, TACE has been applied as a bridge therapy to liver resection or transplantation in early-stage HCC.5 The two main protocols for TACE are con- ventional TACE (cTACE) and drug-eluting beads TACE (DEB-TACE). cTACE uses lipiodol as a chem- otherapy drug carrier to embolize targeting arter- ies, release antitumor medication, and consequent powerful ischemic and cytotoxic effects. However, the rapid decrease of the local antitumor drug con- centration and the high systemic toxicity must be investigated.6 DEB-TACE uses drug-loaded micro- spheres, which can not only load chemotherapy drugs and release them slowly in local regions but also have less systemic side effects; it also embo- lizes the tumor supply vessels permanently.7 Call iSpheres® microspheres (CSM) are the first DEBs in China and have been applied clinically for a few years. Previous studies on DEB-TACE with CSM have focused on the survival rate, safety, and prognostic factors8,9, and most were retrospec- tive studies.10,11 Limited prospective, randomized studies have compared the treatment response of the two methods. A study in 2020 showed that DEB-TACE using CSM loading with arsenic triox is more effective and equally tolerant compared with cTACE in treating unresectable HCC patients. Although the study was prospective, the patients were not randomized and the sample size was not calculated.12 Thus, the present study aimed to pro- spectively and randomized evaluate the efficacy and the safety of DEB-TACE with CSM and cTACE in unresectable HCC patients. Patients and methods Study design This was a prospective, randomized, controlled study designed to compare the efficacy and safe- ty of DEB-TACE with CSM and cTACE. All t he subjects who participated in this study were ran- domized in a 1:1 ratio by a computerized system and enrolled by an investigator physician. The re- sults of allocation were placed in a sealed envelope and delivered to the only assistant aware of DEB- TACE/cTACE progress. All the doctors in charge of surgery and data collection were blinded to the trial design and treatment. All participants were informed about the objective and experimental procedure and allowed to withdraw their consent or discontinue participation without restrictions at any time. Then those who voluntarily partici- pated in the trial signed the written informed con- sent. All p rocedures in the trial were in accordance with the World Medical Association’s Helsinki Declaration. The study plan was approved by the Ethics Committee of the Second Hospital Affiliated Harbin Medical University (No. KY2020- 267) and has been registered at Chictr.org.cn (No. ChiCTR2100044528). Patients From March 2020 to March 2021, 115 HCC pa- tients from the Department of Interventional Radiography of the Second Hospital Affiliated Harbin Medical University were recruited in this study through advertisements or physicians. The inclusion criteria were as follows: (1) Patie nts di- agnosed as primary HCC by pathological findings or clinical features and radiographic examinations according to American Association for the Study of the Liver Diseases (AASLD) guidelines; (2) Tumor location and extent not amenable to elective re- section or ablation; (3) Patients undergoing treat- ment for the first time and were willing to accept CSM DEB-TACE or cTACE; (4) Digital substrac- tion sngiography (DSA) showed that the tumor was abundant in blood supply with no hepatic/ portal vein invasion; (5) Patients > 18 years old; (6) Patients met Child-Pugh stage A or B and BCLC stage A or B and with an Eastern Cooperative Oncology Group (ECOG) performance status ≤ 1; (7) Life expectancy > 6 months. The exclusion cri- teria were as follows: (1) Patients with a history of liver transplantation or other malignancies; (2) Patients with coagulation dysfunction or massive ascites; (3) Patients complicated with severe liver or renal dysfunction; (4) Patients with iodine al- lergy; (5) Patients with cognitive impairment or refusals. Prepara tion before the procedure Each pa tient had fasted for 6 h and water-deprived for 2 h. The patient was placed in a supine position, and a peripheral intravenous line was established before the procedure. The patient was given in- haled oxygen at 3 L/min with a nasal catheter. Vital signs, including ECG, blood pressure, and oxygen saturation, were monitored. Preoperatively, 3 mg granisetron was injected intravenously over 15 s to prevent nausea and vomiting. Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma72 TACE procedure The gro in was prepared in a sterile fashion, and after local anesthesia, percutaneous right common femoral artery was punctured using the modified Seldinger method. Then, a 5F (Terumo, Tokyo, Japan) /4F (Cordis, USA) RH catheter was introduced through a 5F vas- cular sheath (Merit Medical, USA), and placed into the celiac trunk under DSA guidance to perform celiac angiography to identify the tumor feeding ar- tery. If the blood vessels in a certain area of the liver are sparse or the tumor is not fully displayed during celiac trunk angiography, look for the extrahepatic tumor blood supply artery, such as superior mesen- teric artery, inferior phrenic artery, and right adre- nal artery, right inferior intercostal artery, internal mammary artery, etc. After confirming the tumor- feeding artery, a superselective (segmental or sub- segmental) approach was used whenever possible using a 2.4 F microcatheter (Merit Medical, USA) for embolization. The tip of the catheter was advanced into the hepatic artery and feeding branch if the size, location, and blood supply were optimal. The cTA CE group was injected an emulsi- fied mixture containing 10 mL lipiodol (Jiangsu Hengrui Medicine Co., Ltd, China) and 40 mg pira- rubicin (Shenzhen Wanle Pharmaceutical Co., Ltd, China) into the tumor feeding artery through a mi- crocatheter under fluoroscopic monitoring to avoid reflux of the emulsion. The volume of embolization emulsion was based on the size of the focus. Then according to the blood flow velocity of tumor blood vessels, gelatin sponge particles are appropriately selected to strengthen the embolization until the tumor staining was disappeared. Patients in the DEB-TACE group received CSM (100-300 μm or 300-500 μm in diameter; Jiangsu Hengrui Medicine Co. Ltd, China) loaded with 40 mg pirarubicin. TACE upon portal vein visualization achieved “near stasis,” with a further pause of 5 minutes to allow redistribution of the embolic agents within the lesion and their distal propulsion by the blood inflow. The second time angiography was conduct- ed to detect the presence of the remaining blushed nodules. The endpoint of the treatment was com- plete satiation of the tumor vessels with drug and the disappearance of the tumor blush on subse- quent angiographic imaging. Postprocedure management The punct ure wound was bandaged with pres- sure, and all patients were told to rest in bed in the supine position for at least 6 h post-embolization. The blood supply and temperature of the affected leg should be under intensive focus. The patients were given routine treatment after the operation: (1) Reduced glutathione for liver protection; (2) Granisetron for postoperative nausea and vom- iting; (3) Ibuprofen for fever; (4) Non-steroidal anti-inflammatory drugs (Flurbiprofen Axetil) or opioids (morphine) for analgesia; (5) Antibiotics to prevent infection. Assessmen t of treatment response All patie nts underwent blood test (liver function indexes and alpha-fetoprotein) and imaging exam- ination in the first month after TACE. Patients, who showed tumor progression in the first month after TACE, would receive a second TACE according to the previous grouping. Patients with stables dis- ease were followed with imaging every 3 months. The treatment response was assessed by two experienced radiologists based on enhanced com- puted tomography (CT) or magnetic resonance im- aging (MRI) according to the modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria11 at 1 month, 3 months, 6 months post-treat- ment: complete response (CR), partial response (PR), stables disease (SD), and progressive disease (PD). In addition, the objective response rate (ORR) was calculated and defined as CR + PR / total × 100%, and the disease control rate (DCR) was cal- culated and defined as CR + PR + SD / total × 100%. Although CT response evaluation differentia- tion between Lipiodol and contrast agent is lim- ited, the tendency to overestimate treatment re- sponse was avoided in the cTACE group through comparison of tumor enhancement in the arterial phase and the extent of lipiodol accumulation in the unenhanced phase. Assessment of overall survival and progression-free survival The survival status of the two groups was evalu- ated by overall survival (OS) and progression-free survival (PFS). The OS was defined as the interval between the date of the first TACE treatment and the date of patients’death from any cause or cen- sored at the date of the last follow-up. The PFS was defined as the interval between the start of the first TACE treatment date and the first radiological pro- gression date, patients’ death from any cause, or censored at the date of the last follow-up.12 Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma 73 Safety The safety of the two groups was evaluated by the liver function indexes and post-embolization syndrome. The liver function indexes, including alanine aminotransferase (ALT), aspartate ami- notransferase (AST), albumin (ALB), and total bili- rubin (TBIL), were assessed before the procedure (baseline), at 1 week, and 1 month post-procedure. The post-embolization symptoms, including fa- tigue, fever, abdominal distension, abdominal pain, and nausea/emesis, were assessed during the procedure and within 1 month after the procedure. The degree of pain was evaluated by the numeric rating scale (NRS) (0-10), where a score of 0 means no pain and a score of 10 indicates the maximum level of intolerable pain.11 Sample size According to the ORR at 3 months after TACE, as described previously (73.7% in the DEB-TACE group and 42.5% in the cTACE group ( P = 0.005))11, a sample size of at least 39 subjects in each group was required to provide 80% power to detect differ- ences at an α level of 0.05, indicating significance. However, to prevent a 15% attrition rate, we even- tually recruited 90 patients with 45 in each group. Data analysis GraphPad Prism 8.0.2 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis and generating graphs. The nor- mally distributed continuous data were presented as the means ± standard deviation, while skewed FIGURE 1. Flow diagram of the study. ALB = albumin; ALT = alanine aminotransferase; AST = aspartate aminotransferase; cTACE = conventional transarterial chemoembolization; DEB- TACE = drug-eluting beads transarterial chemoembolization; HCC = hepatocellular carcinoma; TBIL = total bilirubin Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma74 distributed continuous variables were presented as median (25th–75th quantiles) and compared using a t-test. The enumeration data were presented as frequencies and percentages and compared using the chi-squared test. Kaplan–Meier (K–M) method was applied for making the survival curves, and the comparison of OS and PFS between the two groups was estimated by Log-rank test. Statistical significance was set at P < 0.05. Results Study flow and baseline demographic data A total of 115 HCC patients were recruited in this trial, 18 did not meet the inclusion criteria, and 7 declined to participate. Finally, 90 patients fulfilled the inclusion criteria and were randomly divided into DEB-TACE or cTACE group (n = 45). The study selection is illustrated in Figure 1. All pa- tients for analysis did not undergo any other treat- ment previously, including surgery, radiofrequen- cy ablation, TACE, and systematic chemotherapy. At 1 week, 1 month, 3 months, and 6 months all patients in each group were analyzed except 1 pa- tient in the DEB-TACE group died in the 5th month after the procedure. The mean age of the cohort was 58.9 ± 7.1 years in th e DEB-TACE group and 60.6 ± 6.5 years in the cTACE group (P = 0.229). In addition, the DEB- TACE group had 40, while the cTACE group con- sisted of 39 males (P = 0.747). The history of alco- ho l consumption and viral hepatitis, Child–Pugh stage, BCLC stage, ECOG performance status, liver function, tumor location and distribution, and di- ameter of the largest tumor in the two groups did not differ significantly. The detailed baseline char- acteristics of patients are listed in Table 1. Treatment response between the two groups The treatment response is shown in Table 2. At 1 month after the procedure, the ORR in the DEB- TACE group was significantly higher than that in cTACE group (P = 0.031), while there was no sig- nificant difference of the CR and DCR between the two groups. At 3 months after the procedure, a sig- nificant difference was observed in the treatment response between the two groups. CR, ORR, and DCR in the DEB-TACE group were significantly higher than those in the cTACE group (P = 0.036, P = 0.003, P = 0.025), while PD in the DEB-TACE group was lower than that in the cTACE group (P = 0.025). At 6 months after the procedure, CR presented no difference between the two groups, but ORR and DCR were significantly elevated (P = 0.002, P = 0.031), and PD was significantly reduced in the DEB-TACE group compared to the cTACE group (P = 0.031). Comparison of OS and PFS between the two groups The final date of survival analysis was March 2022. All patients were followed up until death, or the end of the study and the last follow-up time of the assessment with the median follow-up duration was 365 days (95% CI:150-730 days), which was es- timated by reverse Kaplan-Meier method. Log-rank test revealed that, the median OS of the DEB-TACE group (534 days, 95% CI: 458-634 days) was significantly longer than the cTACE group (367 days, 95% CI: 321-562 days, P = 0.027, Figure 2A). The median PFS of the DEB-TACE TABLE 1. Baseline characteristics of patients Parameters DEB-TACE (n = 45) cTACE (n = 45) P value Gender (male/female) 40/5 39/6 0.747 Age (years) 58.9 ± 7.1 60.6 ± 6.5 0.229 History of alcohol consumption (n/%) 11 (24.4) 15 (33.3) 0.352 History of viral hepatitis (n/%) 31 (68.9) 27 (60.0) 0.378 Child-Pugh stage (n/%) A B 33 (73.3) 12 (26.7) 29 (64.4) 16 (35.6) 0.326 - BCLC stage (n/%) A B 17 (37.8) 28 (62.2) 14 (31.1) 31 (68.9) 0.506 - ECOG performance status (n/%) 0 1 17 (37.8) 28 (62.2) 13 (28.9) 32 (71.1) 0.371 - Liver function ALT (U/L) AST (U/L) ALB (g/L) TBIL (μmol/L) 38.0 ± 20.5 40.3 ± 16.8 39.9 ± 8.2 19.1 ± 6.1 35.7 ± 18.7 40.1 ± 14.2 40.1 ± 6.2 20.7 ± 7.0 0.585 0.946 0.886 0.246 Tumour location (n/%) Unilobar Bilobar 35 (77.8) 10 (22.2) 32 (71.1) 13 (28.9) 0.468 - Tumour distribution (n/%) Unifocal Multifocal 36 (80.0) 9 (20.0) 34 (75.6) 11 (24.4) 0.612 Diameter of the largest tumour (cm) 7.2 ± 2.4 6.8 ± 2.3 0.397 Data were presented as mean ± standard deviation or count (%). Comparisons between two groups were determined by t-test, Wilcoxon rank sum test or Chi-square test. Statistical significance was set at P < 0.05. ALT = alanine aminotransferase; ALB, albumin; AST, aspartate aminotransferase; BCLC = Barcelona Clinic Liver Cancer; cTACE = conventional transarterial chemoembolization; DEB- TACE = drug-eluting beads transarterial chemoembolization; ECOG = Eastern Cooperative Oncology Group; TBIL, total bilirubin Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma 75 group (352 days, 95% CI: 301-467 days) was signifi- cantly longer than the cTACE group (278 days, 95% CI: 247-324 days, P = 0.004, Figure 2B). Comparison of safety between the two groups No significant difference was noted in the base- line value of the liver function indexes (AST, ALT, ALB, and TBIL) between the two groups (Figure 3 A–D). At 1 week after the procedure, the AST, ALT, and TBIL indexes were significantly higher in both groups, while the ALB level was lower than the baseline (Table 3). The comparison at 1 week after the procedure between the two groups showed that, the AST, ALT, and TBIL content in the DEB- TACE group was higher than that in cTACE group (Figure 3 A, B, D). However, there was no signifi- cant difference of the ALB between the two groups (Figure 3 C). At 1 month after the procedure, all the liver function indexes in the two groups returned to the baseline values (Table 3) and had no differ- ence between the two groups (Figure 3 A–D). For post-embolizat ion syndrome, the incidence of fatigue, abdominal distension, abdominal pain, and nausea/emesis were similar in the two groups within 1 month after the operation; however, the FIGURE 2. The comparison of median overall survival (OS) and progression-free survival (PFS) between the two groups. (A) the median OS of the drug-eluting beads transarterial chemoembolization (DEB-TACE) group was significantly longer than the conventional transarterial chemoembolization (cTACE) group (P = 0.027). (B) the median PFS of the DEB-TACE group was significantly longer than the cTACE group (P = 0.004). A A B B C D FIGURE 3. Comparison of the liver function between the two groups. The aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) levels were higher in the drug-eluting beads transarterial chemoembolization (DEB-TACE) group than in the conventional transarterial chemoembolization (cTACE) group at 1 week (P < 0.05) after the operation, and all the liver function indexes had no difference between the 2 groups at 1 month after the operation. Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma76 incidence of fever was higher in the DEB-TACE group than the cTACE group (P = 0.031, Table 4). Notably, the NRS of abdominal pain in the DEB- TACE group (4.4 ± 1.9) was higher than that in cTA- CE group (3.6 ± 1.6, P = 0.037, Table 4). Strikingly, the fever alleviated by drinking excess water and taking ibuprofen, and abdominal pain alleviated by intravenous injection of flurbiprofen axetil or by taking in morphine in 2-3 days. In addition, no serious adverse events, such as liver abscess, acute liver function failure, severe infection, gastrointes- tinal/intratumoral bleeding, and hepatorenal syn- drome, occurred in either group. Discussion In the present prospective study, we compared the treatment response, the survival, and the safety of unresectable HCC patients treated with DEB- TACE with CSM and cTACE. The results indicated that DEB-TACE was significantly superior to cTA- CE in the following aspects: (1) The higher ORR at 1 month, 3 months, and 6 months, and the higher DCR at 3 and 6 months in the DEB-TACE group; (2) The higher CR at 3 months in DEB-TACE group; (3) The lower PD at 3 and 6 months in the DEB-TACE group; (4) The longer OS and PFS in the DEB-TACE group. Although, the liver function indexes (ALT, AST, and TBIL) injury were elevated in the DEB- TACE group at 1 week, but the indexes recovered to the preoperative level at 1 month after treatment in both groups. Moreover, DEB-TACE with CSM increases the incidence of fever and causes severe abdominal pain, which could be controlled by drugs. One patient died 5 months after operation in the DEB-TACE group due to disease progression rather than serious complications of DEB-TACE. In clinical practice, DEB-TACE and cTACE have become the first-line therapeutic selection for in- TABLE 2. Comparison of treatment response evaluated at 1-month, 3-month, and 6-month after treatment between the two groups Parameters 1-month 3-month 6-month DEB-TACE group (n = 45) cTACE group (n = 45) P value DEB-TACE group (n = 45) cTACE group (n = 45) P value DEB-TACE group (n = 44) cTACE group (n = 45) P value CR (%) 8 (17.8) 6 (13.3) 0.561 10 (22.2) 3 (6.7) 0.036 7 (15.9) 3 (6.7) 0.168 PR (%) 24 (53.3) 16 (35.6) 0.090 23 (51.1) 16 (35.6) 0.137 23 (52.3) 13 (28.9) 0.025 ORR (%) 32 (71.1) 22 (48.9) 0.031 33 (73.3) 19 (42.2) 0.003 30 (68.2) 16 (35.6) 0.002 SD (%) 10 (22.2) 18 (40.0) 0.069 6 (13.3) 11 (24.4) 0.178 10 (22.7) 17 (37.8) 0.123 DCR (%) 42 (93.3) 40 (88.9) 0.459 39 (86.7) 30 (66.7) 0.025 40 (90.9) 33 (73.3) 0.031 PD (%) 3 (6.7) 5 (11.1) 0.459 6 (13.3) 15 (33.3) 0.025 4 (9.1) 12 (26.7) 0.031 Data were presented as count (%). Comparison between 2 groups was determined by Chi-square test. Statistical significance was set at P < 0.05, and were shown in boldface. CR = complete response; cTACE = conventional transarterial chemoembolization; DCR = disease control rate; DEB-TACE = drug-eluting beads transarterial chemoembolization; ORR = objective response rate; PR = partial response; SD = stables disease; PD = progression disease TABLE 3. Compared with the baseline, the changes of liver function at 1-week and 1-month after procedure in the two groups Parameters DEB-TACE (n = 45) cTACE (n = 45) Baseline 1-week P value 1-month P value Baseline 1-week P value 1-month P value ALT (U/L) 38.0 ± 20.5 65.0 ± 18.3 < 0.001 35.3 ± 19.8 0.431 35.7 ± 18.7 55.2 ± 19.8 <0.001 37.4 ± 13.5 0.558 AST (U/L) 40.3 ± 16.8 61.3 ± 19.7 < 0.001 39.1 ± 12.2 0.722 40.1 ± 14.2 52.4 ± 20.0 0.003 38.2 ± 11.7 0.534 ALB (g/L) 39.9 ± 8.2 36.7 ± 5.5 0.004 40.3 ± 7.21 0.758 40.1 ± 6.2 37.2 ± 5.4 0.027 39.1 ± 6.9 0.508 TIBL (umol/L) 19.1 ± 6.1 30.0 ± 10.2 < 0.001 18.0 ± 5.2 0.399 20.7 ± 7.0 24.8 ± 10.4 0.003 19.0 ± 7.0 0.328 Data were presented as mean ± standard deviation and were determined by t-test or Wilcoxon rank sum test. Statistical significance was set at P < 0.05, and were shown in boldface ALT = alanine aminotransferase; ALB = albumin; AST = aspartate aminotransferase; cTACE = conventional transarterial chemoembolization; DEB-TACE = drug-eluting beads transarterial chemoembolization; TBIL = total bilirubin Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma 77 termediate and advanced stage HCC according to the BCLC staging system.13 Although DEBs have the ability to load chemotherapeutic agents and release them in a controlled mode, the evidence to show that DEB-TACE is superior to cTACE is in- sufficient. Presently, CSM is the first novel DEB product in China that has been applied clinically only in the last several years. Some studies have compared the tumor response of DEB-TACE with CSM and cTACE for HCC; however, most of them are retrospective studies, and the results were in- consistent. For example, Wu et al.10 indicated that CR, ORR, and DCR rates in the DEB-TACE group were significantly higher than those in the cTACE group at 3 and 6 months. Liang et al.14 showed that compared to cTACE, DEB-TACE with CSM treat- ment had a higher ORR within 6 months and a higher DCR at 3 and 6 months. Different from the above results, Zhang et al.15 demonstrated that ORR was not different between the two groups, while DCR was significantly higher in the cTACE group than in the DEB-TACE group at 1 month and 3 months. Notably, the articles comparing the efficacy and safety of DEB-TACE with CSM and cTACE are mostly retrospective studies. A recent study adopted prospective design to compare the efficacy and safety of cTACE and DEB-TACE with CSM, and the results were similar to ours. But an uncommon cytotoxic drug (arsenic trioxide) was used in DEB-TACE group, so that the significance of clinical guidance is uncertain.12 Herein, we prospectively compared the treat- ment response within 6 months, OS and PFS of cTACE and DEB-TACE using CSM with pirarubicin (a commonly cytotoxic drug). The results exhibited that DEB-TACE with CSM displayed superior CR (at 3 months), ORR (at 1, 3, and 6 months), and DCR (at 3 and 6 months) over cTACE treatment. The im- proved treatment responses could be attributed to the fact that CSM has the ability to load chemo- therapeutic agents and release them in a controlled pattern, thus maintaining a higher concentration of chemotherapeutic drugs and better efficacy on reducing diameters of the tumor tissues than cTACE.16,17 In addition, calibrated CSM has shown permanent embolization, which improves the tu- mor responses in the DEB-TACE treatment with CSM group.18 This might explain the increased CR and decreased PD in the DEB-TACE group at 3 months in this study. Interestingly, with decreased PD in the DEB-TACE group, HCC patients experi- enced a reduced frequency of operations and thus economic burden. The higher CR at 3 months in patients receiving DEB-TACE treatment with CSM could be ascribed to the fact that CSM achieves a high concentration of chemotherapeutic agents in 3 months. According to the survey, most of the HCC pa- tients are in intermediate and advanced stages at diagnosis.19 Hence, short-term efficacy and the safety of the treatment are critical for the HCC pa- tients. A recent multi-center, retrospective registry cohort study8 showed higher CR and ORR, while the DCR was similar in the DEB-TACE group com- pared to the cTACE group. A meta-analysis report- ed that DEB-TACE with CSM displays superior treatment response, which was consistent with our results.20 As we know, the most important outcomes in oncology trials are OS and PFS. Accumulating evi- dence showed that DEB-TACE acquired long-term survival profile (such as OS and PFS) than cTACE in HCC patients.21,22 The patients in DEB-TACE group showed longer OS and PFS in our study, indicating that the patients underwent DEB-TACE with CSM got more survival benefits compared to patients in cTACE group. While a small amount of litera- ture showed no difference in OS or PFS between DEB-TACE and cTACE groups.11,23 The difference of survival results may be related to the different types of EDBs, heterogeneity of included patients, and the different types of research. It can be seen that our prospective randomized controlled study is very necessary. After TACE treatment, liver function injury is one of the major safety concerns in HCC patients. Some studies compared the liver function indexes before and after treatment between DEB-TACE and cTACE and revealed that AST, ALT, and TBIL increased substantially 7 days after TACE and re- TABLE 4. Comparison of post-embolization syndrome Adverse events DEB-TACE (n = 45) cTACE (n = 45) P value Fatigue (n/%) 5 (11.1) 7 (15.6) 0.535 Fever (n/%) 23 (51.1) 13 (28.9) 0.031 Abdominal distension (n/%) 9 (20) 7 (15.6) 0.581 Abdominal pain (n/%) Numeric Rating Scale (NRS) 42 (93.3) 4.4 ± 1.9 43 (95.6) 3.6 ± 1.6 0.645 0.037 Nausea/emesis (n/%) 10 (22.2) 12 (26.7) 0.624 Data were presented as mean ± standard deviation or count (%). Comparisons between two groups was determined by t-test, Wilcoxon rank sum test or Chi-square test. Statistical significance was set at P < 0.05, and were shown in boldface. cTACE = conventional transarterial chemoembolization; DEB-TACE = drug-eluting beads transarterial chemoembolization Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma78 turned to baseline in 1 month.15,24,25 In this study, the liver function was damaged in both DEB-TACE and cTACE groups at 1 week after the treatment, in- cluding the increased ALT, AST, and TBIL and the decreased ALB content. Different from other stud- ies, the current results showed that the ALT, AST, and TBIL in the DEB-TACE group was higher than that in the cTACE group at 1 week. Similar to stud- ies, the indexes recovered to the preoperative level at 1 month after treatment. This phenomenon indi- cated that DEB-TACE with CSM had more serious damage of liver function compared to cTACE in the short term (about 1 week) and could return to base- line in 1 month. The results also showed that DEB- TACE with CSM and cTACE had parallel effects on the liver function at 1 month, and HCC patients can tolerate both procedures satisfactorily. This obser- vation was consistent with previous studies.13,25 Postembolization syndrome is the most com- mon adverse event. Partially in line with these studies15,24,25, adverse events in the current study included fatigue, abdominal distension, abdomi- nal pain, and nausea/emesis, which were similar between the two groups. However, DEB-TACE with CSM resulted in a high incidence of fever. Although no significant difference was observed in the incidence of abdominal pain between the two groups, the NRS of DEB-TACE group was higher. This might be related to the following effects: (1) The tumors in the DEB-TACE group achieved substantial tumor necrosis and results in severe abdominal pain; (2) Patients in the DEB-TACE group had significant tumor necrosis induced by treatment with CSM, and the incidence of inflam- mation could be enhanced by substances released from the necrotic tumor tissue. Thus, the patients in the DEB-TACE group experienced severe pain and a high risk of fever. However, the fever was al- leviated by ibuprofen, and the pain was alleviated by intravenous injection of flurbiprofen axetil or morphine in 2-3 days in both groups. Also, no seri- ous adverse events, such as liver abscess, acute liv- er function failure, and severe infection, occurred in either of the groups, which could be attributed to the superselective technique, and the selected patients were controlled in BCLC stages A and B. This is a randomized controlled trial on the efficacy and safety of DEB-TACE with CSM vs. cTACE; hence, confounding factors were mini- mal, providing accurate evidence to the clinicians. Nevertheless, the current study has some limita- tions: (1) This is a single-center study with small sample size. However, according to the statistical analysis, 39 patients in each group showed varied ORR between the two groups, with 80% power and a 5% significance level; (2) The tumor re- sponses were followed up for only 6 months, and for survival analysis was not based on the death of all patients. At the last follow-up, 64.4% (29/45) of patients in cTACE group and 60.0% (27/45) of pa- tients in DEB-TACE group died. Therefore, a long- er follow-up is necessary in the future study; (3) The amount of pirarubicin administered between DEB-TACE group and cTACE group was not com- pared, which may have an impact on the results, and further study should compare the dose of the pirarubicin between the two groups. In conclusion, this study demonstrated that DEB-TACE with CMS had a better tumor response in some aspects (higher CR at 3 months, ORR at 1, 3, and 6 months) than the cTACE group. The liver function injury was more serious in DEB-TACE group at 1 week but returned to the baseline at 1 month in the cTACE group. The increased inci- dence of fever and severe abdominal pain in the DEB-TACE group could be relieved by sympto- matic treatment. References 1. Chen C, Qiu H, Yao Y, Zhang Z, Ma C, Ma Y, et al. Comprehensive predictive factors for CalliSpheres® microspheres (CSM) drug-eluting bead-transarterial chemoembolization and conventional transarterial chemoembolization on treatment response and survival in hepatocellular carcinoma pa- tients. Clin Res Hepatol Gastroenterol 2021; 45: 101460. doi: 10.1016/j. clinre.2020.05.008 2. Roberts LR, Sirlin CB, Zaiem F, Almasri J, Prokop LJ, Heimbach JK, et al. Imaging for the diagnosis of hepatocellular carcinoma: a systematic review and meta-analysis. Hepatology 2018; 67: 401-21. doi: 10.1002/hep.29487 3. Della Corte C, Triolo M, Iavarone M, Sangiovanni A. Early diagnosis of liver cancer: an appraisal of international recommendations and future perspec- tives. Liver Int 2016; 36: 166-76. doi: 10.1111/liv.12965 4. Kishore S, Friedman T, Madoff DC. Update on embolization therapies for hepatocellular carcinoma. Curr Oncol Rep 2017; 19: 40. doi: 10.1007/ s11912-017-0597-2 5. Park JW, Chen M, Colombo M, Roberts LR, Schwartz M, Chen PJ, et al. Global patterns of hepatocellular carcinoma management from diagnosis to death: the BRIDGE Study. Liver Int 2015; 35: 2155-66. doi: 10.1111/liv.12818 6. de Baere T, Arai Y, Lencioni R, Geschwind JF, Rilling W, Salem R, et al. Treatment of liver tumors with lipiodol TACE: technical recommendations from experts opinion. Cardiovasc Intervent Radiol 2016; 39: 334-43. doi: 10.1007/s00270-015-1208-y 7. Sun J, Zhou G, Xie X, Gu W, Huang J, Zhu D, et al. Efficacy and safety of drug- eluting beads transarterial chemoembolization by CalliSpheres(®) in 275 hepatocellular carcinoma patients: results from the Chinese CalliSpheres(®) Transarterial Chemoembolization in Liver Cancer (CTILC) study. Oncol Res 2020; 28: 75-94. doi: 10.3727/096504019x15662966719585 8. Peng Z, Cao G, Hou Q, Li L, Ying S, Sun J, et al. The comprehensive analysis of efficacy and safety of CalliSpheres(®) drug-eluting beads transarterial chem- oembolization in 367 liver cancer patients: a multiple-center, cohort study. Oncol Res 2020; 28: 249-71. doi: 10.3727/096504019x15766663541105 9. Shi Q, Liu J, Li T, Zhou C, Wang Y, Huang S, et al. Comparison of DEB- TACE and cTACE for the initial treatment of unresectable hepatocellular carcinoma beyond up-to-seven criteria: a single-center propensity score matching analysis. Clin Res Hepatol Gas 2022; 46: 101893. doi: 10.1016/j. clinre.2022.101893 Radiol Oncol 2023; 57(1): 70-79. Shi Z et al. / DEB-TACE vs. cTACE in HCC in hepatocellular carcinoma 79 10. Wu B, Zhou J, Ling G, Zhu D, Long Q. CalliSpheres drug-eluting beads versus lipiodol transarterial chemoembolization in the treatment of hepatocellular carcinoma: a short-term efficacy and safety study. World J Surg Oncol 2018; 16: 69. doi: 10.1186/s12957-018-1368-8 11. Ma Y, Zhao C, Zhao H, Li H, Chen C, Xiang H, et al. Comparison of treatment efficacy and safety between drug-eluting bead transarterial chemoembo- lization with CalliSpheres(®) microspheres and conventional transarterial chemoembolization as first-line treatment in hepatocellular carcinoma pa- tients. Am J Transl Res 2019; 11: 7456-70. PMID: 31934293 12. Duan XH, Ju SG, Han XW, Ren JZ, Li FY, Chen PF, et al. Arsenic trioxide- eluting Callispheres beads is more effective and equally tolerant com- pared with arsenic trioxide/lipiodol emulsion in the transcatheter arterial chemoembolization treatment for unresectable hepatocellular carcinoma patients. Eur Rev Med Pharmaco 2020; 24: 1468-80. doi: 10.26355/eur- rev_202002_20206 13. Xiang H, Long L, Yao Y, Fang Z, Zhang Z, Zhang Y. CalliSpheres drug-eluting bead transcatheter arterial chemoembolization presents with better ef- ficacy and equal safety compared to conventional TACE in treating pa- tients with hepatocellular carcinoma. Technol Cancer Res Treat 2019; 18: 1533033819830751. doi: 10.1177/1533033819830751 14. Liang B, Xiang H, Ma C, Xiong B, Ma Y, Zhao C, et al. Comparison of chem- oembolization with CalliSpheres(®) microspheres and conventional chem- oembolization in the treatment of hepatocellular carcinoma: a multicenter retrospective study. Cancer Manag Res 2020; 12: 941-56. doi: 10.2147/ cmar.S187203 15. Zhang L, Sun JH, Ji JS, Zhong BY, Zhou GH, Song JJ, et al. Imaging changes and clinical complications after drug-eluting bead versus conventional tran- sarterial chemoembolization for unresectable hepatocellular carcinoma: Multicenter study. AJR Am J Roentgenol 2021; 217: 933-43. doi: 10.2214/ ajr.20.24708 16. Zhang S, Huang C, Li Z, Yang Y, Bao T, Chen H, et al. Comparison of pharmacokinetics and drug release in tissues after transarterial chem- oembolization with doxorubicin using diverse lipiodol emulsions and CalliSpheres beads in rabbit livers. Drug Deliv 2017; 24: 1011-7. doi: 10.1080/10717544.2017.1344336 17. Facciorusso A. Drug-eluting beads transarterial chemoembolization for hepatocellular carcinoma: current state of the art. World J Gastroenterol 2018; 24: 161-9. doi: 10.3748/wjg.v24.i2.161 18. Raoul JL, Forner A, Bolondi L, Cheung TT, Kloeckner R, de Baere T. Updated use of TACE for hepatocellular carcinoma treatment: how and when to use it based on clinical evidence. Cancer Treat Rev 2019; 72: 28-36. doi: 10.1016/j.ctrv.2018.11.002 19. Elshaarawy O, Gomaa A, Omar H, Rewisha E, Waked I. Intermediate stage hepatocellular carcinoma: a summary review. J Hepatocell Carcinoma 2019; 6: 105-17. doi: 10.2147/jhc.S168682 20. Liang B, Makamure J, Shu S, Zhang L, Sun T, Zheng C. Treatment response, survival, and safety of transarterial chemoembolization with CalliSpheres(®) microspheres versus conventional transarterial chemoembolization in hepatocellular carcinoma: a meta-analysis. Front Oncol 2021; 11: 576232. doi: 10.3389/fonc.2021.576232 21. Liu YS, Lin CY, Chuang MT, Lin CY, Tsai YS, Wang CK, et al. Five-year outcome of conventional and drug-eluting transcatheter arterial chemoembolization in patients with hepatocellular carcinoma. BMC Gastroenterol 2018; 18: 124. doi: 10.1186/s12876-018-0848-1 22. Song MJ, Chun HJ, Song DS, Kim HY, Yoo SH, Park CH, et al. Comparative study between doxorubicin-eluting beads and conventional transarterial chemoembolization for treatment of hepatocellular carcinoma. J Hepatol 2012; 57: 1244-50. doi: 10.1016/j.jhep.2012.07.017 23. Facciorusso A, Mariani L, Sposito C, Spreafico C, Bongini M, Morosi C, et al. Drug-eluting beads versus conventional chemoembolization for the treat- ment of unresectable hepatocellular carcinoma. J Gadtroen Hepatol 2016; 31: 645-53. doi: 10.1111/jgh.13147 24. Kloeckner R, Weinmann A, Prinz F, Pinto dos Santos D, Ruckes C, Dueber C, et al. Conventional transarterial chemoembolization versus drug-eluting bead transarterial chemoembolization for the treatment of hepatocellular carcinoma. BMC Cancer 2015; 15: 465. doi: 10.1186/s12885-015-1480-x 25. Ni JY, Xu LF, Wang WD, Sun HL, Chen YT. Conventional transarterial chem- oembolization vs microsphere embolization in hepatocellular carcinoma: a meta-analysis. World J Gastroenterol 2014; 20: 17206-17. doi: 10.3748/ wjg.v20.i45.17206 Radiol Oncol 2023; 57(1): 80-85. doi: 10.2478/raon-2022-0026 80 research article Does concurrent gynaecological surgery affect infectious complications rate after mastectomy with implant-based reconstruction? Nina Pislar1,2, Barbara Peric1,2, Uros Ahcan2,3, Romi Cencelj-Arnez1,2, Janez Zgajnar1,2, Andraz Perhavec1,2 1 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Plastic Surgery and Burns, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(1): 80-85. Received 24 April 2022 Accepted 23 May 2022 Correspondence to: Asst. Prof. Andraz Perhavec, M.D., Ph.D., Department of Surgical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: aperhavec@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. Women who undergo breast cancer surgery often have an indication for gynaecological procedure. The aim of our study was to compare infectious complications rate after mastectomy with implant-based reconstruc- tion in patients with and without concurrent gynaecological procedure. Patients and methods. We retrospectively reviewed clinical records of 159 consecutively operated patients after mastectomy with implant-based reconstruction. The patients were divided in 2 groups: 102 patients without (Group 1) and 57 with (Group 2) concurrent gynaecological procedure. Infectious complications rates between the groups were compared using χ2-test. Logistic regression was performed to test for association of different variables with infec- tious complications. Results. There were 240 breast reconstructions performed. Median follow-up time was 297 days (10–1061 days). Mean patient age was 47.2 years (95% CI 32.8–65.9); 48.2 years (95% CI 46.1–50.3) in Group 1 and 45.8 years (95% CI 43.2–48.3) in Group 2; p = 0.002). Infectious complications rate was 17.6% (17.6% vs. 17.5%, p = 0.987), implant loss occurred in 5.7% (4.9% vs. 7.0%, p = 0.58). Obesity (body mass index [BMI] > 30 kg/m2), age, previous breast conserv- ing treatment (BCT) with radiotherapy (RT) were identified as risk factors for infectious complications in univariate analysis. Obesity (adjusted odds ratio [aOR] 3.319, 95% CI 1.085–10.157, p = 0.036) and BCT with RT (aOR 7.481, 95% CI 2.230–25.101, p = 0.001) were independently associated with infectious complications in multivariate model. Conclusions. Concurrent gynaecological procedure for patients undergoing mastectomy with implant-based re- construction did not carry an increased risk for infectious complications. Key words: breast cancer; infectious complications; implant-based reconstruction; concurrent surgical manage- ment; implant loss Introduction Combining a clean surgery that involves prosthetic material with a clean-contaminated surgery has always been controversial.1 Women who undergo mastectomy with implant reconstruction for risk reduction or cancer often have an indication for a gynaecological procedure.2 In premenopausal women with hormone receptor-positive tumours, ovarian suppression with surgical oophorectomy has been recognised as part of the treatment strat- egy for more than a century.3 In high-risk women, Radiol Oncol 2023; 57(1): 80-85. Pislar N et al. / Concurrent gynaecological surgery and infections after implant-based reconstruction 81 surgical intervention with prophylactic bilateral mastectomy reduces breast cancer risk by up to 95%, while bilateral salpingo-oophorectomy re- duces both breast and ovarian cancer risks by around 50% and 80%, respectively.4,5 It is also as- sociated with improved survival.6–8 When immediate implant-based breast recon- struction is planned, skin-sparing mastectomy (SSM) is most commonly performed, but nipple- sparing mastectomy (NSM) can be a safe option in selected cases.9–11 Infectious complications in implant-based re- constructions can cause prolonged antibiotic treat- ment and can result in implant removal.12,13 This may delay adjuvant treatments for breast cancer and cause scarring that can affect functional as well as aesthetic outcome. Therefore, a low infec- tious complications rate is important.14 Infectious complications rate varies between centres and is around 20%.15 Due to increased operating time and an intraab- dominal procedure, coordinated surgical manage- ment of the breast with a concurrent gynaecologi- cal procedure could increase the likelihood of in- fectious complications.16 On the other hand, com- bining the procedures adds to patient satisfaction and optimises the time and cost management.17 The aim of our study was to compare infectious complications rate after mastectomy with implant- based reconstruction in a group of patients with and without concurrent gynaecological procedure. Patients and methods Study cohort and data collection We conducted a retrospective analysis of infec- tious complications in patients after implant-based reconstruction with or without concurrent gy- naecological procedure and followed them until the date of complication, expander-prosthesis ex- change surgery, or until last follow-up visit. We retrospectively reviewed records of 159 women (and 240 breast reconstructions) that were con- secutively operated at the Institute of Oncology Ljubljana, Slovenia between February 2014 and June 2020 for a new or previously diagnosed breast cancer and/or had an increased risk for developing breast cancer, mainly due to a recognised BRCA1/2 mutation. Unilateral or bilateral mastectomy was performed, either SSM or NSM, followed by breast reconstruction with either tissue expander or pros- thesis. Fifty-seven patients had a laparoscopic gynaecological procedure (salpingectomy, oopho- rectomy, hysterectomy or a combination) during the same anaesthesia. Postoperative complications were tracked reviewing follow-up visits with sur- gical oncologist and reconstructive surgeon. We recorded infectious complications requiring the use of oral or parenteral antibiotics, infectious and wound healing complications requiring surgical treatment under general anaesthesia (necrectomy, debridement) and implant loss due to infection. Treatment protocol As part of standard treatment protocol at our cen- tre immediate reconstruction is offered after pro- phylactic or therapeutic mastectomy, either au- tologous or implant-based. Patients’ cases are dis- cussed prior to surgery at a multidisciplinary team meeting between a surgical oncologist, a radiation therapist and a reconstructive surgeon. The pa- tients are operated under general anaesthesia with perioperative antibiotic prophylaxis. Two grams of cephazolin are given for prophylaxis and the an- tibiotics are continued post-operatively, typically until drains are removed. If a gynaecological pro- cedure is planned, it is performed first, followed by breast surgery. The gynaecological procedure is performed laparoscopically. Mastectomy with or without axillary lymph node surgery is performed by a surgical oncologist, followed by the recon- structive procedure that is performed by a recon- structive surgeon. A tissue implant is inserted in a pocket, which consists of pectoralis major and serratus anterior muscle. Prior to implant inser- tion, the area is irrigated with antibiotic solution. The drainage stays in place until less than approxi- mately 50 ml discharge daily for two consecutive days. After the drains are removed and the wounds heal, tissue expanders are filled gradually with sa- line solution in an outpatient setting every two to three weeks. Statistical analysis Patients’ characteristics were compared between the two groups (with and without gynaecological procedure) with χ2 test or Fisher’s exact test for categorical variables and Student t-test for continu- ous variables. Data were reported as counts and frequencies for categorical and as median with sample range or mean with 95% confidence inter- val (CI) for continuous variables. Univariate binary logistic regression was performed to test for asso- ciation of different variables with infectious com- plications. Variables with a statistical significance Radiol Oncol 2023; 57(1): 80-85. Pislar N et al. / Concurrent gynaecological surgery and infections after implant-based reconstruction82 of p < 0.1 were included in a multivariate binary logistic regression model. Data were analysed us- ing IBM SPSS Statistics software (Statistical pack- age for the Social Sciences Statistical Software, IBM Corporation, Armonk, NY, USA). Statistical signifi- cance was set at p < 0.05. The study was reviewed and approved by the Institutional Review Board and Ethics Committee. Results In 159 patients, 240 breast reconstructions were performed with 214 tissue expanders and 26 pros- theses. All patients were women. Median follow- up time was 297 days (10–1061 days), 321 days (14–712 days) in Group 1 and 273 days (10–1061 days) in Group 2. Expander-prosthesis exchange surgery was mostly performed within a year from initial surgery, median 333.5 days (74 – 712 days). Fifty-seven patients (35.8%) had a concurrent laparoscopic gynaecological procedure (Group 2). These patients were younger at the time of surgery (Group 1 48.2 years, 95% CI 46.1–50.3 vs. Group 2 45.8 years, 95% CI 43.2–48.3, p = 0.002) and more likely to have been previously treated with breast conserving therapy (BCT) including radiotherapy (RT) for breast cancer (Group 1 7.8% vs. Group 2 36.8%, p = 0.001). Patients without combined proce- dures (Group 1) were more likely treated with neo- adjuvant chemotherapy (NACT) (21.6% vs. 5.3%, p = 0.007) and more likely received adjuvant RT (31.4% vs. 5.3%, p = 0.001). We present the patients’ characteristics in Table 1. TABLE 1. Patients characteristics Variable All Group 1 Group 2 p N = 159 N = 102 (64.2%) N = 57 (35.8%) Age (years) 47.2 (95% CI 32.8–65,9) 48.2 (95% CI 46.1–50.3) 45.8 (95% CI 43.2–48.3) 0.002 BMI (kg/m2) 24.7 (95% CI 18.9–34,8) 24.4 (95% CI 23.4–25.4) 25.8 (95% CI 24.1–27.4) 0.189 Smoking 37 (23.3%) 22 (21.6%) 15 (26.3%) 0.497 Diabetes mellitus 3 (1.9%) 3 (2.9%) 0 0.191 ASA score 0.622 1 31 (19.5%) 24 (23.5%) 7 (12.3%) 2 80 (50.3%) 56 (54.9%) 24 (42.1%) 3 11 (6.9%) 7 (6.9%) 4 (7.0%) Unknown 37 (23.3%) 15 (14.7%) 22 (38.6%) Previous BCT with RT 29 (18.2%) 8 (7.8%) 21 (36.8%) 0.001 NACT 25 (15.7%) 22 (21.6%) 3 (5.3%) 0.007 Adjuvant RT 35 (22.0%) 32 (31.4%) 3 (5.3%) 0.001 ACT 33 (20.8%) 23 (22.5%) 10 (17.5%) 0.455 Group 1: Patients without gynaecological procedure; Group 2: Patients with gynaecological procedure. ACT = adjuvant chemotherapy; ASA = American Society of Anaesthesiology; BC = breast cancer; BCT = breast conserving therapy; BMI = body mass index; NACT = neoadjuvant chemotherapy; RT = radiotherapy TABLE 2. Surgical site infections after implant reconstruction All (%) Group 1 (%) Group 2 (%) p-value All* 28 (17.6) 18 (17.6) 10 (17.5) 0.987 Surgical intervention needed** 13 (8.2) 8 (7.8) 5 (8.8) 0.84 Implant loss*** 9 (5.7) 5 (4.9) 4 (7.0) 0.58 Group 1: Patients without gynaecological procedure; Group 2: Patients with gynaecological procedure. *all surgical site infectious complications requiring oral or i.v. antibiotic, surgical debridement under general anaesthesia or implant removal surgery**surgical intervention requiring general anaesthesia ***tissue-expander or prosthesis removal due to infection. Radiol Oncol 2023; 57(1): 80-85. Pislar N et al. / Concurrent gynaecological surgery and infections after implant-based reconstruction 83 Overall infectious complication rate in our co- hort of 159 women was 17.6% and did not signifi- cantly differ between the groups (17.6% vs. 17.5%, p = 0.987). Tissue implants had to be removed due to infection in 5.7% (4.9% vs. 7.9%, p = 0.58). We present the comparison between groups in Table 2. Several covariates were tested for association with overall infectious complications in the en- tire cohort. Obesity (body mass index [BMI] > 30 kg/m2), age and previous BCT with RT for breast cancer were identified as risk factors for infec- tious complications. Concurrent gynaecological procedure, smoking, diabetes, American Society of Anaesthesiology (ASA) score, neo-/adjuvant systemic therapy and adjuvant RT were not sig- nificantly associated with infectious complications (Table 3). Age at the time of surgery, BMI and previous BCT with RT were included in the multivariate model. Obesity (BMI > 30 kg/m2) and previous BCT with RT were independently associated with infec- tious complications. Women with a history of BCT and RT for breast cancer had approximately three times higher odds for infectious complications compared to those without previous BCT with RT (adjusted odd ratio [aOR] 3.319, 95% CI 1.085– 10.157, p = 0.036). Obese patients (BMI > 30 kg/m2) had about 7.5-times higher odds for infectious complications compared to women who had a BMI in the normal range between 19 and 25 kg/m2 (aOR 7.481, 95% CI 2.230–25.101, p = 0.001) (Table3). Discussion In presented retrospective single centre series of 159 women, who underwent mastectomy with implant-based reconstruction there was no asso- ciation between infectious complications rate and concurrent gynaecological procedure. The results are consistent with other studies. In a group of seventy breast cancer patients that under- went laparoscopic oophorectomy, among which 29 had a concurrent breast surgery, Willshire et al. have shown it is safe to carry out the gynaeco- logical procedure in a combined setting. However, only four patients in this cohort had mastectomy with implant-based reconstruction and the focus on postoperative complications was the gynaeco- logical procedure.2 For 62 high-risk women that opted for breast and ovarian risk-reducing sur- gery, post-operative complications rate was no different between sequential vs. coordinated surgi- cal management. The study included autologous reconstructions.18 Furthermore, a new approach has been described for performing laparoscopy via a transmammary route to improve aesthetic out- come and avoid abdominal scars.19 In a recently published study, the rates of post- operative complications for implant-based recon- structions were comparable between 141 patients with concurrent gynaecological and 29 patients without gynaecological procedure.20 The complica- tions only represent the perioperative period, but the sample size is comparable to our study. Overall, infectious complications rate in our co- hort was 17.6% and implant loss occurred in 5.7%. In a recent case series of 16 patients with coordi- nated surgical management, a 37% 30-day postop- erative complication rate was observed, but minor complications, such as seroma and excessive drain- age were also included.21 In a subgroup of 19 co- ordinately managed patients with implant-based reconstruction, implant loss was observed in two women (11%). In larger series, implant loss rates are comparable to our centre.22 TABLE 3. Variables associated with infectious complications Variable OR P aOR p Gynaecological procedure 1.116 (0.486–2.564) 0.796 NA NA BMI < 25 1 1 25–30 3.000 (0.974–9.239) 0.056 2.552 (0.777–8.382) 0.122 > 30 8.100 (2.540–25.826) < 0.001 7.481 (2.230–25.101) 0.001 Age > 45 2.707 (1.118–6.552) 0.027 1.939 (0.497–3.907) 0.529 Smoking 1.061 (0.413–2.724) 0.903 NA NA Diabetes 2.286 (0.200-26.094) 0.506 NA NA ASA 1 1 2 1.821 (0.560-5.921) 0.319 NA NA 3 0.675 (0.067-6.789) 0.739 NA NA Previous BCT with RT 3.802 (1.546-9.354) 0.004 3.319 (1.085–10.157) 0.036 NACT 0.345 (0.076-1.553) 0.165 NA NA ACT 0.995 (0.369-2.687) 0.992 NA NA Adj. RT 0.909 (0.338-2.442) 0.849 NA NA aOR = adjusted odds ratio; ASA = American Society of Anaesthesiology; ACT = adjuvant chemotherapy; BC = breast cancer; BCT = breast conserving therapy; BMI = body mass index; NACT = neoadjuvant chemotherapy; RT = radiotherapy Radiol Oncol 2023; 57(1): 80-85. Pislar N et al. / Concurrent gynaecological surgery and infections after implant-based reconstruction84 In our study, patients in the two groups were different for age, history of BCT with RT, NACT and adjuvant RT. Patients that had combined pro- cedures were younger, which is consistent with the fact that gynaecological risk reduction surgery has greater survival gain if performed earlier.23 A higher proportion of women with a history of BCT in Group 2 is also reasonable, as they would more often have an indication for either endocrine or prophylactic gynaecological procedure.2 Obesity, defined as BMI > 30 kg/m2 is an estab- lished risk factor for surgical site infection and our study results are in accordance with this.12,24 Confidence intervals are relatively large due to low absolute number of obese patients in our study co- hort. We can explain the low numbers with the fact that obese patients are more often advised against breast reconstruction at the multidisciplinary team meeting. They often have other comorbidities that can be associated with complications during and after surgery. The association with obesity in our study is statistically significant and displays more than seven times higher odds for infectious com- plications compared to baseline BMI. Obesity is also a risk factor for implant loss and reduces self- image after reconstructive procedure.25 Similar is known for age; however, the effect in our cohort was small in univariate and lost in multivariate analysis. This could be because median age was below 50 years and patients in our cohort did not have many comorbidities. Smoking has also been recognised as a risk factor for complications, but in our cohort, no association was observed. The data on smoking was inconsistent due to retrospective data recollection and loose definition of smoking status. A history of BCT with RT has been associated with an increased complication rate after tissue expander surgery in previous studies and our study shows similar results.26,27 Postoperative RT is also often recognised as a risk factor for infection and implant loss.15,28 In a large systematic review, Momoh et al. reported no difference in reconstruc- tion failure rates between patients with a history of BCT with RT and postoperative RT.29 In our cohort, adjuvant RT was not associated with an increased risk for infectious complications. In univariate analysis, it even displayed a protective effect, al- though not statistically significant, and was there- fore not included in the multivariate analysis. Neither NACT nor adjuvant chemotherapy were associated with infectious complications and the results are consistent with other studies.30 In a large meta-analysis, NACT was shown to slightly increase implant loss rates, but no delay in starting adjuvant treatment was observed.31 The main limitation of our study is retrospective data collection, including quality of data and selec- tion bias. Patients that were at higher risk for com- plications, were more likely advised against co- ordinated surgical management in the first place. Sample size was sufficient, but small numbers in subcategories resulted in large confidence inter- vals. The study was conducted at the only referral centre for breast cancer cases requiring reconstruc- tion in Slovenia. Follow-up is continued in the out- patient setting and patients are seldom lost during follow-up. Other strengths of the study are recent data and a long follow-up time; most patients have been followed until expander-prosthesis exchange surgery. Concurrent laparoscopic gynaecological pro- cedure for patients undergoing mastectomy with implant-based reconstruction was safe and did not carry an increased risk for postoperative infectious complications. Obesity and previous BCT with RT were independent risk factors for infectious com- plications. Acknowledgments The authors acknowledge the financial support from the Slovenian Research Agency (research core funding No. P3-0352 (C). References 1. Altemeier W, Burke J, Pruitt B, Sandusky W. Manual on control of infection in surgical patients. 2nd edition. Philadelphia: JB Lippincott; 1984. 2. Willsher P, Ali A, Jackson L. Laparoscopic oophorectomy in the manage- ment of breast disease. ANZ J Surg 2008; 78: 670-2. doi: 10.1111/j.1445- 2197.2008.04614.x 3. McDonald Wade S, Hackney MH, Khatcheressian J, Lyckholm LJ. Ovarian suppression in the management of premenopausal breast cancer: methods and efficacy in adjuvant and metastatic settings. Oncology 2008; 75: 192- 202. doi: 10.1159/000163059 4. Fatouros M, Baltoyiannis G, Roukos DH. The predominant role of surgery in the prevention and new trends in the surgical treatment of women with BRCA1/2 mutations. Ann Surg Oncol 2008; 15: 21-33. doi: 10.1245/ s10434-007-9612-4 5. Ludwig KK, Neuner J, Butler A, Geurts JL, Kong AL. Risk reduction and sur- vival benefit of prophylactic surgery in BRCA mutation carriers, a systematic review. Am J Surg 2016; 212: 660-9. doi: 10.1016/j.amjsurg.2016.06.010 6. Rebbeck TR, Kauff ND, Domchek SM. Meta-analysis of risk reduction esti- mates associated with risk-reducing salpingo-oophorectomy in BRCA1 or BRCA2 mutation carriers. J Natl Cancer Inst 2009; 101: 80-7. doi: 10.1093/ jnci/djn442 7. Li X, You R, Wang X, Liu C, Xu Z, Zhou J, et al. Effectiveness of prophylactic surgeries in BRCA1 or BRCA2 mutation carriers: a meta-analysis and sys- tematic review. Clin Cancer Res 2016; 22: 3971-81. doi: 10.1158/1078-0432. CCR-15-1465 Radiol Oncol 2023; 57(1): 80-85. Pislar N et al. / Concurrent gynaecological surgery and infections after implant-based reconstruction 85 8. Domchek SM. Association of risk-reducing surgery in BRCA1 or BRCA2 mu- tation carriers with cancer risk and mortality. JAMA 2010; 304: 967-75. doi: 10.1001/jama.2010.1237 9. Kronowitz SJ, Kuerer HM. Advances and surgical decision-making for breast reconstruction. Cancer 2006; 107: 893-907. doi: 10.1002/cncr.22079 10. Petit JY, Rietjens M, Lohsiriwat V, Rey P, Garusi C, Lorenzi F De, et al. Update on breast reconstruction techniques and indications. World J Surg 2012; 36: 1486-97. doi: 10.1007/s00268-012-1486-3 11. Jakub JW, Peled AW, Gray RJ, Greenup RA, Kiluk JV, Sacchini V, et al. Oncologic safety of prophylactic nipple-sparing mastectomy in a population with BRCA mutations: A multi-institutional study. JAMA Surg 2018; 153: 123-9. doi: 10.1001/jamasurg.2017.3422 12. Reichman DE, Greenberg JA. Reducing surgical site infections: a review. Rev Obstet Gynecol 2009; 2: 212-21. doi: 10.3909/riog0084 13. Peled AW, Stover AC, Foster RD, McGrath MH, Hwang ES. Long-term re- constructive outcomes after expander-implant breast reconstruction with serious infectious or wound-healing complications. Ann Plast Surg 2012; 68: 369-73. doi: 10.1097/SAP.0b013e31823aee67 14. Mioton LM, Jordan SW, Hanwright PJ, Bilimoria KY, Kim JYS. The relationship between preoperative wound classification and postoperative infection: a multi-institutional analysis of 15,289 patients. Arch Plast Surg 2013; 40: 522-9. doi: 10.5999/aps.2013.40.5.522 15. Jagsi R, Jiang J, Momoh AO, Alderman A, Giordano SH, Buchholz TA, et al. Complications after mastectomy and immediate breast reconstruction for breast cancer: a claims-based analysis. Ann Surg 2016; 263: 219-27. doi: 10.1097/SLA.0000000000001177 16. Kryger ZB, Dumanian GA, Howard MA. Safety issues in combined gyneco- logic and plastic surgical procedures. Int J Gynecol Obstet 2007; 99: 257-63. doi: 10.1016/j.ijgo.2007.05.028 17. Khadim MF, Eastwood P, Price J, Morrison P, Khan K. Multidisciplinary one-stage risk-reducing gynaecological and breast surgery with immediate reconstruction in BRCA-gene carrier women. Eur J Surg Oncol 2013; 39: 1346-50. doi: 10.1016/j.ejso.2013.09.018 18. Chapman JS, Roddy E, Panighetti A, Hwang S, Crawford B, Powell B, et al. Comparing coordinated versus sequential salpingo-oophorectomy for BRCA1 and BRCA2 mutation carriers with breast cancer. Clin Breast Cancer 2016; 16: 494-9. doi: 10.1016/j.clbc.2016.06.016 19. Perabò M, Fink V, Günthner-Biller M, Von Bodungen V, Friese K, Dian D. Prophylactic mastectomy with immediate reconstruction combined with simultaneous laparoscopic salpingo-oophorectomy via a transmammary route: a novel surgical approach to female BRCA-mutation carriers. Arch Gynecol Obstet 2014; 289: 1325-30. doi: 10.1007/s00404-013-3133-0 20. Jayaraman AP, Boyd T, Hampton SN, Haddock NT, Teotia SS. The impact of combined risk-reducing gynecological surgeries on outcomes in DIEP flap and tissue-expander breast reconstruction. Plast Surg 2020; 28: 112-6. doi: 10.1177/2292550320925905 21. D’Abbondanza JA, George R, Kives S, Musgrave MA. Concurrent prophy- lactic mastectomy, immediate reconstruction, and salpingo-oophorec- tomy in high-risk patients: a case series. Plast Surg 2020; 28: 243-8. doi: 10.1177/2292550320928551 22. Nahabedian MY, Tsangaris Theodore, Momen B, Manson PN. Infectious complicatious following breast reconstruction with expanders and im- plants. Plast Reconstr Surg 2003; 112: 467-76. doi: 10.1097/01. PRS.0000070727.02992.54 23. Finch APM, Lubinski J, Møller P, Singer CF, Karlan B, Senter L, et al. Impact of oophorectomy on cancer incidence and mortality in women with a BRCA1 or BRCA2 mutation. J Clin Oncol 2014; 32: 1547-53. doi: 10.1200/ JCO.2013.53.2820 24. Elmi M, Azin A, Elnahas A, McCready DR, Cil TD. Concurrent risk-reduction surgery in patients with increased lifetime risk for breast and ovarian cancer: an analysis of the National Surgical Quality Improvement Program (NSQIP) database. Breast Cancer Res Treat 2018; 171: 217-23. doi: 10.1007/ s10549-018-4818-7 25. Kern P, Zarth F, Kimmig R, Rezai M. Impact of age, obesity and smoking on patient satisfaction with breast implant surgery - a unicentric analysis of 318 implant reconstructions after mastectomy. Geburtshilfe Frauenheilkd 2015; 75: 597-604. doi: 10.1055/s-0035-1546171 26. Cordeiro PG, Snell L, Heerdt A, McCarthy C. Immediate tissue expander/im- plast breast reconstruction after salvage mastectomy for cancer recurrence following lumpectomy/irradiation. Plast Reconstr Surg 2012; 129: 341-50. doi: 10.1097/PRS.0b013e318205f203 27. Chetta MD, Aliu O, Zhong L, Sears ED, Waljee JF, Chung KC, et al. Reconstruction of the radiated breast: a national claims-based assessment of postoperative morbidity. Plast Reconstr Surg 2017; 139: 783-92. doi: 10.1097/PRS.0000000000003168 28. Jugenburg M, Disa JJ, Pusic AL, Cordeiro PG. Impact of radiotherapy on breast reconstruction. Clin Plast Surg 2007; 34: 29-37. doi: 10.1016/j. cps.2006.11.013 29. Momoh AO, Ahmed R, Kelley BP, Aliu O, Kidwell KM, Kozlow JH, et al. A systematic review of complications of implant-based breast reconstruction with prereconstruction and postreconstruction radiotherapy. Ann Surg Oncol 2014; 21: 118-24. doi: 10.1245/s10434-013-3284-z 30. Voineskos SH, Frank SG, Cordeiro PG. Breast reconstruction following con- servative mastectomies: predictors of complications and outcomes. Gland Surg 2015; 4: 484-96. doi: 10.3978/j.issn.2227-684X.2015.04.13 31. Varghese J, Gohari SS, Rizki H, Faheem I, Langridge B, Kümmel S, et al. A systematic review and meta-analysis on the effect of neoadjuvant chemo- therapy on complications following immediate breast reconstruction. Breast 2021; 55: 55-62. doi: 10.1016/j.breast.2020.11.023 Radiol Oncol 2023; 57(1): 86-94. doi: 10.2478/raon-2022-0048 86 research article The spine and carina as a surrogate for target registration in cone-beam CT imaging verification in locally advanced lung cancer radiotherapy Jasna But-Hadzic1,2, Karmen Strljic1, Valerija Zager Marcius1,3 1 Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Faculty of Health Sciences, University of Ljubljana, Department of Medical Imaging and Radiotherapy, Ljubljana, Slovenia Radiol Oncol 2023; 57(1): 86-94. Received 8 May 2022 Accepted 19 October 2022 Correspondence to: Valerija Žager Marciuš, Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-100 Ljubljana, Slovenia E-mail: valerija.zager@zf.uni-lj.si; vzager@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Th is 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 accuracy of volumetric lung image guidance using the spine or carina as a surrogate to target for image registration, as the best approach is not established. Patients and methods. Cone beam computed tomography images from the 1st, 10th, 15th, and 20th fraction in 40 lung cancer patients treated with radical radiotherapy were retrospectively registered to planning CT, using three approaches. The spine and carina alignment set-up deviations from a reference (tumour/lymph nodes) registration in the lateral (LAT), longitudinal (LONG) and vertical (VRT) directions were analysed and compared. Tumour location and nodal stage influence on registration accuracy were explored. Results. The spine and carina mean set-up deviation from reference were largest in the LONG, with the best match in the VRT and LAT, respectively. Both strategies were more accurate in central tumours, with the carina being more precise in 50% LAT and 66% LONG mean deviations. For all measurements in all patients a carina vs. spine registration comparison showed improved carina accuracy in LAT and LONG. In comparative subgroup analysis the carina was superior compared to spine in LAT and LONG in centrally located tumours, N2 and N3. Both strategies were compa- rable for peripheral tumours and N0. Conclusions. Carina registration shows greater accuracy compared to spine in the LAT and LONG directions and is superior in central tumours, N2 and N3. The spine and carina surrogates are equally accurate for peripheral tumours and N0. We propose the carina as a surrogate to target for CBCT image registration in locally advanced lung cancer. Key words: locally advanced lung cancer; volumetric image verification; tumour registration; carina registration; spine registration; adaptive radiotherapy. Introduction Recent advances in systemic and radiotherapy treatment have resulted in improved survival for patients with inoperable locally advanced lung cancer.1 But still, nearly half of the patients will ex- perience locoregional relapse.2 The accuracy of dif- ferent steps in radiotherapy treatment preparation and execution have a strong impact on local con- trol.3 With the introduction of computed tomogra- phy (CT), positron emission computed tomogra- phy (PET CT) and four-dimensional (4D) CT simu- lation, the target delineation accuracy increased.4 Modern radiation techniques have enabled a more conformal dose delivery, the dose to normal tis- sue was reduced and the radical treatment of Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance 87 more advanced N3 disease and/or dose escalation has become possible.5 Because of the steeper dose gradient and smaller safety margins, the accuracy of treatment delivery became increasingly impor- tant. For daily treatment position verification, cone beam CT (CBCT) largely replaced electronic portal imaging because of better soft-tissue visibility. The alignment of the treatment image with the plan- ning CT (pCT) is usually performed by radiation therapists (RTTs), who are not trained in target de- termination. The fast interpretation of CBCT is also challenging due to the lower quality of CBCT im- ages (no i.v. contrast) and changes with or adjacent to the tumour during treatment.6 Manual tumour matching is subjected to strong inter-observer var- iability even among radiation oncologists.7 The op- timal surrogate structure for image matching has not yet been established. Spine alignment is feasi- ble and reproducible, but shows poor correlation with tumour position.8 Recently, the carina surro- gate alignment was explored and showed superior reproducibility compared to spine alignment.7 We performed a retrospective study to deter- mine the accuracy of the carina vs. spine regis- tration compared to target (primary tumour and lymph nodes) registration as a reference. To avoid interobserver variability, reference registration was performed by individual thoracic radiation oncologist. To consider tumour and normal struc- ture variations during the course of treatment and their possible impact on image registration, the 1st, 10th, 15th, and 20th fraction CBCT were included in the registration analysis. Materials and methods Patient selection We retrospectively included 40 consecutive lung cancer patients treated with on-line cone-beam computer tomography (CBCT) image guided radi- cal radiotherapy from September 2018 to February 2019. All the patients had a visible tumour and/or lymph nodes on CT. They were treated with con- ventional, or hypofractionated volumetric modu- lated arc therapy on the Elekta Synergy linear accelerator (Elekta Synergy, Stockholm, Sweden). Clinical and treatment details were retrieved from medical records. Simulation and planning The planning CT scan was performed on the Big Bore CT simulator (Philips N.V., Eindhoven, NL), the Somatom Definition AS CT simulator (Siemens, Erlangen, D) and, in 6 patients, on Siemens Biograph mCT 40 (Siemens, Erlangen, D). The pa- tients were immobilised on a Posirest-2 (Civco, Coralville, USA) with the arms abducted above the head (36 patients) or with a long thermoplastic mask (4 patients). The gross tumour volume (GTV) was delineated as the visible tumour and patho- logic lymph nodes on free breathing pCT (with i.v. contrast). Additionally, 4D pCT was used for inter- nal target volume (ITV) delineation in 10 tumours. Planning was performed on the Monaco treatment planning system using the Monte Carlo calculation algorithm. Conventional fractionation (1.8–2 Gy daily dose) was used in 37 patients, and hypofrac- tionation in 3 (2.2, 2.2 and 2.75 Gy daily dose). The plan, pCT images and the delineated (target) con- tours were exported to the Elekta Synergy X-ray Volumetric Imaging (XVI) System. Imaging Kilovoltage gantry mounted CBCT systems were used for daily on-line CBCT treatment verification. According to physician instructions, localisation was based on automatic spine or carina matching between CBCT and pCT with additional manual translation correction by RTT. All set-up errors were corrected before treatment delivery. Study procedure Retrospective rigid image registration was done in the Elekta XVI System. We used the first treatment verification CBCT image from the 1st, 10th, 15th, and 20th fraction. The CBCT image was retrospectively registered with pCT based on three different strategies: (a) bony registration on the spine, (b) soft tissue regis- tration on the carina and (c) target (tumour/lymph node) matching on GTV/ITV. For 40 patients, we analysed 160 registration images and recorded 1440 corrections in the x (lateral – LAT), y (longi- tudinal – LONG) and z (vertical – VRT) directions. First two retrospective registrations on the spine and carina were performed by RTT and were based on automatic registration using a clip box (Figure 1). Residual translation errors were corrected manually, if necessary. Next, reference registra- tion on target (tumour/lymph node) matching was performed by an experienced thoracic radiation oncologist. Following automatic bone registra- tion, translational misalignments were manually Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance88 corrected based on the visual adjustment of the GTV (ITV) contour on the CBCT image, to provide the best match for all known gross disease. If the lymph nodes were not visible, close anatomical surrogates were used. Translation corrections for target matching were recorded and used for the reference position. Spine and carina corrections were compared to the reference position and de- viations in LAT, LONG and VRT measurements were analysed. Statistics Microsoft Excel 2010 and the Statistical Package for the Social Sciences, version 25.0 (SPSS Inc., Chicago, IL, USA) were used. General data were presented with descriptive statistics. Kolmogorov-Smirnov and Shapiro-Wilk tests rejected normal data distri- bution. The Nonparametric Mann Whitney U test (MW), Wilcoxon Signed Ranks test and nonpara- metric Kruskal-Wally’s test (KW) were used for the analysis of the set-up deviation. The Wilcoxon Signed Ranks Test for dependent samples was used for comparison (pairwise). The KW and MW were used when we analysed the differences in a certain measurement according in two (MW) or into multiple groups (KW). A p value ≤ 0.05 was considered statistically significant. Results Patients, tumour and treatment characteristics The patients, disease and treatment characteris- tics are summarised in Table 1. Radiation was the primary treatment of “de novo” lung cancer in 80% of patients. One patient was treated for local progression of epidermal growth factor receptor (EGFR)+ adenocarcinoma and one had postop- erative regional recurrence. Five patients received reirradiation for local/regional recurrence. There were three plan adaptations after the 21st or 22nd fraction due to atelectasis (developing, resolution and worsening). Spine to target registration set-up deviation analysis The set-up deviation in the LAT, LONG and VRT directions for the spine according to target regis- tration for the 1st, 10th, 15th, and 20th fractions were analysed (Figure 2A). The best registration match was in the VRT direction, with a mean set-up devi- ation between 1.2 and 1.68 mm. The biggest devia- tion was detected in the LONG direction (2.03–2.73 mm). Deviation differences from the 1st through 20th fractions were not statistically significant in any direction. There was no time trend detected. Comparison of deviations between directions on the 20th fraction showed a significant set-up dif- ference in deviation between LAT vs. LONG (p = 0.002) and VRT vs. LONG (p = 0.000). The mean de- viation for all set-up measurements was 1.39 mm in LAT (SD 0.9, range 0–4.0 mm), 2.44 mm in LONG (SD 1.6, range 0.5–8.0 mm) and 1.36 mm in VRT direction (SD 1.04, range 0–4.75 mm). Carina to target registration set-up deviation analysis Analysis of the set-up carina registration devia- tions from the target set-up measurements for the 1st, 10th, 15th, and 20th fractions were also analysed (Figure 2B). The smallest set-up difference was in the LAT direction (from 0.9 to 1.8 mm). The larg- est discrepancies were detected in the LONG di- rection (from 1.53 to 2.15). The difference in de- viations for different fractions was not significant in any direction. There was no time trend in the deviations. Calculated from all the measurements, the mean deviation for the carina set-up deviations from the reference was 1.03 mm in LAT (SD 0.75, FIGURE 1. Automatic clip box carina registration with manual alignment check. Target contours (gross tumour volume [GTV] inner contour, planning target volume [PTV] outer contour) are imported for target registration. Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance 89 range 0–3.75mm), 1.78 mm in LONG (SD 1.5, range 0–6.75mm) and 1.33 mm in the VRT direction (SD 1.25, range 0–5.25). Comparison of spine to target and carina to target set-up deviation differences The differences in the spine/target and carina/tar- get mean set-up deviations were compared indi- vidually for the different fractions and the mean for all measurements in all directions (Table 2). The registration on carina was more accurate ac- cording to the reference in all measurements ex- cept in the vertical direction on the 1st and 20th frac- tions. The only significant difference was found on the 10th fraction VRT direction, with the smallest deviation for carina registration. For all the measurements, registration on the ca- rina was significantly closer to the reference regis- tration in the LAT and LONG direction (p = 0.003 and p = 0.002, respectively). We found no differ- ence in the set-up deviation in the VRT direction for all measurements. The impact of tumour location (central/ peripheral) and N stage on the spine/ target and carina/target registration deviation Analysis of the spine registration deviations from the reference showed a significantly better regis- tration match for centrally located tumours in LAT on the 1st and 10th fractions, in LONG on the 1st fraction and in VRT on the 10th fraction (Table 3). The carina/target registration comparison showed significantly smaller differences for cen- tral tumours in 50% LAT measurements (10th and 15th fraction), in 2/3 LONG measurements (10th –20th fraction), but not in the VRT direction (Table 4). We found no impact of the node stage on the spine/target registration deviations. When the carina was used for alignment, the differences were significantly smaller for N2 and N3 in the LAT 15th, LAT 20th, LONG 15th and VRT 20th frac- tion (p = 0.034, 0.028, 0.025 and 0.034, respectively) (Supplementary Tables S1–S2). Possible time trend for spine/target LAT deviation difference was de- tected for central tumours (Table 3). TABLE 1. Patients, disease, and treatment characteristics N = 40 Gender Female 16 (40 %) Male 24 (60 %) Age (years) Median (range) 67 (53–81) Tumour location* RUL 15 (37.5%) RML 3 (7.5%) RLL 7 (17.5%) LUL 8 (20%) LLL 8 (20%) Central (C)/ peripheral (P) tumour location** C 17 (42.5%) P 22 (55%) Histology NSCLC 31 (77.5%) SCLC 9 (22.5%) Disease treated De novo lung cancer 32 (80%) Local progression 1 (2.5%) Local recurrence reirradiation 2 (5%) Regional recurrence 1 (2.5%) Locoregional recurrence reirradiation 4 (10%) Systemic treatment Concurrent chemotherapy 13 (32.5%) Sequential chemotherapy 16 (40%) Target therapy 1 (2.5%) None 10 (25%) Tumour (T) stage T0 1 (2.5%) T1 4 (10%) T2 14 (35%) T3 8 (20%) T4 13 (32.5%) Lymph nodes (N) stage N0 9 (22.5%) N1 1 (2.5%) N2 14 (35%) N3 16 (40%) Fractionation Conventional 37 (92.5%) Hypofractionation 3 (7.5%) Radiation technique VMAT 40 (100%) * In one patient, two synchronous tumours were treated (RUL and LUL). **In one patient, only the lymph nodes were treated (regional recurrence). LLL = left lower lobe; LUL = left upper lobe; NSCLC = non-small cell cancer; RML = right middle lobe; RLL = right lower lobe; RUL = right upper lobe; SCLC = small cell lung cancer; VMAT = volumetric modulated arc therapy Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance90 FIGURE 2. Spine to target (A) and carina to target (B) registration set-up deviation in the lateral (LAT), longitudinal (LONG) and vertical (VRT) directions. A B Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance 91 The impact of tumour location (central/ peripheral) and N stage on the spine/ target vs. carina/target deviation differences The deviation differences analysis between the spine/target and carina/target registration accord- ing to tumour location and N stage is shown in Supplementary tables S3–S5. For peripherally lo- cated tumours, the carina registration deviation was only smaller compared to the spine set-up de- viation in the 1st fraction LONG with a deviation difference of 1.91mm (p = 0.034). In centrally located tumours, the registration on carina was found to be significantly more accurate on the 15th and 20th frac- tions LAT, (p = 0.012 and 0.048, respectively), the 15th and 20th fractions LONG (p = 0.010 and 0.011, respectively) and for all LAT and LONG measure- ments (p = 0.003). The deviation differences were 0.76, 0.94, 1.59, 1.35, 0.5 and 0.71mm, respectively. There was no difference in the set-up deviation in the VRT direction regardless of tumour location. Comparison of the deviation differences ac- cording to the N stage showed a better correlation between the carina and target registration for N2 and N3 disease. We found deviation differences TABLE 2. Mean set-up deviation difference (DD) comparison according to spine/target vs. carina/target registration Fraction LAT deviation mean (mm) p value LONG deviation mean (mm) p value VRT deviation mean (mm) p valueSpine/ target Carina/ target DD Spine/ target Carina/ target DD Spine/ target Carina/ target DD 1st 1.28 0.90 0.38 NS 2.65 1.80 0.85 NS 1.20 1.43 -0.23 NS 10th 1.30 1.08 0.22 NS 2.03 1.53 0.50 NS 1.68 1.10 0.58 0.04 15th 1.48 1.08 0.4 NS 2.38 1.65 0.73 0.05 1.38 1.38 0 NS 20th 1.53 1.05 0.48 NS 2.73 2.15 0.58 NS 1.2 1.43 -0.23 NS All measurements 1.39 1.03 0.37 0.003 2.44 1.78 0.66 0.002 1.36 1.33 0.03 NS DD = deviation difference; LAT = lateral; LONG = longitudinal; NS = non-significant (p>0.05); target = primary tumour and lymph nodes; VRT = vertical TABLE 3. Spine/target registration deviation according to tumour location Fraction LAT mean (mm) p value LONG mean (mm) p value VERT mean (mm) p value Central Peripheral Central Peripheral Central Peripheral 1st 0.71 1.77 0.048 1.18 3.91 0.002 1.12 1.32 NS 10th 0.76 1.77 0.008 1.53 2.41 NS 1.41 1.86 0.017 15th 1.47 1.55 NS 2.59 2.32 NS 1.35 1.45 NS 20th 1.94 1.27 NS 2.29 3.14 NS 1.29 1.09 NS LAT = lateral; LONG = longitudinal; NS = non significant (p>0.05); target = primary tumour and lymph nodes; VRT = vertical TABLE 4. Carina/target registration deviation according to tumour location Fraction LAT mean (mm) p value LONG mean (mm) p value VERT mean (mm) p value Central Peripheral Central Peripheral Central Peripheral 1st 0.65 1.14 NS 1.65 2.00 NS 1.06 1.77 NS 10th 0.53 1.55 0.002 1.18 1.86 0.041 0.82 1.32 NS 15th 0.71 1.41 0.049 1.00 2.23 0.027 0.88 1.77 NS 20th 1.00 1.14 NS 0.94 3.14 0.019 0.88 1.82 NS LAT = lateral, LONG = longitudinal, NS = non significant (p>0.05); VRT = vertical; target = primary tumour and lymph nodes Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance92 of 0.63mm N2 and 1.17mm N3 in LONG, 0.48mm N3 in LAT for all measurements and 0.94mm N3 10th fraction LONG (p = 0.013, 0.015, 0.002 and 0.007, respectively). A small but significant 0.5mm differ- ence in favour of the carina registration was found for N0 in all measurements LAT (p = 0.034). Because only 1 patient had the N1 stage, we excluded his measurements from this analysis (Supplementary Table S5). Discussion Since the introduction of CBCT into treatment verification, new insights to target position uncer- tainties have evolved6, suggesting that image guid- ance could currently be the weakest link in the ra- diotherapy procedure.3,9 Only a few studies have explored the carina as a surrogate in volumetric lung image guidance7,10,11 and there is a lack of knowledge in this field. Here we present new data on the accuracy of the carina vs. spine surrogate compared to the target alignment as a reference in image registration. A separate analysis of the spine and carina reg- istration set-up differences compared to the ref- erence (target) registration showed the smallest differences in the VRT and LAT directions, with the largest deviation in the LONG direction for both strategies (Fig. 2). The differences were not significantly different in time and no time trend was detected for the cohort, so we evaluated all the measurements together and again showed a good registration match in the VRT and LAT directions, but in LONG direction, the range of misalignment reached above 5 mm in both registration strategies. The greatest area of uncertainty in the LONG di- rection was also shown in the study by Ottosson et al. where they compared the spine and target reg- istration in free-breathing and breath-hold CBCT in locally advanced lung cancer patients. The largest intra- and inter-fractional misalignments were found in the LONG direction, independent of the registration method.12 Although we found a smaller mean LONG misalignment in the carina vs. spine registration (1.78 vs. 2.44 mm), uncertain- ties in image registration should be considered for both strategies, with the enlargement of the PTV in the craniocaudal (CC) direction. To determine the best registration match com- pared to the reference, we compared differences in the set-up deviations for the spine and carina alignment. Both strategies were equally accurate in the VRT direction, but the carina was more accu- rate in the LONG and LAT directions in all meas- urements. The accuracy of the spine vs. carina reg- istration was also tested in a study by a Canadian group, where four independent observers auto- matically and manually aligned the first fraction CBCT with the pCT in 30 lung cancer patients.7 They used spine, carina and tumour registration strategies. Automatic spine and carina registra- tions provided similar tumour coverage, with the tumour inside the ITV in 60% of observations. The same group, in a second study, verified the tumour (T) and lymph node (LN) coverage following spine and carina registration for initial, middle, and final fraction CBCT. Both strategies improved the com- bined target coverage throughout the treatment course compared to tattoo alignment. Carina bet- ter improved the combined coverage and showed significantly superior nodal coverage compared to the spine, without compromising primary tumour coverage.10 With a significantly better registration match in the LONG and LAT directions, our data supports the suggestion from the Canadian group that the carina may be superior to the spine in the image guidance of locally advanced lung cancer. The second Canadian study showed similar pri- mary tumour coverage, regardless of the registra- tion strategy.10 But in our study, the registration ac- curacy was influenced by tumour location. Both the spine and carina alignment showed smaller set-up differences for centrally located tumours with the difference being more significant in the carina reg- istration LONG (66% fractions) and LAT (50% frac- tions). For peripheral tumours, we found no differ- ence in the accuracy of spine vs. carina alignment, but for centrally located tumours, the carina was more accurate in the LAT and LONG directions. Our data suggests that the carina is a better sur- rogate for centrally located tumours. Importantly, we also showed that the carina is as accurate as the spine for registration in peripherally located tumours and can be proposed as a registration sur- rogate regardless of the tumour location. According to our data, carina matching can also be used regardless of the nodal stage. We found no differences in the spine, but a better match for ca- rina alignment in N2 and N3 disease. In advanced nodal disease, the carina showed superior registra- tion vs. the spine in LAT and LONG. The finding is in concordance with the second Canadian study, where carina matching improved the node cover- age compared to spine registration.10 Importantly, we found no set-up difference for N0 disease sug- gesting that the carina and spine can equally be used as a surrogate in this stage. As there was Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance 93 only a single case of early nodal disease, we can- not make any conclusions for N1. The reliability of spine matching for LN coverage was investigated in the study by Mohammed et al., where an equal geographical target miss was found for spine vs. combined target matching, but inferior LN cover- age in the case of tumour matching, based on a weekly 4D CBCT registration.9 Because the same geographical miss was shown for hilar and medi- astinal lymph nodes, we can hypothesize that ca- rina matching could safely be used in N1 as well. Target alignment should be “the ground truth” and have an impact on the therapeutic ratio,3,12 but is rather difficult to implement clinically. Due to the poor soft tissue contrast on CBCT and tu- mour changes during the course of treatment, only half of the tumours can clearly be contoured using CBCT.13 For these reasons, we used con- toured-based registration for reference. A similar approach was used for target coverage and geo- graphical miss assessments studies9,10 and target registration in the study by Ottosson et al.12 Direct tumour registration is also impractical because it is time-consuming and unreliable due to high intra-observer variability.7 In contrast, the carina is clearly visible on the CBCT and automatic ca- rina alignment shows high reproducibility, supe- rior even to spine alignment.7 Although the carina shows some respiratory movement, especially in the CC direction, it is still an excellent surrogate for directly adjacent mediastinal lymph nodes. Preliminary data also suggests a good correlation between the carina and GTV motion.7,11 We acknowledge the limitations of our study, which was retrospective and relatively small. We also present a heterogeneous group of patients, though they represent real everyday radiothera- pists’ lung cancer patients. In our study, CBCT im- age changes were not systematically determined. Because the majority of changes appear in the first five weeks of radiotherapy,14 we analysed CBCT images from the 1st, 10th, 15th and 20th fractions. Maybe the 25th and 30th fraction CBCT should have been included since we found cases for plan modi- fication in the 6th week of treatment. Nevertheless, we detected no significant set-up differences in time. The rate of plan adaptation was low (7.5%), suggesting the need for a “traffic light” protocol implementation.3 In conclusion, carina registration was shown to be feasible, fast, and reproducible. Our data shows that, compared to target registration, carina is equally as accurate as spine registration, with su- perior accuracy in the LONG and LAT directions. The carina is a better surrogate than the spine for alignment in centrally located tumours, N2 and N3 disease. Although spine alignment can equally be used in N0 and peripheral tumours, for simplic- ity we propose that the carina should be the pri- mary surrogate for the target in image guidance in locally advanced lung cancer. The next step should be to incorporate carina registration uncertainties into the PTV margin. Acknowledgement This research was supported by Slovenian re- search programme for comprehensive cancer con- trol SLORApro (P3-0429). Reference 1. Spigel DR, Faivre-Finn C, Gray JE, Vicente D, Planchard D, Paz-Ares LG, et al. Five-year survival outcomes with durvalumab after chemoradiotherapy in unresectable stage III NSCLC: an update from the PACIFIC trial. J Clin Oncol 2021; 39: 8511-11. doi: 10.1200/JCO..39.15_suppl.8511 2. Vrankar M, Stanic K, Jelercic S, Ciric E, Vodusek AL, But-Hadzic J. Clinical outcomes in stage III non-small cell lung cancer patients treated with durvalumab after sequential or concurrent platinum-based chemoradio- therapy - single institute experience. Radiol Oncol 2021; 55: 482-90. doi: 10.2478/raon-2021-0044 3. Tvilum M, Khalil AA, Møller DS, Hoffmann L, Knap MM. Clinical outcome of image-guided adaptive radiotherapy in the treatment of lung cancer patients. Acta Oncol (Madr) 2015; 54: 1430-7. doi: 10.3109/0284186X.2015.1062544 4. Mercieca S, Belderbos JSA, van Herk M. Challenges in the target volume definition of lung cancer radiotherapy. Transl Lung Cancer Res 2021; 10: 1983-98. doi: 10.21037/tlcr-20-627 5. Chan C, Lang S, Rowbottom C, Guckenberger M, Faivre-Finn C. Intensity-modulated radiotherapy for lung cancer: current status and future developments. J Thorac Oncol 2014; 9: 1598-608. doi: 10.1097/ JTO.0000000000000346 6. Clarke E, Curtis J, Brada M. Incidence and evolution of imaging changes on cone-beam CT during and after radical radiotherapy for non-small cell lung cancer. Radiother Oncol 2019; 132: 121-6. doi: 10.1016/j.ra- donc.2018.12.009 7. Higgins J, Bezjak A, Franks K, Le LW, Cho BC, Payne D, et al. Comparison of spine, carina, and tumor as registration landmarks for volumetric image- guided lung radiotherapy. Int J Radiat Oncol Biol Phys 2009; 73: 1404-13. doi: 10.1016/j.ijrobp.2008.06.1926 8. Purdie TG, Bissonnette J-P, Franks K, Bezjak A, Payne D, Sie F, et al. Cone- beam computed tomography for on-line image guidance of lung ste- reotactic radiotherapy: localization, verification, and intrafraction tumor position. Int J Radiat Oncol Biol Phys 2007; 68: 243-52. doi: 10.1016/j. ijrobp.2006.12.022 9. Mohammed N, Kestin L, Grills I, Shah C, Glide-Hurst C, Yan D, et al. Comparison of IGRT registration strategies for optimal coverage of primary lung tumors and involved nodes based on multiple four-dimensional CT scans obtained throughout the radiotherapy course. Int J Radiat Oncol Biol Phys 2012; 82: 1541-8. doi: 10.1016/j.ijrobp.2011.04.025 10. Lavoie C, Higgins J, Bissonnette J-P, Le LW, Sun A, Brade A, et al. Volumetric image guidance using carina vs spine as registration landmarks for conven- tionally fractionated lung radiotherapy. Int J Radiat Oncol Biol Phys 2012; 84: 1086-92. doi: 10.1016/j.ijrobp.2012.02.012 Radiol Oncol 2023; 57(1): 86-94. But-Hadzic J et al. / Accuracy of volumetric lung image guidance94 11. van der Weide L, van Sörnsen de Koste JR, Lagerwaard FJ, Vincent A, van Triest B, Slotman BJ, et al. Analysis of carina position as surrogate marker for delivering phase-gated radiotherapy. Int J Radiat Oncol Biol Phys 2008; 71: 1111-7. doi: 10.1016/j.ijrobp.2007.11.027 12. Ottosson W, Rahma F, Sjöström D, Behrens CF, Sibolt P. The advantage of deep-inspiration breath-hold and cone-beam CT based soft-tissue registra- tion for locally advanced lung cancer radiotherapy. Radiother Oncol 2016; 119: 432-7. doi: 10.1016/j.radonc.2016.03.012 13. Lim G, Bezjak A, Higgins J, Moseley D, Hope AJ, Sun A, et al. Tumor regression and positional changes in non-small cell lung cancer dur- ing radical radiotherapy. J Thorac Oncol 2011; 6: 531-6. doi: 10.1097/ JTO.0b013e31820b8a52 14. Elsayad K, Kriz J, Reinartz G, Scobioala S, Ernst I, Haverkamp U, et al. Cone- beam CT-guided radiotherapy in the management of lung cancer: diagnostic and therapeutic value. Strahlenther Onkol 2016; 192: 83-91. doi: 10.1007/ s00066-015-0927-y Radiol Oncol 2023; 57(1): 95-102. doi: 10.2478/raon-2023-0004 95 research article Effects of gold fiducial marker implantation on tumor control and toxicity in external beam radiotherapy of prostate cancer Matthias Moll, Magdalena Weiß, Vladimir Stanisav, Alexandru Zaharie, Gregor Goldner Department of Radiation Oncology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Radiol Oncol 2023; 57(1): 95-102. Received 13 07 2022 Accepted 26 10 2022 Correspondence to: Dr Matthias Moll, M.D., Department of Radiation Oncology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria. E-mail: matthias.moll@meduniwien.ac.at 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. Evidence regarding the effects of fiducials in image-guided radiotherapy (IGRT) for tumor control and acute and late toxicity is sparse. Patients and methods. Patients with primary low- and intermediate-risk prostate cancer, 40 with and 21 without gold fiducial markers (GFM), and treated between 2010 and 2015 were retrospectively included. The decision for or against GFM implantation took anaesthetic evaluation and patient choice into account. IGRT was performed using electronic portal imaging devices. The prescribed dose was 78 Gy, with 2 Gy per fraction. Biochemical no evidence of disease (bNED) failure was defined using the Phoenix criteria. Acute and late gastrointestinal (GI) and genitourinary toxicity (GU) were assessed using the Radiation Therapy Oncology Group criteria. Results. Most patients did not receive GFM due to contraindications for anaesthesia or personal choice (60% and 25%). Regarding tumor control, no significant differences were found regarding bNED and overall and disease-spe- cific survival (p = 0.61, p = 0.56, and p > 0.9999, respectively). No significant differences in acute and late GI (p = 0.16 and 0.64) and GU toxicity (p = 0.58 and 0.80) were observed. Conclusions. We were unable to detect significant benefits in bNED or in early or late GI and GU side effects after GFM implantation. Key words: prostate cancer; IGRT; fiducials; tumor control; toxicity Introduction Prostate cancer can be treated by external beam ra- diotherapy (EBRT) brachyradiotherapy or radical prostatectomy; for low-risk disease and highly se- lected intermediate disease, active surveillance can be offered.1-5 Important factors in patient decision making are, therefore, the duration of treatment, circumstances of treatment, and treatment-induced side effects. Regarding side effects in EBRT, the use of intensity-modulated radiotherapy (IMRT) has been shown to reduce treatment-associated side ef- fects compared with 3D-conformal radiotherapy.6,7 Further reduction of side effects is expected us- ing image-guided radiotherapy (IGRT)8, in which fiducials, ultrasound, MRI, CT scans, or electro- magnetic responders are used to locate the pros- tate before and during radiotherapy.9 However, to our knowledge, there are as yet no data regarding IGRT in the form of randomized controlled trials, although IGRT is recommended by the NCCN1, EAU5, British NICE10, and German S34 guidelines. With this paper, we explore the role of gold fidu- cial markers (GFM) used for IGRT in primary pros- tate cancer treatment. There are only a few stud- ies comparing patients with and without GFM. For example, a retrospective study by Zelefsky et al.11 described an advantage after implanting GFM Radiol Oncol 2023; 57(1): 95-102. Moll M et al. / Effects of gold fiducial markers96 in regard to genitourinary (GU) side effects in all patients and biochemical no evidence of disease (bNED) rates in high-risk prostate cancer patients. However, this study compared a group of pa- tients treated with IGRT, but without IMRT with an IMRT-only group, making its results difficult to apply to modern day radiotherapy, in which IMRT is always recommended.1 The same applies to Sveistrup et al.12, Zapatero et al.13 and Wortel et al.7, which compared patients treated with fiducial- marker IGRT and IMRT with patients treated with 3D conformal radiotherapy.12 Singh et al. compared the use of GFM IGRT with no IGRT et al.14 However, to our knowledge, only one study has compared the use of IGRT with and without GFM15, and it showed no significant benefits regarding tumor control and toxicity. In our department, we use cone-beam CT scans and ExacTrac (Brainlab, Munich, Germany), as well as routinely implanted GFM for image guid- ance. As GFM are implanted with the use of anes- thesia, the benefit of the GFM has to be larger than the risks from anesthesia, including, but not lim- ited to, nausea, allergic reactions, intraoperative awareness, or death16,17, and the risk of the inter- vention itself, such as infections or injuries to adja- cent organs. Local anaesthesia reduces the risks of anaesthesia. Some patients have contraindications against anesthesia or refuse the intervention. A more precise patient positioning due to GFM can be expected to lead to greater treatment precision and hence, possibly, to reduced toxicity and in- creased tumor control. These assumed benefits can justify its routine application in patients with pros- tate cancer. Without these advantages, the routine use of GFM would not be justified. Therefore, we wanted to compare a group of patients receiving IGRT with GFM with a group receiving only IGRT to evaluate the potential benefits of GFM regard- ing tumor control and GI and GU toxicity. Patients and methods Design, setting, and participants Our study protocol was approved by the ethical review board of our university according to local regulations (EK Nr: 1533/2020). We included all pa- tients between 2010 and 2015 with localized prima- ry prostate cancer treated with EBRT by the use of volumetric modulated arc therapy (VMAT) with or without GFM implantation. Patients treated before 2010 were excluded, as routinely prescribed doses were below the currently recommended 75.6 Gy with 1.8 Gy per fraction.1 Due to the implementa- tion of moderate hypofractionation in our depart- ment, patients treated after 2015 were excluded to keep the fractionation uniform among patients. The stage had to be cN0/x and cM0/x, with a risk of lymph node involvement < 15% according to the Roach formula18, and a clinical T category of 1 or 2. Interventions Target volumes were defined using CT and MRI for planning. The prescribed doses were 78 Gy with 2 Gy per fraction. Doses were prescribed to 95% of the planning target volume (PTV) according to International Commission on Radiation Units and Measurements report 83.19 Safety margins were 7 mm after GFM implantation and 10 mm without. Irradiation was performed with the patient in su- pine position. All patients received a rectal balloon20 for prostate immobilization. Cone-beam CT con- trol scans were performed daily for the first week, followed by daily ExacTrac (Brainlab, Munich, Germany) controls for the rest of treatment. The clinical target volume (CTV) included the prostate for low-risk tumors and additionally the base of the seminal vesicles for intermediate-risk tumors. GFM implantation was recommended to all pa- tients. Reasons for not receiving GFM were con- traindications against anesthesia, such as pre-ex- isting pulmonary illness, coronary heart disease, or myocardial infarction, or refusal by the patient. Implantation itself was performed transperineally with ultrasound guidance by the radiation oncolo- gist, under mask narcosis performed by an anaes- thesiologist. In each patient, 3 GFM were implant- ed, one on each side of the prostate and one in the apex. Ciprofloxacin 250 mg 1-0-1 was prescribed as postinterventional prophylaxis for 5 days. If patients received androgen deprivation therapy, it was prescribed by the caretaking urologist. Outcome measurements Patient follow-ups were immediately after treat- ment, 3 months after treatment, 12 months after treatment, and every 12 months from then on. At each follow-up, PSA levels and GI and GU side ef- fects were assessed by the physician. bNED failure was defined using the Phoenix criteria (PSA nadir + 2 ng/mL).21 Acute and late GI and GU side effects were assessed using the RTOG criteria.22 Survival data were collected using the local death registry. Dose-volume histogram (DVH) data were col- lected using our database. They included the vol- Radiol Oncol 2023; 57(1): 95-102. Moll M et al. / Effects of gold fiducial markers 97 umes, Dmax/D1, and Dmean of the CTV, PTV, rec- tum, and bladder, as well as the Dmin of the CTV and the V70 and V60 for the rectum and V70, V50, and V30 for the bladder, as these data were used for plan evaluation. Statistical analysis The statistical analysis was performed using GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA, USA) and SPSS 28.0.1.0 (IBM, Armonk, NY, USA). A p-value of < 0.05 was considered sta- tistically significant. bNED, overall survival, and disease-free survival were compared using the Kaplan-Meier method. Side effects were analyzed by use of the Mann-Whitney U test, the Kruskal- Wallis test, and a Cox regression analysis for the onset of late toxicity grade 2 or higher, as well as bNED and overall survival. Results Patient characteristics are displayed in Table 1. We also analyzed the reasons for patients not receiving GFM implantation. Among them, most patients (12, 60%) had medical contraindications for anes- thesia, 5 patients (25%) refused GFM implantation, and for 3 patients (15%) who did not receive GFM, the reason was unknown. bNED rates are displayed in Figure 1. bNED ratios after 5 years for patients with and without GFM implantation were 92% and 100%, respective- FIGURE 1. Biochemical no evidence of disease (bNED) rates of patients with and without gold fiducial marker (GMF) implantation (p = 0.61). TABLE 1. Patient characteristics GFM % No GFM % n 41 100% 20 100% cT category 1 23 56% 16 80% 2 18 44% 4 20% iPSA in μg/l, median (IQR) 7 (5.3/8.7) 5.9 (4.4/10.6) Gleason score 6 26 63% 18 90% 7a 12 29% 1 5% 7b 3 7% 1 5% Risk group Low risk 16 39% 10 50% Intermediate risk 25 61% 10 50% ADT prescribed 14 34% 3 15% Median duration of ADT in months (IQR) 9 (3/18) 6 (6/8) Median age at RT (IQR) 74 (70/77) 72 (67/72) Median follow-up in months (IQR) 72 (49/84) 68 (36/86.5) ADT = androgen deprivation therapy; cT = clinical tumor extension; GFM = gold fiducial markers; iPSA = initial prostate-specific antigen; IQR = interquartile range; RT = radiotherapy low risk = PSA < 10 μg/L and Gleason Score 6; intermediate risk = PSA ≥ 10 μg/L or Gleason Score 7a/b Radiol Oncol 2023; 57(1): 95-102. Moll M et al. / Effects of gold fiducial markers98 ly. After 8 years, bNED rates were 87% and 86%, respectively (p = 0.61). We also analyzed disease- specific survival (DSS) and overall survival (OS). Regarding DSS, we did not find a single death due to prostate cancer (p > 0.9999). For OS, survival pro- portions for patients with and without GFM after 5 years were 93% and 95%, and after 8 years 90% and 95% (p = 0.56). Maximum acute and late gastrointestinal side effects are displayed in Tables 2 and 3. No signifi- cant differences between the two groups were de- tected. It is noteworthy that at no point was a grade 4 toxicity detected. We also compared late GI and GU toxicity in the two groups after 3, 12, 24, 36, 48, 60, 72, 84, and 96 months, and did not find a significant difference at any point. For DVH data, we started by comparing the two groups regarding volumes, Dmax/D1, and Dmean of the CTV, PTV, rectum, and bladder, as well as the Dmin of the CTV and the V70 and V60 for the rectum and V70, V50, and V30 for the blad- der. Significant differences were found in the PTV (median 124.7 cm³ with GFM and 157.1 cm³ with- out, p = 0.004), the Dmin of the CTV (median 76.0 Gy with GFM and 76.5 Gy without, p = 0.04), the rectal Dmean (median 36.0 Gy with GFM and 39.3 Gy without, p = 0.01), V70 (median 11.4 Gy with GFM and 16.1 Gy without, p < 0.001), and V60 (me- dian 20.0 Gy with GFM and 25.3 Gy without, p < 0.001). An overview of the DVH data is presented in Table 4. We also performed a univariable, and if more than one variable was significant, a multivariable Cox regression analysis regarding bNED and OS, as displayed in Table 5, as well as the onset of late GI or GU toxicity grade 2 or higher. The results of our Cox regression analysis regarding bNED and OS are displayed in Table 5. As no patient died of prostate cancer, we did not model a Cox regression for DSS. Analyses in- cluding rectal or bladder DVH variables regarding bNED and OS were performed, and none of the variables was significant. Therefore, to improve clarity, we did not add them to Table 5. The results regarding toxicity are displayed in Table 6. Discussion As GFM implantation is an invasive procedure, the benefits of implantation have to outweigh the po- tential risks. There are two categories of risks. The first is the risk due to the implantation itself. Citing our information sheet, these risks are bleeding, in- juries of the bladder and urethra, infection includ- ing abscess formation, and allergic reactions to the prescribed antibiotic. The second category is risks due to the anaesthesia. Even when only a breathing mask is used to administer anaesthesia, these risks include tissue damage due to patient positioning, allergic reactions, malignant hyperthermia, sub- sequent confusion, aspiration, and regaining con- sciousness during anesthesia.16,17 While these side effects occur rarely in our clinical experience, as well as in the literature23, they have to be consid- ered in evaluating GFM implantation, aside from its influence on tumor control and side effects. Several retrospective studies comparing fidu- cial IGRT with a control group exist. However, most of them compare a 3D conformal group with either an IGRT 3D conformal group14,24 or an IGRT IMRT group12, making them hard to apply to today’s treatment due to their being outdated or their comparison of two different treatment mo- dalities with inherent differences regarding out- comes. While many international guidelines1,4,10 suggest the use of IGRT, no study covering this topic is mentioned in the NCCN1 or NICE10 guide- lines. Napieralska et al.15, to our knowledge the TABLE 2. Maximum acute side effects in patients with and without gold fiducial marker implantation Grade Gastrointestinal toxicity Genitourinary toxicity Gold fiducial markers No gold fiducial markers Gold fiducial markers No gold fiducial markers 0 27% 15% 17% 5% 1 66% 65% 44% 55% 2 7% 20% 39% 40% No significant differences for acute gastrointestinal and genitourinary side effects were detected (p = 0.15 and p = 0.58, respectively). TABLE 3. Maximum late side effects in patients with and without gold fiducial marker implantation Grade Gastrointestinal toxicity Genitourinary toxicity Gold fiducial markers No gold fiducial markers Gold fiducial markers No gold fiducial markers 0 59% 50% 37% 35% 1 12% 15% 27% 25% 2 24% 35% 34% 35% 3 5% 0% 2% 5% No significant differences for late gastrointestinal and genitourinary side effects were detected (p = 0.64 and p = 0.80, respectively). Radiol Oncol 2023; 57(1): 95-102. Moll M et al. / Effects of gold fiducial markers 99 only other study comparing patients with and without fiducial marker implantation and IMRT, found no significant differences regarding either bNED, with the exception of improved OS in inter- mediate patients, or late toxicity when comparing fiducial marker guidance and bone structure guid- ance. A significant difference was found regarding acute GU toxicity. Our results are similar, with no significant differences in acute toxicity. However, the aforementioned study did not include DVH data. Looking at our DVH data, we were unable to translate the differences regarding PTV and rectal variables into differences related to tumor control or toxicity, most likely due to DVH constraints in both groups. It is quite likely that the most crucial effect re- garding potential outcomes in our department is the safety margin reduction by 3 mm in GFM pa- tients, which is absent from the aforementioned studies. The effect of reducing the safety margin by 3 mm can be displayed using math. Assume a spherical form for the prostate, with a radius be- tween 2 and 2.5 cm and safety margins of 7 mm and 10 mm. The radii, including the PTV safety margins, are 2.7 cm and 3 cm, with an initial ra- dius of 2 cm and 3.2 cm, respectively, and 3.5 cm with an initial radius of 2.5 cm. With 4/3*π*r³ being the formula for the volume of a sphere, reducing the safety margins by 3 mm leads to a decrease in the PTV of 27% for the 2-cm initial radius and 24% for the 2.5-cm initial radius (2.7³/3³ and 3.2³/3.5³). In our data, the difference in the median volume is 21%, similar to the expected difference. Although this calculation is far from perfect, as the prostate is not a perfect sphere, it allows one to imagine the effect of safety margin variation through the third power of the radius. Zelefsky’s conclusion in his IGRT study11 was that safety margins should be reduced. However, to our knowledge, he has yet to publish any data comparing bNED rates before and after margin reduction. When comparing our bNED rates with those of groundbreaking studies like those of Peeters et al.25 and Pasalic et al.26, using 78 Gy, and Dearnaley et al.27, using 74 Gy, with bNED rates after 5 years ranging from 64% for Peeters to above 90% for Pasalic, we are leaning toward the top end, with bNED rates after 5 years of 92% and more, while only including patients with low- and intermedi- ate-risk tumors. Regarding late side effects, while there were no significant differences between the two groups, our data, with 29%–35% of maximum GI toxicity at RTOG grade 2 or higher toxicity and approximately 36%–40% of GU toxicity of RTOG TABLE 4. Dose-volume histograms for patients with and without gold fiducial markers (GFM) GFM No GFM PTV prostate cm³ median (IQR) 124.72 (98.68–152.83) 157.12 (128.34–173.66) PTV Dmax/D1 median (IQR) 81.55 (80.55–82.75) 81.15 (80.46–82.06) PTV Dmean median (IQR) 78.10 (77.41–78.70) 78.10 (77.00–78.43) CTV prostate cm³ median (IQR) 44.14 (34.75–64.70) 43.91 (34.40– 55.42) CTV Dmax/D1 median (IQR) 81.24 (80.64–82.57) 81.22 (80.35– 81.85) CTV Dmean median (IQR) 78.58 (77.97–79.47) 78.63 (77.94–79.04) CTV Dmin median (IQR) 76.00 (75.36–76.32) 76.49 (75.88–77.05) Rectal volume median (IQR) 113.02 (100.22–134.55) 114.53 (105.57–136.38) Rectal Dmax median (IQR) 80.06 (78.88–81.98) 80.24 (79.23–81.25) Rectal Dmean median (IQR) 35.99 (32.94–39.61) 39.30 (37.81–42.99) Rectal V70 Gy % median (IQR) 11.41 (9.41–13.04) 16.12 (14.55–18.66) Rectal V70 Gy cm³ median (IQR) 13.21 (11.33–15.93) 18.66 (16.55–21.39) Rectal V60 Gy % median (IQR) 19.95 (16.39–22.37) 25.25 (23.77–29.26) Rectal V60 Gy cm³ median (IQR) 23.36 (19.93–25.98) 29.23 (26.74–33.13) Bladder volume median (IQR) 196.93 (113.66–282.94) 155.76 (127.30–296.44) Bladder Dmax/D1 median (IQR) 78.78 (77.01–80.53) 79.53 (77.04–80.44) Bladder Dmean median (IQR) 21.11 (16.92–30.06) 28.10 (18.99–35.20) Bladder V70 Gy % median (IQR) 7.50 (5.44–11.78) 9.43 (6.06–14.02) Bladder V70 Gy cm³ median (IQR) 14.14 (10.53–21.16) 19.01 (12.00–22.20) Bladder V50 Gy % median (IQR) 17.14 (12.00–26,40) 20.81 (13.60–30.86) Bladder V50 Gy cm³ median (IQR) 34.00 (23.17–42.23) 37.58 (27.16–44.42) Bladder V30 Gy % median (IQR) 28.24 (21.74–41.73) 41.31 (25.78–51.83) Bladder V30 Gy cm³ median (IQR) 58.71 (43.43– 78.57) 65.38 (48.97–83.61) CTV = clinical target volume; GFM = gold fiducial marker; IQR = interquartile range; PTV = planning target volume; RT = radiotherapy grade ≥ 2, are worse than the results presented by Napieralska15, showing 12% grade ≥ 2 GU side ef- fects and 15%–19% grade ≥ 2 GI side effects, possi- bly due to differences in side-effect assessment be- tween institutions. Zelefsky reports lower toxicity rates, using CTCAE criteria, complicating a direct comparison. One weakness of our study is its retrospective nature. On top of that, there is no group of patients with GFM implantation and a safety margin of 10 Radiol Oncol 2023; 57(1): 95-102. Moll M et al. / Effects of gold fiducial markers100 TABLE 5. Uni- and multivariable Cox regression of biochemical no evidence of disease (bNED) and overall survival (OS) bNED Univariable analysis Multivariable analysis p-value exp(HR) exp(HR) (95% conf.) p-value exp(HR) exp(HR) (95% conf.) Use of GFM 0.603 1.788 0.200–16.018 - - - Age at RT 0.960 0.996 0.857–1.157 - - - PTV prostate 0.789 1.003 0.980–1.027 - - - PTV Dmax/D1 0.556 1.133 0.747–1.719 - - - PTV Dmean 0.960 0.978 0.410–2.332 - - - CTV prostate 0.006 1.020 1.006–1.035 0.862 0.996 0.947–1.046 CTV Dmax/D1 0.014 0.814 0.690–0.960 0.224 2.583 0.560–11.925 CTV Dmean 0.005 0.858 0.770–0.956 0.298 0.188 0.008–4.393 CTV Dmin 0.005 0.875 0.797–0.960 0.453 2.067 0.310–13.770 OS Univariable analysis p-value exp(HR) exp(HR) (95% conf.) Use of GFM 0.564 1.907 0.213–17.063 - - - Age at RT 0.235 1.149 0.913–1.446 - - - PTV Prostate 0.723 1.004 0.984–1.024 - - - PTV Dmax/D1 0.300 0.705 0.364–1.366 - - - PTV Dmean 0.153 0.426 0.132–1.373 - - - CTV Prostate 0.833 1.002 0.980–1.025 - - - CTV Dmax/D1 0.775 0.964 0.747–1.242 - - - CTV Dmean 0.946 0.992 0.791–1.245 - - - CTV Dmin 0.879 1.024 0.758–1.383 - - - bNED = biochemical no evidence of disease; CTV = clinical target volume; GFM = gold fiducial marker; HR = hazard ratio; OS = overall survival; PTV = planning target volume; RT = radiotherapy mm, complicating the direct comparison of our two groups, as GFM were implanted and the safety margin was reduced to 7 mm together. However, retrospective studies should be used to generate hypotheses, not prove them, and with this study we have generated the hypothesis that the use of GFM for IGRT provides no benefits, but only risks, and should not be performed regularly if other, non-invasive tools for IGRT are in use. Another weakness is the limited sample size. Regarding strengths, we analyzed a homogene- ous patient collective to address this question, with in-depth data including bNED, OS, DSS, and acute and late toxicity, as well as DVH data. Besides, our patient collectives were recruited in parallel, and this reduced any potential bias due to changing treatment modalities over time. Conclusions With this study, we have developed the hypothesis that the use of GFM in IGRT does not provide a sub- stantial benefit regarding tumor control or toxicity when other modalities for IGRT are used. To clarify the role of GFM in IGRT, prospective studies based on this hypothesis are needed to possibly reduce the number of unnecessary medical interventions in the treatment of men’s most common cancer. Acknowledgments Language editing was performed by San Francisco Edit, 1755 Jackson Street, Suite 610, San Francisco, CA 94109, USA. Radiol Oncol 2023; 57(1): 95-102. Moll M et al. / Effects of gold fiducial markers 101 TABLE 6. Uni- and multivariable Cox regression of the onset of late gastrointestinal or genitourinary toxicity grade 2 or higher GI Univariable analysis Multivariable analysis Variable p-value exp(HR) exp(HR) (95% conf.) p-value exp(HR) exp(HR) (95% conf.) Use of GFM 0.89 0.936 0.368–2.378 - - - Acute GI grade 2 0.09 2.642 0.875–7.981 - - - Age at RT 0.03 1.124 1.010–1.252 0.101 1.092 0.983-1.213 PTV prostate 0.06 0.987 0.974–1.000 - - - PTV Dmax/D1 0.76 1.037 0.824–1.305 - - - PTV Dmean 0.49 1.158 0.767–1.747 - - - CTV prostate 0.03 0.966 0.936–0.996 0.073 0.972 0.942–1.003 CTV Dmax/D1 0.55 1.056 0.884–1.263 - - - CTV Dmean 0.51 1.090 0.843–1.410 - - - CTV Dmin 0.64 1.042 0.877–1.236 - - - Rectal Volume 0.29 1.007 0.994–1.020 - - - Rectal Dmax 0.29 1.125 0.906–1.397 - - - Rectal Dmean 0.25 0.951 0.973–1.036 - - - Rectal V70 Gy % 0.59 0.971 0.875–1.078 - - - Rectal V70 Gy cm³ 0.42 1.040 0.945–1.144 - - - Rectal V60 Gy % 0.42 0.969 0.899–1.046 - - - Rectal V60 Gy cm³ 0.50 1.024 0.955–1.099 - - - GU Univariable analysis Variable p-value exp(HR) exp(HR) (95% conf.) Use of GFM 0.93 1.041 0.441–2.459 - - - Acute GU grade 2 0.09 2.042 0.892–4.673 - - - Age at RT 0.50 1.026 0.952–1.106 - - - PTV prostate 0.29 1.006 0.995–1.017 - - - PTV Dmax/D1 0.14 0.808 0.611–1.068 - - - PTV Dmean 0.04 0.573 0.342–0.961 - - - CTV prostate 0.93 0.999 0.989–1.010 - - - CTV Dmax/D1 0.93 1.006 0.895–1.130 - - - CTV Dmean 0.67 1.025 0.916–1.147 - - - CTV Dmin CTV 0.58 1.035 0.918–1.165 - - - Bladder volume 0.72 0.999 0.997–1.002 - - - Bladder Dmax/D1 0.24 0.973 0.696–1.094 - - - Bladder Dmean 0.89 0.997 0.960–1.036 - - – Bladder V70 Gy % 0.49 0.977 0.913–1.045 - - - Bladder V70 Gy cm³ 0.23 0.969 0.921–1.020 - - - Bladder V50 Gy % 0.54 0.988 0.951–1.027 - - - Bladder V50 Gy cm³ 0.29 0.985 0.957–1.013 - - - Bladder V30 Gy % 0.79 0.997 0.974–1.020 - - - Bladder V30 Gy cm³ 0.70 0.997 0.980–1.014 - - - CTV = clinical target volume; GFM = gold fiducial marker; GI = gastrointestinal; GU = genitourinary; PTV = planning target volume; RT = radiotherapy Radiol Oncol 2023; 57(1): 95-102. 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Lancet Oncol 2007; 8: 475-87. doi: 10.1016/S1470-2045(07)70143-2 Radiol Oncol 2023; 57(1): 103-110. doi: 10.2478/raon-2023-0014 103 research article The five-year KRAS, NRAS and BRAF analysis results and treatment patterns in daily clinical practice in Slovenia in 1st line treatment of metastatic colorectal (mCRC) patients with RAS wild-type tumour (wtRAS) – a real- life data report 2013–2018 Tanja Mesti1,2, Martina Rebersek1,2, Janja Ocvirk1,2 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(1): 103-110. Received 6 September 2022 Accepted 30 September 2022 Correspondence to: Prof. Janja Ocvirk, M.D., Ph.D., Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. E-mail: jocvirk@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 preformed a Phase IV non-interventional study to assess KRAS, NRAS and BRAF status in metastatic colorectal cancer (mCRC) patients suitable for 1st line treatment and to evaluate the decisions for 1st line treatment considering the treatment goals in the RAS wild type (wt) patients. The aim of our study was also to evaluate the influ- ence of a waiting period for biomarkers analysis on the start of first-line treatment. Patients and methods. Patients with histologically confirmed mCRC adenocarcinoma suitable for first-line treat- ment fulfilling all inclusion criteria were included in the study. The KRAS, NRAS and BRAF analysis was performed from tissue samples of primary tumor site or metastatic site. All included patients have given consent to participate in the study by signing the informed consent form. Results. From April 2013 to March 2018 at the Institute of Oncology Ljubljana 650 patients were included, 637 of them were treated with first- line systemic treatment according to RAS and BRAF status. Remaining 13 patients with mCRC did not receive systemic first-line treatment. The distribution of patients with KRAS mutated and wild-type tumors, was almost equal, 48.8% and 47.9% respectively, 89 % of the patients had wt NRAS tumours and 86.1% had wt BRAF tu- mours. The most frequently prescribed treatment was bevacizumab-based therapy (53.1%), either in combination with doublet chemotherapy or with mono-chemotherapy. EGFR inhibitors cetuximab and panitumumab were prescribed in wt RAS mCRC patients (30.9%). The waiting period for biomarkers analysis was two weeks. Conclusions. Our real-world data, single centre 5-year analysis showed that the distribution between wild type and mutated type tumors of the patients with mCRC was approximately the same, as worldwide, so the Slovenian popula- tion with mCRC has the same ratio distribution of KRAS, NRAS and BRAF wild and mutated genes. We concluded that a two-week waiting period for biomarkers analysis did not influence the first line treatment decision, so it was in the accordance with the worldwide treatment guidelines based on evidence-based medicine. Key words: metastatic colorectal cancer; RAS and BRAF biomarkers; systemic treatment Introduction Colorectal cancer (CRC) is one of the most com- mon cancers and one of the leading causes of cancer death in the world and also in Slovenia. According to the Cancer Registry of Slovenia 2021, 1349 new patients were diagnosed with CRC in 2018.1 Approximately 25% of patients present with Radiol Oncol 2023; 57(1): 103-110. Mesti T et al. / Metastatic colorectal cancer and RAS and BRAF biomarkers104 metastatic disease at diagnosis, and about 50% of patients with CRC will eventually develop metas- tases.2,3 Metastatic disease is still incurable, with 5% five-year overall survival (OS) without treat- ment. Until 1996 five-fluorouracil (5-FU) was the only approved drug for this disease. Since then, five new agents have been approved in the United States and in Europe for this disease, among them irinotecan, oxaliplatin and the targeted therapies cetuximab, bevacizumab and panitumumab.2-16 With the current management of metastatic dis- ease, with chemotherapy with oxaliplatin and irinotecan in combination with biologicals, tar- geting epidermal growth factor mediated growth regulatory pathway and the vascular endothelial growth factor mediated angiogenesis pathway, the progression-free survival (PFS) and OS of these patients can be prolonged. The CRC-related 5-year survival rate approaches 60%.2,3 CRC represents a heterogeneous group of dis- eases. They are promoted by environmental risk factors and various molecular pathways, which in- fluence individual susceptibility to cancer. About 70% of CRCs are sporadic, while 20–30% have a hereditary component, such as Lynch syndrome and familial adenomatous polyposis (FAP).2,3,15-17 Classification of CRC can be divided in anatomic, genetic and molecular transcriptomic classifica- tion. The race, foods, nutrients, carcinogenic agents and increasingly more important the gut microbi- ome act in a specific manner determined by the primary tumour location to promote carcinogen- esis right-sided tumours (RCC) account about to 35% of cases, while left-sided (LCC) and rectal can- cer represent about 65% of cases.17,18 According to genetic classification approximately 85% of CRC, comprises of non-hypermutated, microsatellite stable (MSS) tumours with chromosomal instabil- ity with a high frequency of DNA somatic altera- tions.17,18 Several oncogenes play key roles in promot- ing colorectal cancer.17-20 Among them firstly in clinical practice of mCRC management, onco- genic mutations of Rat sarcoma virus (RAS) and b-Raf murine sarcoma viral oncogene homolog B (BRAF), which activate the mitogen-activated protein kinase (MAPK) signalling pathway, are the most important.21-24 Oncogenic RAS mutations have historically been present in approximately 40–50% of CRC patients. According to additional subsequent analysis, the prevalence of RAS mu- tations in mCRC has been shown to be higher, in 55.9% with mutations in KRAS exon 2 being the most common, they represent 42.6%, followed by KRAS exon 3 in 3.8%, KRAS exon 4 in 6.2%, NRAS exon 2 in 2.9%, NRAS exon3 in 4.2% and NRAS exon4 in 0.3% mutations.16 NRAS mutant tumours are more frequently in older patients and located on the proximal colon. KRAS and NRAS mutant tumours exhibit similar metastasis patterns. RAS mutations are encountered in approximately 50% of the total population worldwide but it also dem- onstrates geographical variations. Therefore, such a country-by-country determination of the mu- tation status of the RAS gene in mCRC patients would be meaningful for personalised treatment decision-making. Selection of patients for anti-epidermal growth factor receptor (EGFR) antibodies based on mo- lecular characteristics of the tumour is very im- portant, because the activity of the anti-EGFR antibodies was confined to wtKRAS tumours (tra- ditionally mutations on codon 12 and 13 of exon 2).22-26 Than the testing was expanded to the other more rare RAS mutations: codon 61 of exon 3 and codon 117 and 146 of exon 4 of KRAS and exons 2, 3 and 4 of N-Rat sarcoma virus (NRAS), which are also predictive to response to anti-EGFR anti- bodies.2 Exon 2 KRAS mutations occur in 40% of CRC cases, and the other KRAS and NRAS muta- tions in 10%–15% of CRC patients. Those muta- tions influence the response to the combinations of cetuximab or panitumumab alone or with irinotecan- and oxaliplatin-based regimens.27-42 Treatment with anti-EGFR antibodies may even harm patients with a RAS mutation, especially when combined with oxaliplatin.4 BRAF mutations (mt BRAF) are present in about 10–15% of CRCs, with mt BRAFV600E mutations being the most frequent.15,16 BRAF encodes a ser- ine/threonine protein kinase, a downstream ef- fector of the KRAS protein, and mt BRAF results in constitutive activation of the MAPK signal- ling pathway. RAS mutations (mt RAS) and mt BRAF mutations are usually mutually exclusive. The most common mutation of the BRAF gene is V600E.15-18 The same was reported in our previ- ous study carried on Slovenian patients with CRC where the mt BRAF V600E was found in 5.1% of pa- tients.22,43,44 A mt BRAF is a strong negative prog- nostic biomarker. The patients with a mt BRAF mCRC have a very poor prognosis. In prognostic analyses, patients with mt BRAF had significantly shorter PFS (7.0 months vs. 12.2 months, HR 2.78; p < 0.001) and OS (13.4 months vs. 37.9 months, HR 5.67; p < 0.001) compared with BRAF wild-type (wt BRAF) patients, whilst patients with mt RAS experienced no difference in PFS (11.0 months vs. Radiol Oncol 2023; 57(1): 103-110. Mesti T et al. / Metastatic colorectal cancer and RAS and BRAF biomarkers 105 12.2 months, HR 1.15; p = 0.241); however, OS was significantly shorter compared with wtRAS pa- tients (26.3 months vs. 37.9 months, HR 1.44; p = 0.015).2,3,41,44 Other, non-V600 mt BRAF are a very rare and occur in 2% of metastatic CRC patients, most frequently in younger male patients, with pa- tients, mostly males. Tumours are generally well differentiated tumours, more frequently harbour concurrent mt RAS. Survival of these patients was shown to be improved compared to metastatic CRC patients with V600E mt BRAF or RAS wild- type (wt RAS).15,16 As the role of targeted therapy for treatment of advanced or mCRC has become increasingly imported, so currently, determination of tumour gene status for KRAS/NRAS and BRAF mutations, as well as HER2 amplifications and MSI/MMR sta- tus, neurotrophic tyrosine receptor kinase (NTRK) fusions are recommended for patients with mCRC. Testing may be carried out for individual genes or as part of an NGS panel.2,3 In order to get the real-life data for Slovenian population with mCRC treated at the Oncology Institute of Ljubljana from 30 April 2013 to 5 March 2018 we have performed a Phase IV non- interventional study to assess KRAS, NRAS and BRAF status in mCRC patients suitable for 1st line treatment. This prospective cohort study was approved by the Institutional Review Board Committee (Approval number: KESOPKR-6 and 03-Z/ KESOPKR-6) and was conducted following the ethical standards defned by the Declaration of Helsinki. The study was conducted with the un- derstanding and the consent of the patients. Prior to treatment patients have signed an informed consent for treatment and that their data could be used for scientific purposes. The aim of this prospective cohort study was to determine in our mCRC patient population the biomarkers status and distribution in the RAS and BRAF genes, and to determine the time from re- ceipt of the histological sample to the determina- tion of the molecular status. Patients and methods We performed a prospective, single-arm, single- centre, non-comparative, case-based, observation- al study from 30 April 2013 to 5 March 2018. The study was comprised of one centre which covers the treatment of all mCRC patients and reflects Slovenian demographic distribution. Originally, all samples for biomarker examination were to be evaluated for KRAS status by the certificated Molecular laboratory at the Institute of Oncology Ljubljana. The data was to be recorded in a collec- tive data base to provide reliability. As from 2013 before initiating treatment with cetuximab, the testing for RAS status included not only KRAS, but also NRAS mutations by an experienced labora- tory using validated test methods for detection of KRAS and NRAS. According to this in November 2013 in addition to KRAS we included also NRAS mutations analysis in all patients included in this study. RAS testing had previously been performed for all patients already included. In this study, 650 mCRC patients treated at the Institute of Oncology Ljubljana were enrolled. Initially, the planned number of patients for the study was 300. Between April 2013 and February 2014, 300 patients were recruited and underwent KRAS testing. The protocol was then amended in view of full RAS testing becoming standard clini- cal practice. The protocol was amended again in November 2015, to include an extra 350 above the original plan – a total of 650, and BRAF testing was added to the list of biomarkers analysed. From November 2015 until March 2018, 350 additional patients were recruited, making a total recruit- ment of 650. Thirteen patients that were included and signed consent to be part of this study, and underwent RAS and BRAF testing, did not present for the first-line systemic treatment afterwards, re- sulting in complete data for 637 patients. The basic data about each patient were record- ed, as date of birth, gender, performance status Eastern Cooperative Oncology Group (ECOG) performance status and smoking habits. The dis- ease characteristics were also included, including date of diagnosis of the primary tumour, tumour location – colon or rectum, initial stage at the time of diagnosis (according to the TNM (tumour T, nodes N, and metastases M) classification sys- tem), the treatment option used for the primary tumour, (radiotherapy or surgery, followed or not by adjuvant chemotherapy, with the duration time treatment and the type of the adjuvant chemo- therapy (capecitabine, 5-FU or oxaliplatin). The date of metastatic diagnosis was also included in the analysis, the sites of metastatic spread and the treatment option used, as surgery and systemic treatment, with systemic treatment comprising the treatment goals – curative, potentially curative or palliative, liver and/or lung metastases potentially respectable, depending upon tumour burden. The systemic first line treatment options included vari- Radiol Oncol 2023; 57(1): 103-110. Mesti T et al. / Metastatic colorectal cancer and RAS and BRAF biomarkers106 ous combinations of irinotecan, oxaliplatin based therapy, 5-FU, capecitabine, FOLFOXIRI protocol, cetuximab in a weekly or biweekly manner, beva- cizumab, panitumumab or other. The tumour characteristics were assessed re- garding with KRAS/RAS and BRAF status. The date of the request for the biomolecular analysis was included, and the date of the analysis receipt. DNA for molecular analysis was extracted from formalin-fixed, paraffin-embedded tumour tissue of primary tumours or metastases with at least 70% of tumour cells. Molecular testing was per- formed with RT-PCR KRAS Mutation Analysis Kit (EntroGen, Inc.), RT-PCR NRAS Mutation Analysis Kit (EntroGen, Inc.) and RT-PCR BRAF Mutation Analysis Kit (EntroGen, Inc.), according to man- ufacturer’s instructions. The KRAS and NRAS analyses were assessed on exons 2, 3 and 4, and for BRAF on exon 15. Statistical methods In this study 95% confidence intervals were calcu- lated to indicate the degree of certainty of KRAS, NRAS and BRAF status frequency as being wild type or mutant type. A sample size of 551-634 patients was neces- sary for a two-sided 95% confidence interval with a width of 4% anticipating a BRAF mutant rate of 5.5% to 6.5%. The planned sample size was in- creased to 650 patients, to consider non-evaluable biomarker test results and missing data. Results In total, 650 patients of the whole mCRC patients treated at Oncology Institute of Ljubljana were en- rolled in this prospective clinical study. Patient’s characteristics and treatment The demographic data about each patient were recorded, such as date of birth, gender, perfor- mance status (WHO) and smoking habits. Two thirds of the patients were male and most (84.9%) of the patients initially had a very good (ECOG 0–1) performance status. The main localization of the primary tumour was colon in 60.8% of the pa- tients. All of included patients in this study had pathological confirmation of the carcinoma of the colon or rectum. Almost one third (27.9%) of the patients had clinical signs for CRC, before being pathologically confirmed, including occult bleed- ing or were asymptomatic and detected in the Slovenian national program for the early detec- tion of the colorectal cancer named SVIT. For more than half (56.7%) of patients the diagnosis was con- firmed pathologically, after biopsy was performed at colonoscopy, and for the others after the surgery. TABLE 1. Patients baseline characteristics Patients baseline characteristics Number (%) Gender female male 233 (36.6) 404 (63.4) WHO clasiffication: 0 1 2 241 (37.8) 300 (47.1) 72 (11.3) Tumor location: colon rectum 387 (60.8) 250 (39.2) Clinical signs of the primary colorectal cancer present before pathological confirmation 178 (20.9) Primary metastatic 361 (56.7) Liver metastases 218 (34.3) * Some pathohistological reports from external hospital had unclear description, about the TNM status and risk factors TABLE 2. Disease characteristics Disease characteristics Number (%) pT4 of primary tumor 186 (29.2) Affected regional lymph nodes (N): N0 (no affected regional lymph nodes) N1 (1 to 3 affected regional lymph nodes) N2 (more than 3 affected regional lymph nodes) Missing data* 110 (17.3) 209 (32.8) 239 (37.5) 62 (11.3) Vascular invasion 114 (17.9) Perineural invasion 95 (27.9) Lymphangiosis 115 (18.1) Grade of differentiation: G1 (well) G2 (medium) G3 (poorly) G4 (no differentiated) Missing data* 19 (3) 162 (25.4) 53 (8.3) 1 (0.2) 402 (63.2) Resection of primary tumour R0 R1 R2 63 (9.9) 10 (1.6) 16 (2.5) * Other metastatic locations: brain, ovaries, suprarenal glands, local recurrence, muscular dissemination Radiol Oncol 2023; 57(1): 103-110. Mesti T et al. / Metastatic colorectal cancer and RAS and BRAF biomarkers 107 Complete resection (R0) of operated primary tu- mour was achieved in 9.9%. Most of the included patients were non-smokers (62.6%). According to the TNM characteristics, initially at the first presentation of the primary CRC, in the external hospitals, 53.7% of patients had T3 tu- mours, 31.6% of patients had N1 (31,6%) and 34.5% of patients had N2. One third of patients had ini- tially no metastatic disease (33.4%). Mostly, the primary CRC had pathohistological characteristics as follows: grade 2, with one third having vascu- lar invasion, perineural invasion or lymphangi- osis. Primary metastatic disease was confirmed in 56.7% of patients. The most common sites of metastases were liver and lung. Some of patients with mCRC in liver and lungs had surgery of the metastasis before the mCRC treatment, 6.8% and 2.0% respectively. In 10% of the mCRC patients R0 resection (70.8% of the total number of resections) was achieved. The metastatic sites were included in the analysis, and the main sites of CRC dis- semination were liver (34.2%), lungs (11.0%), lymph nodes (5.3%), peritoneum (5.0%). Sixty-two patients were no previous smokers, and only 9% of patients were smoking at the time of our analysis. Patients’ baseline characteristics and are shown in Table 1, disease characteristics are shown in Table 2, the most frequent metastatic site are shown in Table 3, smoking habits of included patients are shown in Table 4. The treatment goals were in oligometastatic disease as follows: respectable and potentially re- sectable, or palliative for patients with high bur- den of the disease. More than a half (58.7%) of the patients were initially candidates for the palliative treatment, and 24.2% were potentially resectable at presentation. Treatment goal options at presenta- tion of the mCRC patients are shown in Figure 1. More than half (58.7%) of patients were initially candidates for palliative systemic treatment, and 24.2% were potentially resectable at presentation. Of all 637 mCRC patients had the following possible first line systemic treatment options: chemotherapy - fluoropyrimidine based systemic therapy combined with oxaliplatin and/or irinote- can plus, for subjects with KRAS/RAS wild type tumours, the EGFR inhibitors cetuximab or pani- tumumab, or the VEGF inhibitor bevacizumab for those with KRAS/RAS mutated tumours. Most TABLE 3. The most frequent metastatic sites The most frequent metastatic sites Number (%) Total 637 (100) Liver 218 (34.3) Lungs 70 (11.0) Lymph nodes 34 (5.3) Peritoneum 115 (18.1) Bones 4 (0.6) Other* 18 (2.8) Multiple locations 261 (41.0) Palliative/sequential treatment 374 pts (58.7%) Early tumour shrinkage (ETS) 109 pts (17.1 %) Liver±Lung- potentially resectable 154 pts (24.2%) FIGURE 1. Treatment goal options at presentation of the metastatic colorectal cancer (mCRC) patients. TABLE 4. Treatment regimen decision for the first line treatment of the metastatic colorectal cancer (mCRC) Systemic therapy options Number (%) Fluoropirimidines/Irinotecan/Cetuximab* 73 (11.4) Fluoropirimidines/Oxaliplatin/Cetuximab* 49 (7.7) Fluoropirimidines/Irinotecan/Panitumumab* 17 (2.7) Fluoropirimidines/Oxaliplatin/Panitumumab* 12 (1.9) EGFR inhibitors other** 46 (7.2) Fluoropirimidines/Irinotecan/Bevacizumab 126 (19.8) Fluoropirimidines/Oxaliplatin/Bevacizumab 191 (30.0) VEGF inhibitor other*** 21 (3.3) Chemotherapy only 102 (16.0) *EGFR inhibitors for RAS (NRAS and KRAS) wild type only **EGFR inhibitors monotherapy ± one chemotherapy ***VEGF inhibitor monotherapy ± one chemotherapy Radiol Oncol 2023; 57(1): 103-110. Mesti T et al. / Metastatic colorectal cancer and RAS and BRAF biomarkers108 frequently used was bevacizumab-based therapy (53.1%), either in combination with doublet chemo- therapy or with one cytostatic or as monotherapy. EGFR inhibitors cetuximab and panitumumab were used in RAS (KRAS/NRAS) wild type mCRC subjects (30.9%). Data presented in the Table 9. Fifty-one subjects with RAS (KRAS/NRAS) wild type tumours received chemotherapy alone or in combination with Bevacizumab, as first line treat- ment. The first line treatment decision is made by the oncologist in accordance with NCCN and ESMO guidelines and consider the patient’s pref- erences. Some patients found skin side effects and higher parenteral application frequency important parameters for a possible decrease of their quality of life. Most frequently prescribed treatment pro- tocols are shown in Table 4. Biomarker status The assessment of the KRAS/RAS and BRAF status was performed for all 650 patients. The RAS and BRAF status analysis were performed simultane- ously, and for the group of subjects from the first Amendment, after the Amendment II was added to the protocol, the NRAS and BRAF data were added to the database. Data of the RAS/KRAS and BRAF status distribution are shown in Table 5. The distribution of patients with KRAS mutated and wild-type tumours, was almost equal, 48.8% and 47.9% respectively. For 3.3% of patients of the as- sessed population the KRAS status analysis was unsuccessful, due to unrepresentative tumour samples (Table 6). For 2.0% and 0.7% of the subjects the NRAS and BRAF genotyping was not possi- ble due to unrepresentative tumour samples. The KRAS and NRAS analyses were assessed on ex- ons 2, 3 and 4, and for BRAF on exon 15. The most frequent mutation was in KRAS in codon 12 and codon 13, and the same was true for NRAS and for the BRAF V600E mutation. The most frequent mutations, as follows were: pGly12Asp (c35 G>A) 7.8%, pGly 12Val (c35G>T) 6.6%, pGly13Asp (c38 G>A) 4.7% and pGly12Cys (c34 G>T) 2.4%. The median time for biomarkers status assess- ment was 14 days. Mostly direct sequencing was used for KRAS/ RAS and BRAF status assessment, but also oth- er methods, as pyrosequencing and qPCR. The Entrogen RT PCR KRAS/RAS and BRAF mutation Kit was used. Methods used for KRAS/RAS and BRAF analysis are shown in Table 6. Discussion The aim of the observational mCRC population- based study was to determine the biomarker sta- tus in the RAS and BRAF genes, to determine their distribution in the mCRC patient Slovenian popu- lation, and to determine the time from receiving the histological sample to the determination of the biomarker status and its impact on the initiation of systemic oncological therapy. This study was performed in order to achieve a real perspective of the bio-markers status of the patients with metastatic colorectal cancer in comparison with worldwide data. The analysis of KRAS/NRAS and BRAF status was a relatively new approach in the treatment of the patients with metastatic colorectal cancer at the time of conduct- ing of our clinical study, so we also wanted to get real insight of the time frame needed for such anal- ysis and its possible effect on the initiation of the first-line treatment. The status of mutations in the RAS gene is a mo- lecular predictive factor for response to treatment with EGFR inhibitors inmCRC. Determination of mutational status in KRAS gene has been stand- ard clinical practice since 2008. Additional muta- tions in the codons of 61 and 146 of KRAS gene, and in codons 12, 13, 61 and 146 of NRAS gene are determined at our Institute of Oncology Ljubljana TABLE 5. The RAS/KRAS and BRAF status distribution The RAS/KRAS and BRAF status distribution Number (%) KRAS testing KRAS mutated KRAS wild type Not possible 637 (100) 311 (48.8) 305 (47.9) 21 (3.3) NRAS testing NRAS mutated NRAS wild type Not possible 637 (100) 57 (9.0) 567 (89.0) 13 (2.0) BRAF testing BRAF mutated BRAF wild type Not possible 637 (100) 84 (13.2) 548 (86.1) 5 (0.7) TABLE 6. Methods used for KRAS/RAS and BRAF analysis Methods used for KRAS/RAS and BRAF analysis Number (%) Direct sequencing 223 (35.0) Pyrosequencing 322 (50.5) qPCR 92 (14.5) Radiol Oncol 2023; 57(1): 103-110. Mesti T et al. / Metastatic colorectal cancer and RAS and BRAF biomarkers 109 since autumn 2013 and are being standard in inter- national clinical practice since 2014. According to the literature data, are about 15%. In our prospective cohort study KRAS muta- tions were found in 48.8% of patients, and addi- tionally 9% of NRAS mutations were determined. So approximately 60% of patients had RAS mutat- ed mCRC, which is consistent with reports from previous literature.2,3,22,45 In our retrospective anal- ysis we found 17% of additional mutations in RAS gene, which is a higher percentage than in our pro- spective cohort study, probably due to characteris- tics of patient population included.45 Also, higher, 13.2% BRAF mutations, were de- termined in our cohort study. According to the lit- erature, the frequency of this mutation is from 5 to 10%. 2,3,24 According to our previous retrospective analysis, the V600E mutation in the BRAF gene was also present at a similar percentage of patients, in 7.4%.46 It is assumed that this difference in the per- centage of BRAF mutations in clinical studies is due to the characteristics of the patients included in the analysis. So, according to our study analysis data, the distribution between wild type and mutated type tumours of mCRC patients was approximately the same, as worldwide, so the Slovenian popula- tion with mCRC has the same ratio distribution of KRAS, NRAS and BRAF wild and mutated genes. The two-week time period from the initial pres- entation of the patient until the biomarker status analysis report did not affect the starting of the systemic treatment or the treatment decision, as usually mCRC patients are given a brief period of time for psychical and physical preparation for the systemic treatment, at which time supportive care – nutritional and symptomatic support, is given. As for the time spent on biomarkers analysis, we concluded that the two-week period was not influ- encing the first line treatment decision, because patients started with systemic chemotherapy im- mediately and after 2 weeks have already received a biologic drug at the 2nd cycle of chemotherapy ac- cording to molecular genetic testing, And also in Slovenia we do not have waiting lists and cancer patients, who need treatment are referred to the oncology treatment facility shortly after the diag- nosis, so this brief period of time is usual for psy- chical and physical preparation for the systemic treatment, at which time supportive care – nutri- tional and symptomatic support, is given. In conclusion, we have proven that the bio- markers distribution in Slovenian population with mCRC was approximately the same as worldwide, so the decision treatment approach should be in the accordance with the worldwide treatment guidelines based on evidence-based medicine. 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BRAF mutation in metastatic colorectal cancer. N Engl J Med 2009; 361: 98-9. doi: 10.3978/j.issn.2078-6891.2015.077 44. Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, et al. Poor survival associatedwith the BRAF V600E mutation in microsat- ellite-stable colon cancer. Caner Res 2005; 65: 6063-9. doi: 10.1158/0008- 5472.CAN-05-0404 45. Rebersek M, Mesti T, Boc M, Ocvirk J. Molecular biomarkers and histological parameters impact on survival and response to first- line systemic therapy of metastatic colorectal cancer patients. Radiol Oncol 2019; 53: 85-95. doi: 10.2478/raon-2019-0013 46. Rebersek M, Boc M, Škerl P, Benedik J, Hlebanja Z, Volk N, et al. Efficacy of First- line systemic treatment in correlation with BRAF V600E and differ- ent KRAS mutations colorectal cancer - a single institution retrospective analysis. Radiol Oncol 2011; 45: 285-91. doi: 10.2478/v10019-011-0039-y Radiol Oncol 2023; 57(1): 111-120. doi: 10.2478/raon-2023-0003 111 research article Association of OPRM1, MIR23B, and MIR107 genetic variability with acute pain, chronic pain and adverse effects after postoperative tramadol and paracetamol treatment in breast cancer Zala Vidic1, Katja Goricar1, Branka Strazisar2, Nikola Besic2, Vita Dolzan1 1 Pharmacogenetics Laboratory, Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 2 Institute of Oncology, Ljubljana, Slovenia Radiol Oncol 2023; 57(1): 111-120. Received 14 September 2022 Accepted 28 September 2022 Correspondence to: Prof. Vita Dolžan, M.D., Ph.D., Pharmacogenetics Laboratory, Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. E-mail: vita.dolzan@mf.uni-lj.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. Tramadol is an opioid analgesic often used for pain management after breast cancer surgery. Its anal- gesic activity is due to the activation of the μ-opioid receptor, encoded by the OPRM1 gene. This study investigated the association of genetic variability in OPRM1 and its regulatory miRNA genes with outcomes of tramadol/paraceta- mol treatment after breast cancer surgery with axillary lymphadenectomy. Patients and methods. The study included 113 breast cancer patients after breast cancer surgery with axillary lymphadenectomy treated with either 75/650 mg or 37.5/325 mg of tramadol with paracetamol for pain relief within the randomized clinical trial KCT 04/2015-DORETAonko/si at the Institute of Oncology Ljubljana. All patients were geno- typed for OPRM1 rs1799971 and rs677830, MIR23B rs1011784, and MIR107 rs2296616 using competitive allele-specific PCR. The association of genetic factors with acute and chronic pain as well as adverse effects of tramadol treatment was evaluated using logistic regression, Fisher’s exact test, and Mann-Whitney test. Results. The investigated OPRM1 related polymorphisms were not associated with acute pain assessed with the VAS scale within four weeks after surgery (all P > 0.05). Carriers of at least one polymorphic OPRM1 rs1799971 allele had a higher risk of constipation in the first four weeks after surgery compared to non-carriers (OR = 4.5, 95% CI = 1.6–12.64, P = 0.004). Carriers of at least one polymorphic OPRM1 rs677830 allele had a higher risk of constipation after third week of tramadol treatment (OR = 3.11, 95% CI = 1.08–8.89, P = 0.035). Furthermore, carriers of two polymorphic MIR23B rs1011784 alleles had a higher risk of nausea after 28 days of tramadol treatment (OR = 7.35, 95% CI = 1.27–42.6, P = 0.026), while heterozygotes for MIR107 rs2296616 allele had a lower risk of nausea after 21 days of tramadol treatment (OR = 0.21, 95% CI = 0.05–0.87, P = 0.031). In carriers of two polymorphic MIR107 rs2296616 alleles, chronic pain was significantly more common than in carriers of two wild-type alleles (P = 0.004). Carriers of at least one polymorphic MIR23B rs1011784 allele experienced more neuropathic pain after adjustment for tramadol dose (OR = 2.85, 95% CI = 1.07–7.59, P = 0.036), while carriers of at least one polymorphic OPRM1 rs677830 allele experienced less neuropathic pain compared to carriers of two wild-type alleles (OR = 0.38, 95% CI = 0.15–0.99, P = 0.047). Conclusions. Genetic variability of OPRM1 and genes coding for miRNAs that could affect OPRM1 expression may be associated with adverse effects of tramadol/paracetamol treatment as well as with chronic and neuropathic pain after breast cancer surgery with axillary lymphadenectomy. Key words: breast cancer surgery; miRNA; OPRM1; pain treatment; tramadol Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer112 Introduction Tramadol is a synthetic centrally acting opioid drug that exhibits its analgesic activity by bind- ing to μ-opioid receptor (MOR).1 It is widely used for the treatment of moderate to severe acute pain after breast cancer surgery with axillary lymphad- enectomy.2 Tramadol is considered a relatively safe opioid drug since it does not cause respiratory de- pression at therapeutic doses and is less likely to cause addiction compared to many other opioids. However, its analgesic efficiency is lesser com- pared to morphine.1-3 MORs, encoded by the OPRM1 gene, are mem- bers of the Rhodopsin family of G-protein coupled receptors.4 MORs are expressed in the cerebral cortex, thalamus, periaqueductal gray, nucleus accumbens, and basolateral amygdala, and are therefore responsible for the occurrence of eu- phoria, dependence, respiratory depression, and activation of the reward system.5 They are crucial for the analgesic effects of the majority of opioid drugs.6 It has been shown that polymorphisms in the OPRM1 gene can affect the efficacy of analgesic ac- tion of several opioid drugs. One of the most stud- ied single nucleotide polymorphisms (SNPs) of the OPRM1 gene is rs1799971 (c.118A>G; p.Asn40Asp).4 A study performed in European subjects on long- term treatment for chronic pain with different opioids, including tramadol, reported a higher frequency of adverse effects in wild-type sub- jects compared to individuals with the AG or GG genotype.7 A study in Chinese population showed greater analgesic action of tramadol combined with paracetamol (acetaminophen) for the treat- ment of peripheral neuropathy in wild-type pa- tients compared to carriers of polymorphic G al- lele.8 Numerous studies focused on investigating the impact of this SNP on response to morphine treatment in different populations. Many of them confirmed the association between rs1799971 and pain relief or morphine dose requirements.9-12 Furthermore, a haplotype of seven OPRM1 SNPs, that included rs1799971 and another non-synony- mous SNP, rs677830 (c.1231C>T, p.Gln411Ter) was associated with postoperative morphine response in Caucasians.12 OPRM1 gene expression may be regulated by non-coding RNAs such as microRNAs (miRNAs) that bind to target mRNAs molecules and prevent protein translation or promote mRNA degrada- tion.13,14 It was shown that several miRNAs can af- fect MOR expression, or lead to opioid dependence or tolerance.15,16,17 Furthermore, opioids can regu- late the expression of several miRNAs.15 miRNA-23b was the first identified miRNA molecule that regulates OPRM1 expression17, how- ever miRNA-107 was shown to influence OPRM1 expression as well.15 Both miR-23b and miR107 were experimentally proven to reduce the expres- sion level of OPRM1 splice variants in mouse or human neuronal cell lines, human transfected em- bryonic kidney cell lines, or mouse animal models. The binding of these miRNAs to 3’ UTR of OPRM1 mRNAs prevented them from binding to ribo- somes without altering the respective mRNAs lev- els. Furthermore, these studies also reported that long-term morphine treatment increased miRNAs expression.15,17,18 The aim of this study was to evaluate the asso- ciation of OPRM1 rs1799971 and rs677830, MIR23B 1011784, and MIR107 rs2296616 polymorphisms with the severity of acute pain and the presence of adverse effects in the first four weeks after breast cancer surgery with axillary lymphadenectomy as well as with chronic and neuropathic pain at one year after surgery. Patients and methods Patients Our study included breast cancer patients that participated in a prospective double-blind- ed randomized clinical trial at the Institute of Oncology Ljubljana from 2015 to 2018 (Trial KCT 04/2015-DORETAonko/si).2,19 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 32/03/15). The main inclusion criteria was treatment with tramadol and paracetamol after breast cancer surgery with axillary lymphadenectomy, while exclusion criteria were: concomitant breast re- construction with a tissue expander or free-flap, hypersensitivity to the drugs used in the study, male gender, pregnancy, high risk of anesthesia according to the criteria of the American Society of Anaesthesiology (ASA over 3), patient’s age under 18 and over 70 years, severe liver or kidney dis- ease, regular use of analgesics or antidepressants, history of opioid abuse or presence of psychiatric illness, such as dementia, schizophrenia, or manic depressive illness. Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer 113 Before the surgery, all patients were examined according to the standard protocol of the Institute of Oncology Ljubljana. Data was collected about the body characteristics, associated diseases or al- lergies, the presence of pain, the use of other medi- cations, and the anaesthesia risk was assessed ac- cording to the criteria of the American Society of Anaesthesiology.20 During surgery, patients received standard postoperative local analgesia. On the first post- operative day, all patients received 37.5/325 mg of tramadol with paracetamol every 8 h, 550 mg of naproxen sodium every 12 h, and 10 mg of meto- clopramide every 8 h. From the second to the 29th postoperative day inclusive, tramadol and par- acetamol dose depended on the group into which a patient was randomized at inclusion in the study. The group with lower analgesia received 37.5/325 mg of tramadol with paracetamol every 8 hours, while the group with higher analgesia received 75/650 mg every 8 hours. The tramadol and par- acetamol doses for each patient were unblinded 15 months after the inclusion of the last patient. For four weeks, all patients also received 550 mg of naproxen sodium twice a day and 20 mg of pan- toprazole once a day. In the case of severe pain de- spite taking tramadol/paracetamol and naproxen sodium, patients received 500 mg of metamizole up to 4 g per day. In the first four weeks after surgery, patients re- corded the severity of the acute pain in the area of the breast and shoulder using the standard visual analog scale (VAS) three times a day. Every day, patients recorded the consumption of medications and the frequency of shoulder exercises. Patients also recorded adverse effects and reported the col- lected data to the healthcare professionals every seven days when they came for the weekly check- ups at the outpatients’ clinic. Before surgery, and again at the final follow-up examination at 12 to 15 months after the surgery, the patients filled in the standardized question- naire of the Institute of Oncology on the presence of chronic pain and the DN4 questionnaire on the presence of neuropathic pain. Methods Bioinformatics analysis First, we searched for potentially functional SNPs in the OPRM1 gene by performing a literature search in PubMed (https://pubmed.ncbi.nlm.nih. gov/), Scholar (https://scholar.google.com/), and SNPedia (https://www.snpedia.com).21 Their mi- nor allele frequency (MAF) was obtained from the dbSNP database from The National Center for Biotechnology Information (NCBI) (https://www. ncbi.nlm.nih.gov/snp/) and linkage disequilib- rium between polymorphisms was evaluated by the LDlink Tool (https://analysistools.cancer.gov/ LDlink/?tab=ldmatrix).22 We identified four mis- sense or nonsense SNPs in the coding regions of the OPRM1 gene with potential function in trama- dol treatment outcome. Among them, only three met the MAF criteria of at least 5% in Caucasians: rs1799971, rs677830 and rs540825. As OPRM1 rs540825 and rs677830 were in high linkage dise- quilibrium (R2 = 0.98), only rs1799971 and rs677830 were included in our study. Next, we searched PubMed, Scholar, and SNPedia for miRNAs with experimentally proven impact on MOR expression or activity. Moreover, we searched the following online databases that can predict the targets of miRNAs or report known associations between a specific gene and miRNA molecules: miRTarBase (http://mirtarbase.cuhk. edu.cn/php/index.php)23, miRDB (http://www. mirdb.org)24, and online tool miRNet (https://www. mirnet.ca)25, to identify miRNAs that can regulate OPRM1. After narrowing down the list of potential miRNAs with impact on MOR function, we iden- tified polymorphisms in genes coding for selected miRNAs with MAF of at least 5% in Caucasians. After the literature search for established roles of these polymorphisms in the expression of OPRM1 or other genes we have selected two polymor- phisms in two miRNA genes for the analysis. In total, four polymorphisms in three genes were selected for inclusion in our study: OPRM1 rs1799971 (NP_000905.3: p.Asn40Asp), OPRM1 rs677830 (NP_001138758.1: p.Gln411Ter), MIR23B rs1011784 (NR_029664.1: n.525G>C), and MIR107 rs2296616 (NR_029524.1: n.-382C>T). Molecular analysis DNA samples were isolated from peripheral blood samples using Qiagen FlexiGene Kit (Qiagen, Hilden, Germany) according to the manufac- turer’s instructions. Genotyping of selected poly- morphisms was performed retrospectively using custom made validated competitive allele-specific polymerase chain reaction (KASP) assays follow- ing the instructions of the manufacturer (LGC Biosearch Technologies, UK). The analysis was repeated for 10% of samples and the results were concordant. Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer114 Statistical analysis Statistical analysis was performed using IBM SPSS v27.0 (IBM Corporation, Armonk, NY, USA). Continuous data were presented with median and interquartile range, while categorical data were presented with frequencies. For each polymor- phism, a standard χ2-test was used to evaluate whether genotype distribution followed Hardy- Weinberg equilibrium (HWE). Only the dominant genetic model was used in all statistical analyses for OPRM1 polymorphisms due to the small num- bers of carriers of two polymorphic alleles, while both dominant and additive genetic model was used in the analyses for polymorphisms in miRNA genes. The association of genetic factors with ad- verse effects, chronic and neuropathic pain was evaluated using binary logistic regression to obtain odds ratios (OR) with corresponding 95-% confi- dence intervals (95% CI). Fisher’s exact test was used if there were no subjects within one of the groups. Mann-Whitney test was used to evaluate the association of polymorphisms with the severity of acute pain. All tests were two-sided and the level of statistical significance was set at 0.05. Results In total, 113 patients were included in the study, 55 (48.7%) received stronger postoperative analgesia and 58 (51.3%) received weaker postoperative an- algesia. Patients’ clinical characteristics are shown in Table 1. Among all patients, 14 (12.4%) patients discontinued tramadol treatment due to severe ad- verse events before the end of four weeks. Adverse TABLE 1. Patients’ clinical characteristics All subjects Stronger postoperative analgesia with tramadol/ paracetamol Weaker postoperative analgesia with tramadol/ paracetamol Sample size N = 113 N = 55 N = 58 Characteristic N (%) N (%) N (%) Age (years) Median (25–75 %) 55 (48–63) 57 (49–64) 53,5 (47.8–61) Weight (kg) Median (25–75 %) 72 (63.5–82) 72 (64–82) 72 (63–82.3) Body Mass Index (kg/m2) Median (25–75 %) 27.1 (23.4–31.0) 26.7 (23.0–30.8) 27.6 (23.5–31.2) Smoking No 87 (77.7) [1] 41 (75.9) [1] 46 (79.3) Yes 25 (22.3) 13 (24.1) 12 (20.7) ASA score 1 22 (19.6) [1] 10 (18.2) 12 (21.1) [1] 2 81 (72.3) 39 (70.9) 42 (73.7) 3 9 (8.0) 6 (10.9) 3 (5.3) Side of the operation Left 62 (54.9) 30 (54.5) 32 (55.2) Right 50 (44.2) 24 (43.6) 26 (44.8) Bilateral 1 (0.9) 1 (1.8) 0 (0.0) Tumor grade 1 4 (3.6) [1] 1 (1.9) [1] 3 (5.2) 1–2 3 (2.7) 2 (3.7) 1 (1.7) 2 39 (34.8) 17 (31.5) 22 (37.9) 2–3 8 (7.1) 2 (3.7) 6 (10.3) 3 58 (51.3) 32 (59.3) 26 (44.8) VAS after 7 days Median (25–75 %) 2 (1–3) [5] 1.3 (1–2) [3] 2 (1–3) [2] VAS after 14 days Median (25–75 %) 1.5 (0–2) [11] 1 (0–2) [9] 2 (1–2) [2] VAS after 21 days Median (25–75 %) 1 (0.4–2) [11] 1 (0–2) [9] 1 (1–2) [2] VAS after 28 days Median (25–75 %) 1 (0–2) [14] 1 (0–1) [10] 1 (1–2) [4] ASA = American Society of Anaesthesiology; N = sample size; [n] = number of missing data; VAS = visual analog scale Genotype frequencies, MAF, and Hardy-Weinberg equilibrium analysis (PHWE) of investigated polymorphisms in all patients enrolled in our study are presented in Table 2. Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer 115 events were therefore evaluated in 99 patients. The vast majority of the patients who discontinued tramadol were from the higher dose group (P = 0.004). A total of 101 patients completed the follow- up visit one year after surgery and were included in the analysis of genetic factors associated with the chronic and neuropathic pain. Within the first four weeks of tramadol/paracet- amol treatment, the severity of the self-perceived acute pain assessed with the VAS scale was not as- sociated with the patient’s genotypes for any of the investigated polymorphisms in none of the time points (all P > 0.05) (Supplementary Table 1). Several patients experienced the side effects that could be related to tramadol treatment at least once within the first four weeks: 36 (36.7%) pa- tients experienced nausea, 9 (9.1%) patients expe- rienced vomiting, 35 (35.4%) patients experienced dizziness, and 48 (48.5%) patients experienced con- stipation. TABLE 2. The investigated polymorphism’s characteristics and frequencies Gene SNP Genotype N (%) PAF (%) MAF HapMap CEU expected (%) PHWE OPRM1 rs1799971 AA 84 (74.3) 14.2 15.6–16.7 0.570 AG 26 (23.0) GG 3 (2.7) rs677830 CC 56 (49.6) 27.4 20.7 0.098 CT 52 (46.0) TT 5 (4.4) MIR23B rs1011784* CC 63 (56.3) [1] 26.8 23.3 0.153 CG 38 (33.9) GG 11 (9.8) MIR107 rs2296616* GG 21 (18.6) 57.1 54.2–55.3 0.944 GA 55 (48.7) AA 37 (32.7) ASA = American Society of Anaesthesiology; CEU = Northern Europeans from Utah; HGVS = Human Genome Variation Society; HWE = Hardy-Weinberg equilibrium; N = sample size; PAF = polymorphic allele frequency; SNP = single nucleotide polymorphism; *KASP assays were designed on the reverse strand TABLE 3. The impact of investigated polymorphisms on constipation anytime during the first four weeks of tramadol treatment (N = 99) SNP Genotype Constipation anytime N (%) OR (95% CI) P OR (95% CI)adj Padj OPRM1 rs1799971 AA 30 (40) Ref. Ref. AG+GG 18 (75) 4.5 (1.6–12.64) 0.004 4.31 (1.52–12.17) 0.006 OPRM1 rs677830 CC 24 (48) Ref. Ref. CT+TT 24 (49) 1.04 (0.47–2.29) 0.922 0.98 (0.44–2.19) 0.964 MIR23B rs1011784 CC 29 (50) Ref. Ref. CG 16 (50) 1 (0.42–2.37) 1.000 0.96 (0.4–2.31) 0.930 GG 3 (37.5) 0.6 (0.13–2.75) 0.510 0.46 (0.1–2.23) 0.338 CG+GG 19 (47.5) 0.9 (0.4–2.03) 0.808 0.84 (0.37–1.91) 0.676 MIR107 rs2296616 GG 11 (57.9) Ref. Ref. GA 23 (48.9) 0.7 (0.24–2.04) 0.511 0.73 (0.25–2.16) 0.566 AA 14 (42.4) 0.54 (0.17–1.68) 0.285 0.53 (0.17–1.67) 0.276 GA+AA 37 (46.3) 0.63 (0.23–1.72) 0.364 0.64 (0.23–1.77) 0.386 adj = adjustment for tramadol dose; N = sample size; OR = odds ratio; SNP = single nucleotide polymorphism; 95% CI = 95% confidence interval Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer116 Carriers of at least one polymorphic OPRM1 rs1799971 allele had a higher risk of constipation in the first four weeks compared to carriers of two wild-type alleles (OR = 4.5, 95% CI = 1.6–12.64, P = 0.004). This association remained significant after the adjustment for the tramadol dose (OR = 4.31, 95% CI = 1.52–12.17, P = 0.006). None of the other polymorphisms were associated with the overall risk for constipation during the first month after surgery (Table 3). Data for constipation for each week separately are shown in Supplementary Table 2. Carriers of at least one polymorphic OPRM1 rs677830 allele had a higher risk of con- stipation after 21 days of tramadol treatment when compared to carriers of two wild-type alleles (OR = 3.11, 95% CI = 1.08–8.89, P = 0.035). The risk of nausea after 28 days of tramadol treatment was significantly higher in carriers of two polymorphic MIR23B rs1011784 alleles com- pared to carriers of two wild-type alleles (OR = 7.35, 95% CI = 1.27–42.6, P = 0.026). Heterozygotes for MIR107 rs2296616 allele had a lower risk of nausea after the third week of tramadol treatment compared to carriers of two wild-type alleles (OR = 0.21, 95% CI = 0.05–0.87, P = 0.031). Similarly, car- riers of at least one polymorphic MIR107 rs2296616 had a lower risk of nausea after the third week of tramadol treatment compared to carriers of two wild-type alleles (OR = 0.30, 95% CI = 0.09–0.97, P = 0.045) (Table 4). None of the investigated pol- ymorphisms were associated with the risk for nausea within the first two weeks after surgery (Supplementary Table 3). One year after surgery 21 (20.8%) patients expe- rienced chronic pain and 25 (24.8%) experienced neuropathic pain. In carriers of two polymorphic MIR107 rs2296616 alleles, chronic pain was signifi- cantly more common compared to carriers of two wild-type alleles (35.3% compared to 0% of patients, P = 0.004). Carriers of at least one polymorphic OPRM1 rs677830 allele experienced less neuro- pathic pain compared to carriers of two wild-type alleles (OR = 0.38, 95% CI = 0.15–0.99, P = 0.047), but the difference was no longer significant after ad- justment for tramadol dose (P = 0.060). Carriers of at least one polymorphic MIR23B rs1011784 allele experienced more neuropathic pain compared to carriers of two wild-type alleles, but the difference was only significant after adjustment for tramadol dose (OR = 2.85, 95% CI = 1.07–7.59, P = 0.036). The other investigated miRNA polymorphism was not associated with the persistence of chronic or neu- ropathic pain (Table 5). Discussion Our study investigated the association of genetic variability of OPRM1 and genes coding for miR- NAs regulating OPRM1 expression with acute and long-term pain management as well as adverse ef- fects of tramadol treatment after surgical treatment of breast cancer and axillary lymphadenectomy. None of the investigated polymorphisms were associated with the intensity of acute pain after the surgery. However, we confirmed the associa- tion between some of the investigated polymor- phisms and the presence of adverse effects as well as chronic and neuropathic pain. In particular, OPRM1 rs1799971 polymorphism increased the risk of constipation in the first month of tramadol treatment, while OPRM1 rs677830 polymorphism TABLE 4. The impact of investigated miRNA polymorphisms on nausea after 21 and 28 days of tramadol treatment (N = 99) SNP Genotype Nausea 21 days N (%) OR (95% CI) P Nausea 28 days N (%) OR (95% CI) P MIR23B rs1011784 CC 9 (16.4) Ref. 4 (7.5) Ref. CG 4 (12.9) 0.76 (0.21–2.7) 0.668 5 (16.1) 2.36 (0.58–9.54) 0.230 GG 2 (25) 1.7 (0.3–9.83) 0.551 3 (37.5) 7.35 (1.27–42.6) 0.026 CG+GG 6 (15.4) 0.93 (0.3–2.86) 0.898 8 (20.5) 3.16 (0.88–11.39) 0.078 MIR107 rs2296616 GG 6 (31.6) Ref. 2 (10.5) Ref. GA 4 (8.9) 0.21 (0.05–0.87) 0.031 4 (8.7) 0.81 (0.14–4.84) 0.817 AA 5 (16.7) 0.43 (0.11–1.69) 0.229 6 (21.4) 2.32 (0.41–12.96) 0.338 GA+AA 9 (12) 0.30 (0.09–0.97) 0.045 10 (13.5) 1.33 (0.27–6.64) 0.730 N = sample size; OR = odds ratio; SNP = single nucleotide polymorphism; 95% CI = 95% confidence interval Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer 117 increased the risk of constipation in the third week of tramadol treatment. OPRM1 rs677830 polymor- phism also reduced the risk of neuropathic pain one year after the surgery. The presence of MIR23B rs1011784 polymorphism increased the risk of nau- sea in the fourth week of tramadol treatment and increased the risk of neuropathic pain one year after the surgery. Lastly, the presence of MIR107 rs2296616 polymorphism reduced the risk of nau- sea in the third week of treatment and increased the risk of chronic pain one year after the surgery. So far, many studies confirmed the impact of OPRM1 rs1799971 polymorphism on pain per- ception, opioid response, the presence of adverse effects, and susceptibility to alcohol or drug de- pendence, but the majority of studies of OPRM1 rs1799971 polymorphisms were conducted on Asian population. According to systematic review and metaanalysis, carriers of at least one polymor- phic OPRM1 rs1799971 allele have higher opioid dose requirements to manage postoperative pain.26 However, after adjustment for different ethnic groups and different opioids, this effect remained significant only for the Asian population and in the group receiving morphine. In the Caucasian population, the association had not been con- firmed. The different ethnic origin of patients is, according to the authors of the review, one of the TABLE 5. The impact of investigated polymorphisms on chronic and neuropathic pain (N = 101) SNP Genotype Chronic painN (%) OR (95% CI) P OR (95% CI)adj Padj OPRM1 rs1799971 AA 15 (20.3) Ref. Ref. AG+GG 6 (22.2) 1.12 (0.39–3.28) 0.831 1.2 (0.4–3.57) 0.739 OPRM1 rs677830 CC 14 (27.5) Ref. Ref. CT+TT 7 (14) 0.43 (0.16–1.18) 0.101 0.44 (0.16–1.21) 0.112 MIR23B rs1011784 CC 9 (15.3) Ref. Ref. CG 10 (32.3) 2.65 (0.94–7.45) 0.065 2.85 (0.99–8.19) 0.052 GG 2 (20) 1.39 (0.25–7.64) 0.706 1.68 (0.29–9.83) 0.563 CG+GG 12 (29.3) 2.3 (0.86–6.11) 0.095 2.58 (0.94–7.1) 0.067 MIR107 rs2296616 GG 0 (0) Ref. Ref. GA 9 (18.4) / 0.099* AA 12 (35.3) / 0.004* GA+AA 21 (25.3) / 0.021* SNP Genotype Neuropathic pain N (%) OR (95% CI) P OR (95% CI)adj Padj OPRM1 rs1799971 AA 19 (25.7) Ref. Ref. AG+GG 6 (22.2) 0.83 (0.29–2.36) 0.722 0.94 (0.32–2.75) 0.915 OPRM1 rs677830 CC 17 (33.3) Ref. Ref. CT+TT 8 (16) 0.38 (0.15–0.99) 0.047 0.4 (0.15–1.04) 0.060 MIR23B rs1011784 CC 11 (18.6) Ref. Ref. CG 12 (38.7) 2.76 (1.04–7.31) 0.042 3.25 (1.17–9.02) 0.023 GG 2 (20) 1.09 (0.2–5.87) 0.919 1.59 (0.27–9.24) 0.607 CG+GG 14 (34.1) 2.26 (0.9–5.68) 0.082 2.85 (1.07–7.59) 0.036 MIR107 rs2296616 GG 3 (16.7) Ref. Ref. GA 11 (22.4) 1.45 (0.35–5.93) 0.607 1.37 (0.33–5.69) 0.666 AA 11 (32.4) 2.39 (0.57–10.02) 0.233 2.34 (0.55–9.97) 0.249 GA+AA 22 (26.5) 1.8 (0.48–6.83) 0.386 1.73 (0.45–6.65) 0.424 adj = adjustment for tramadol dose, N = sample size, OR = odds ratio, SNP = single nucleotide polymorphism, 95% CI = 95% confidence interval; *calculated using Fisher’s exact test Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer118 main reasons for mentioned differences in opioid response among different populations, since MAF for polymorphic OPRM1 rs1799971 allele in the Caucasian population is lower compared to the Asian population, and thus limiting the recogni- tion of the recessive effect of the polymorphism. Besides, it is possible that other gene variants with an impact on MOR activity, that are in linkage disequilibrium with OPRM1 rs1799971 polymor- phism, are the reason for differences in opioid re- sponse.26 Other authors also state that ethnic ori- gin is an important cause for conflicting research results on the impact of OPRM1 rs1799971 poly- morphism on subjects’ response to opioid drugs.4 According to the 1000Genome project, MAF in the Asian population is between 39 and 42%, while MAF in the European population is 16%.27 In our group of patients, MAF for OPRM1 rs1799971 was 14.2%, which coincides with the expected fre- quency for Europeans. Another study conducted on the Caucasian population, investigating the impact of OPRM1 and COMT polymorphisms on postoperative acute and chronic pain could not confirm the impact of OPRM1 rs1799971 polymor- phism on any of these types of pain28, which is consistent with the results of our study. The com- bination of tramadol and paracetamol was more effective for the treatment of neuropathic pain in wild-type homozygotes compared to carriers of OPRM1 rs1799971 polymorphic allele in a group of the Asian patients with oxaliplatin-induced neuropathy.8 Furthermore, wild-type homozy- gotes had lower opioid dose requirements for the treatment of chronic pain compared to carriers of OPRM1 rs1799971 polymorphic allele in European patients.29 Adverse effects, especially nausea and gastro- intestinal events, were more common in a group of people with wild-type genotype compared to the carriers of polymorphic OPRM1 rs1799971 al- lele.7,30 A possible reason for this could be a loss of the N-glycosylation site of MOR in the carriers of polymorphic allele29, since the N-glycosylation site of MOR may be involved in the trafficking into the cell membrane, binding of ligands, and signal transduction.31 On the other hand, OPRM1 rs1799971 polymorphism could impact the expres- sion levels of MOR directly and consequently re- duce the risk of opioid toxicity.29 Contrary to ex- pectations, our results showed an increased risk of constipation in the carriers of the polymorphic OPRM1 rs1799971 allele. Since the expression of MOR is tissue-specific, the impact on constipation cannot be explained by the central action of opi- oids. Instead, we should investigate the expression patterns and mechanisms of action of MOR on the periphery. A study of the association between OPRM1 rs677830 polymorphism and postoperative pain treatment with morphine in the Caucasian popu- lation showed no significant influence of polymor- phism, although an impact of the haplotype of seven polymorphisms of the OPRM1 gene, includ- ing rs1799971 and rs677830, on morphine require- ment has been observed.12 Due to the presence of a stop codon that leads to reduced expression of MOR caused by OPRM1 rs677830 polymorphism32, it was expected that greater severity of acute pain will be reported from the carriers of at least one polymorphic OPRM1 rs677830 allele. As the great- er severity of acute pain is one of the main risk factors for the presence of persistent pain33, we expected increased risk of chronic or neuropathic pain in carriers of at least one polymorphic al- lele, yet our results showed no impact on severity of acute pain and even indicated on reduced risk of neuropathic pain in carriers of polymorphic rs677830 allele. A literature search showed no re- port on the impact of mentioned polymorphism on adverse effects after opioid pain treatment or the presence of chronic or neuropathic pain so far. A study conducted on the mouse and human neuronal cells showed that long-term morphine treatment cannot influence the transcription of OPRM1. On the other hand, it can lead to increased miR-23b levels, which can bind to mRNA for MOR1 and thus prevent the translation of mRNA to the receptor, which causes a drug tolerance.17,34 Tramadol and morphine have a similar molecular structure, thus there is a possibility that MOR ex- pression may be affected by tramadol in the same way. An altered sequence of miR-23b, caused by MIR23B rs1011784 polymorphism, could change its target sequence and therefore prevent binding of miR-23b to mRNA for MOR1. In that case, recep- tor activity would remain unchanged, but we were unable to confirm these assumptions. Similar to miR-23b, long-term morphine expo- sure led to up-regulated miR-107 levels, leading to decreased levels of MOR1A in human neuro- blastoma cells and striatum of morphine-tolerant mouse.15 It would be interesting to evaluate the possible impact of tramadol on miR-107 expression as well. In addition, even though MIR107 rs2296616 polymorphism does not lead to decreased tran- scription of DNA to pre-miRNA, it could affect the processing of pre-miR-107 to mature transcript and consequently levels of miR-107.35 As a result, it Radiol Oncol 2023; 57(1): 111-120. Vidic Z et al. / OPRM1 related genetic variability and postoperative pain treatment in breast cancer 119 could affect MOR expression, but to confirm this we would have to do the quantification of miR-107 and MOR levels. Despite the confirmed association between polymorphisms in miRNA genes and tramadol treatment outcome, it is not sure that these miRNA acts through the altered activity of MOR. miR-23b played a crucial role in neuropathic pain relief in a mouse model after spinal cord injury. When a higher level of pain was present, decreased expres- sion level of miR-23b was measured and therefore increased levels of its target gene NOX4, coding for NADPH oxidase 4, that contributes to reac- tive oxygen species formation. After infusion of ectopic miR-23b molecules, an expression level of NOX4 decreased, and the symptoms improved.36 From the results of the mentioned study, we can conclude that miR-23b can influence the percep- tion of pain through its influence on various genes, not only OPRM1. Additionally, miR-107 paralogue miR-103 that differs in only one nucleotide in 3’-end, therefore regulating overlapping target molecules15, includ- ing mRNA for MOR in mice and humans, was shown to be regulating pain perception in rats and was down-regulated in neuropathic animals. It is responsible for the altered expression level of three subunits of Cav1.2 L-type calcium channels that play a crucial role in the sensation in chronic neuropathic pain.37 It is reasonable to assume that the miR-107 molecule could have the same effect. If polymorphism in MIR107 alters the target se- quence of a mentioned molecule, it would conse- quently lead to the same effect as downregulation of the miR-107, therefore increased neuropathic pain. Yet our results only suggested the impact of miR-107 on chronic pain perception. Our study was one of the few studies inspecting the impact of polymorphisms of the OPRM1 gene on the tramadol treatment outcome in Caucasians. Nevertheless, it has some limitations such as a rela- tively small sample size of the patients and that the genotyping analysis was performed retrospective- ly. Since only a few studies investigated the impact of miRNA on the expression and functionality of MOR, it was a challenge to narrow the selection of miRNA and potentially functional polymor- phisms in genes coding for miRNA. Furthermore, we investigated the impact of polymorphisms on pain, which is always liable to subjective assess- ment, even though we used VAS and other stand- ardized questionnaires to limit this impact. On the other hand, the advantage of our study was an ethnically homogenous group of patients, as well as the unified treatment and follow-up protocol of patients. Overall, it was a longitudinal, prospec- tive, randomized double-blind clinical trial, where neither the healthcare professionals nor the pa- tients knew the dose level of tramadol. Conclusions In this study, we observed for the first time to our knowledge, that the presence of OPRM1 rs677830 polymorphism reduces the risk for the presence of neuropathic pain and increases the risk of con- stipation in response to tramadol pain treatment. Furthermore, we observed for the first time the association of investigated polymorphisms in miR-23b and miR-107 coding genes with tramadol treatment outcome. The results of our study are expanding the knowledge in the field of person- alized medicine that could lead to improved pain management and reduced risk of adverse effects, therefore improving the quality of patient’s life.38 Acknowledgments This work was financially supported by the Slovenian Research Agency (ARRS Grants No. P1- 0170 and P3-0289). References 1. Subedi M, Bajaj S, Kumar MS, Yc M. An overview of tramadol and its usage in pain management and future perspective. Biomed Pharmacother 2019; 111: 443-51. doi: 10.1016/j.biopha.2018.12.085 2. 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Pharmacogenomics 2010; 11: 843-64. doi: 10.2217/ pgs.10.47 Radiol Oncol 2023; 57(1): 121-126. doi: 10.2478/raon-2023-0010 121 research article Treatment of vulvar cancer recurrences with electrochemotherapy – a detailed analysis of possible causes for unsuccessful treatment Gregor Vivod1,2, Tanja Jesenko2,3, Gorana Gasljevic4, Nina Kovacevic1,2,5, Masa Bosnjak3,6, Gregor Sersa3,7; Sebastjan Merlo1,2,8, Maja Cemazar3,9 1 Department of Gynecological Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Medical Faculty Ljubljana, University of Ljubljana, Ljubljana, Slovenia 3 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Department Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Faculty of Health Care Angela Boškin, Jesenice, Slovenia 6 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 7 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 8 Medical Faculty, University of Maribor, Maribor, Slovenia 9 Faculty of Health Sciences, University of Primorska, Izola, Slovenia Radiol Oncol 2023; 57(1): 121-126. Received 21 December 2022 Accepted 11 January 2023 Correspondence to: Assist. Prof. Sebastjan Merlo, M.D., Department of Gynecological Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia. E-mail: smerlo@onko-i.si and Prof. Maja Čemažar, Ph.D., Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia. E-mail: mcemazar@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. Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Most studies have reported the safety and effectiveness of electrochemotherapy for palliative treatment of gynecological can- cers and mostly vulvar squamous cell carcinoma. Some tumors, however, fail to respond to electrochemotherapy. The biological features/determinants for the nonresponsiveness are not determined yet. Patient and methods. A recurrence of vulvar squamous cell carcinoma was treated by electrochemotherapy using intravenous administration of bleomycin. The treatment was performed by hexagonal electrodes according to stand- ard operating procedures. We analyzed the factors that could determine nonresponsiveness to electrochemotherapy. Results. Based on the presented case of nonresponsive vulvar recurrence to electrochemotherapy, we hypothesize that the vasculature of the tumors prior to treatment may predict the response to electrochemotherapy. The histo- logical analysis showed minimal presence of blood vessels in the tumor. Thus, low perfusion may reduce drug delivery and lead to a lower response rate because of the minor antitumor effectiveness of vascular disruption. In this case, no immune response in the tumor was elicited by electrochemotherapy. Conclusions. In this case, of nonresponsive vulvar recurrence treated by electrochemotherapy, we analyzed pos- sible factors that could predict treatment failure. Based on histological analysis, low vascularization of the tumor was observed, which hampered drug delivery and distribution and resulted in no vascular disrupting action of electro- chemotherapy. All these factors could contribute to ineffective treatment with electrochemotherapy. Key words: electrochemotherapy; bleomycin; vulvar cancer; recurrence Introduction Vulvar cancer is the fourth most common gyneco- logical cancer, with an incidence of 2.6 per 100,000 women per year.1 The treatment of vulvar cancer usually involves a combination of surgery and radiotherapy. Systemic treatment is rarely used. Most often, surgery includes radical vulvectomy Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer122 and bilateral lymph groin node dissection or sen- tinel lymph node biopsy.2 Radiotherapy can be used as adjuvant therapy after initial surgery or as part of primary therapy in locally advanced disease. Most recurrences of vulvar cancer occur locally near the surgical margins or in the con- tralateral lymph groin region. The therapeutic mo- dalities used depend on the location, the extent of recurrence and previously used radiotherapy or concomitant chemoradiotherapy.3 The emerging treatment modality for vulvar cancer recurrence is electrochemotherapy. Literature review of electrochemotherapy Electrochemotherapy is a local ablative therapy that uses the application of reversible electric pulses to the tumor to permeabilize the cell mem- brane, hence enabling the entry of cytotoxic drugs into the cells.4 It is most commonly used for the treatment of superficial tumors such as melanoma, sarcoma, squamous cell carcinoma, basal cell car- cinoma, skin metastases from breast cancer and others.5,6 It can also be utilized for the treatment of deep-seated tumors such as primary hepatocellu- lar carcinoma, colorectal cancer, unresectable colo- rectal liver metastases or pancreatic carcinoma.7-12 It is conducted following standard operating pro- cedures, and the method is now used in nearly 180 cancer centers around the world.6 Only a small number of papers describing the use of electrochemotherapy for the palliative treat- ment of gynecological cancers and mostly vulvar squamous cell carcinoma have been presented.13,14 Safety and local efficacy after electrochemothera- py with bleomycin in locoregional cutaneous re- currences of vulvar carcinomas previously treated with chemotherapy, radiotherapy and surgery or unsuitable for standard treatments have been demonstrated.15-19 The effectiveness of the clini- cal cases and studies is presented in Table 1. The success rate of such tumors is 80%, which is lower than the response rate of other skin tumors treated with electrochemotherapy.13,16,18,20-22 The biological predictors of unsuccessful treat- ment have already been reviewed.5 The clinical predictive factors of the tumor response were identified to be the size of the lesions and previ- ous treatment as well as the tumor type. However, the biology behind this process has still not been explored. Indicated were the intrinsic tumor sen- sitivity and tumor stroma, where the vasculariza- tion of the tumors might be the most important factor, in addition to the involvement of the im- mune response. The importance of the vasculature and vascular perfusion of tumors has already been shown to have an important role in the responsive- ness of tumors to electrochemotherapy due to its role in drug delivery and tumor response due to the vascular disrupting action of electrochemo- therapy.23 However, its importance in the response in clinical cases has not yet been discussed. With the aim of better understanding the pathophysiol- ogy of possible causes for unsuccessful treatment with electrochemotherapy in vulvar cancer recur- rences, we present a case report of a 75-year-old woman with vulvar cancer recurrence in whom treatment with electrochemotherapy was ineffec- tive and analyzed the possible causes of failure of such a treatment. A case of unresponsive tumor A 75-year-old woman was diagnosed with recur- rence of vulvar cancer in the clitoral region. At the age of 70, simple vulvectomy of the left la- bium major and sentinel node biopsy (SNB) were TABLE 1. Review of studies evaluating electrochemotherapy in vulvar cancer First author, year published Included no. of patients Average age Histology Response of vulvar cancer OR NR Perrone, 201320 8 84y 8 SCC 6/8 (75%) 2/8 (25%) Perrone, 201521 25 85y 25 SCC 20/25 (80%) 5/20 (20%) Pellegrino, 201618 10 68y 9 SCC1 Paget’s 6/10 (60%) 4/10 (40%) Perrone, 201913 55 79y 57 SCC 3 Paget’s 1 melanoma 46/55 (84%) 9/55 (16%) Corrado, 202016 15 83y 14 SCC 1 CS 12/15 (80%) 3/15 (20%) CS = carcinosarcoma; No = number; NR = no response; OR = objective response; SCC = squamous cell carcinoma; y = year(s) Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer 123 performed because of vulvar squamous cell car- cinoma. The sentinel inguinal nodes were nega- tive, and the tumor was removed with free surgi- cal margins. Five years after primary treatment, recurrence of vulvar squamous cell carcinoma in the clitoral region was diagnosed on regular fol- low up (Figure 1A). Inguinal and distal metasta- ses were excluded after clinical assessment and imaging diagnostics. The patient was presented at the Interinstitutional Tumor Board, which de- cided that electrochemotherapy is a safe and vi- able treatment approach before eventual surgical treatment. The tumor board comprised medical oncologists, radiotherapists, and gynecological on- cologists. The electrochemotherapy protocol was approved by the Institutional Medical Board and Slovenian National Ethical Committee (Number 0120-262/2021/3). Electrochemotherapy with bleo- mycin was performed according to the standard operating procedure.6 The patient underwent re- gional anesthesia. Bleomycin was administered at a dose of 15000 IU/m2 (Bleomycin medac, Medac GmbH, Germany). Eight minutes after intravenous administration of the drug, electric pulses were applied to the tumor in a way that covered all tu- mor nodule, including the safety margin of ~ 1 cm. Hexagonal geometry needle electrodes were used, and electric pulses were generated by Cliniporator (IGEA S.P.A., Italy). Altogether, 7 applications of electric pulses were delivered, and their delivery was verified on the screen of the generator (cur- rent > 1.5 A). The patient was discharged from the hospital the day after electrochemotherapy with no pain and no symptoms of any disturbance. At regular follow-ups one and two months after elec- trochemotherapy, we observed no clinical changes in the tumor (Figure 1B). Histologic analysis of repeated biopsy showed the presence of vulvar squamous cell carcinoma. Wide excision of the tumor was performed with free surgical margins. Eighteen months after vulvar cancer recurrence treatment, there were no visible signs of recur- rence or progression of the disease. Histological examination of the first excisional biopsy performed in 2016 showed well-differ- entiated squamous cell carcinoma arising in the background of differentiated vulvar intraepithe- lial neoplasm (VIN) (Figure 2A). The absence of high-risk HPV was proven by the negative immu- nohistochemical reaction to p16. Staining for p16 was negative in the invasive component as well as in precancerous lesions. Additionally, two sentinel lymph nodes were excised and found to be nega- tive. The patient again underwent an excisional biopsy in 2018 showing differentiated VIN and in 2021 after electrochemotherapy. Histological ex- amination of the post electrochemotherapy biopsy (Figure 2B) showed residual, well to moderately differentiated squamous cell carcinoma, measur- ing approximately 1.3 cm in the largest diameter, invading 0.4 cm in depth. There was no lympho- vascular or perineural invasion present. In the sur- rounding parenchyma, there were some throm- bosed and recanalized blood vessels. Additionally, an immunohistochemical panel consisting of anti CD3, CD20, CD68 PGM1, CD163, and ERG antibod- ies (markers for T and B lymphocytes, macrophag- es and blood vessels) was stained on the primary biopsy from 2016 and the post electrochemothera- py biopsy from 2021. Negligible number of cells on the primary as well as post-electrochemotherapy biopsy was stained positive for CD20, therefore we did not include these sections into analysis. For other markers, five different fields of immu- nohistochemically stained sections were captured with a DP72 CCD camera connected to a BX- 51microscope (Olympus,Hamburg,Germany) and analyzed with AxioVision program (Carl Zeiss, Jena, Germany) to determine the number of posi- tive regions per section. These were then averaged and t-test were performed to determine the sta- FIGURE 1. (A) Local recurrence of vulvar cancer. (B) No response to treatment two months after electrochemotherapy. FIGURE 2. Hematoxylin and eosin stained sections of primary (A) and post- electrochemotherapy (B) biopsy. Scale bar represents 50 μm. A A B B Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer124 A B C D FIGURE 3. Immunohistologically stained sections for lymphocytes CD3 (A), macrophages CD68 PMG1 (B) and CD163 (C), and blood vessels ERG (D), from primary biopsy (Primary Bx) and post-electrochemotherapy biopsy (post-ECT Bx) samples. Scale bar represents 50 μm. The number of positively stained regions ± standard error of the mean (SEM) is presented. Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer 125 tistical significance using GraphPad Prism 9 (La Jolla, CA, USA). Detailed analysis showed signifi- cant differences only in the amount of small blood vessels being more numerous in the first pretreat- ment biopsy in comparison to the posttreatment biopsy (Figure 3). On the other hand, no difference was found in the number of CD3-positive lympho- cytes or CD68-positive (pan macrophages marker) or CD163-positive M2 macrophages between the primary biopsy and post electrochemotherapy bi- opsy. Discussion Response to electrochemotherapy is evaluated in accordance with the modified Response Evaluation Criteria in Solid Tumors (RECIST).24 Complete re- sponse for squamous cell carcinoma is observed in 63% of cases, and objective response is observed in 80%.22 For vulvar cancer, stable disease and pro- gression of disease are observed in 16 to 40% of cases.14 There are many clinical factors contributing to predicting the response to treatment with electro- chemotherapy. The possibility for unsuccessful treatment increases with tumor size, and a marked drop in response rate occurs after chemotherapy or in previously irradiated tissue compared with nonirradiated tissue.22 There are, although, differences in the response rate of different tumor histologies; i.e., melanoma was the most resistant, and basal cell carcinoma was the most sensitive to electrochemotherapy.22 The underlying biological factors have not yet been fully explored. As indicated in the review5, stromal factors may play a significant role. Vasculature has already been shown in preclinical studies to play a crucial role in the perfusion of tumors and conse- quently in drug delivery to tumors.23 To overcome this obstacle in less perfused tumors, intratumoral drug delivery could be an approach to overcome insufficient drug delivery. The vascular component and its destruction by electrochemotherapy can be a significant factor in the tumor response. The vascular disrupting effect of electrochemotherapy is based on the apoptosis of endothelial cells in small vessels, where the abrogation of the blood flow induces hypoxia in tumors and consequently indirect tumor cell death. The bigger vessels, as demonstrated in a study on electrochemotherapy of normal liver in pigs, are not affected by elec- trochemotherapy. The histological analysis was performed 2 and 7 days after electrochemotherapy and no thrombosis or other clinically significant damage to large blood vessels and bile ducts in the liver was observed.25 Another biological factor may contribute to non-responsiveness, tumor cells in low oxygenated parts of tumors are more aggres- sive and are more resistant to therapy, leading also to higher recurrence rate of the treated tumors.26,27 Therefore, in less responsive tumors, lower vascu- larity and slow perfusion could be predictors of a lower response rate. In two clinical studies on elec- trochemotherapy of liver tumors, one on colorectal liver metastases and the other on hepatocellular carcinoma, the effect of vascularity was evident.7,8 A clear difference in the response rate of these two tumor types was observed, although the treat- ment was performed in the same way, even by the same team of experts. Based on the known tumor histology features, colorectal metastases are less perfused tumors than hepatocellular carcinoma. Based on this, it can be deduced that the vascular component is important factor contributing to the response of the tumors. Namely, the vasculature is important for drug delivery and distribution and the vascular disruptive component of electro- chemotherapy. Clinical cases of nonresponsive tumors to elec- trochemotherapy are lacking. This is the first de- tailed analyzed case of recurrent vulvar cancer that has not responded to electrochemotherapy. The analysis of the immune component of the tu- mor stroma showed no significant changes after electrochemotherapy, with lymphocytes present in the margin of the tumor, while macrophages were also distributed in the tumors; however, we can presume that their phenotype was M2, as there were no antitumor effects. In responsive tumors, at least some increase in immune cell infiltration would be expected since electrochemotherapy in- duces immunogenic cell death.28,29 On the other hand, the vascularization of the treated lesion was minimal, which may indicate a poor response to electrochemotherapy due to the minimal delivery of bleomycin after intravenous administration to the tumor site, lack of distribution around the tu- mor and absence of vascular disrupting action of electrochemotherapy. This case clearly indicates that we have to search for biological determinants of failure of electrochemotherapy. As already suggested, the explanations for the heterogeneity in tumor re- sponse may reside in the altered vasculature that occurs with tumor growth and the difference in cell susceptibility or aggressiveness in the hypoxic environment.5 Radiol Oncol 2023; 57(1): 121-126. Vivod G et al. / Electrochemotherapy in treatment of recurrent vulvar cancer126 In conclusion, we propose to analyze the tu- mor vasculature with pathohistological biopsy or ultrasound prior to electrochemotherapy. The investigation of tumor vasculature may allow us to predict the treatment response of vulvar cancer with electrochemotherapy, which will help to de- termine the best individual treatment option and may ultimately improve patient outcomes. Acknowledgement The authors acknowledge the financial support from the state budget by the Slovenian Research Agency, program no. P3-0003. References 1. United States Cancer Statistics (USCS). U.S. Cancer Statistics Data Visualizations Tool. CDC, Centers for Disease Control and prevention. May 24, 2022. [cited 2022 Aug 19]. Available at: https://www.cdc.gov/cancer/ uscs/dataviz/index.htm 2. Merlo S. Modern treatment of vulvar cancer. Radiol Oncol 2020; 54: 371-6. doi: 10.2478/raon-2020-0053 3. Salom EM, Penalver M. Recurrent vulvar cancer. Curr Treat Options Oncol 2002; 3: 143-53. doi: 10.1007/s11864-002-0060-x 4. Cemazar M, Sersa G. Recent advances in electrochemotherapy. Bioelectricity 2019; 1: 204-13. doi: 10.1089/bioe.2019.0028 5. Sersa G, Ursic K, Cemazar M, Heller R, Bosnjak M, Campana LG. Biological factors of the tumour response to electrochemotherapy: review of the evidence and a research roadmap. Eur J Surg Oncol 2021; 47: 1836-46. doi: 10.1016/j.ejso.2021.03.229 6. Gehl J, Sersa G, Matthiessen LW, Tobian M, Soden D, Occhini A, et al. Updated standard operating procedures for electrochemotherapy of cu- taneous tumours and skin metastases. Acta Oncol 2018; 57: 874-82. doi: 10.1080/0284186X.2018.1454602 7. Djokic M, Cemazar M, Bosnjak M, Dezman R, Badovinac D, Miklavcic D, et al. A prospective phase II study evaluating intraoperative electrochemotherapy of hepatocellular carcinoma. Cancers 2020; 12: E3778. doi: 10.3390/can- cers12123778 8. Edhemovic I, Brecelj E, Cemazar M, Boc N, Trotovsek B, Djokic M, et al. Intraoperative electrochemotherapy of colorectal liver metastases: a pro- spective phase II study. Eur J Surg Oncol 2020; 46: 1628-33. doi: 10.1016/j. ejso.2020.04.037 9. Casadei R, Ricci C, Ingaldi C, Alberici L, Di Marco M, Guido A, et al. Intraoperative electrochemotherapy in locally advanced pancreatic cancer: indications, techniques and results-a single-center experience. Updat Surg 2020; 72: 1089-96. doi: 10.1007/s13304-020-00782-x 10. Spallek H, Bischoff P, Zhou W, de Terlizzi F, Jakob F, Kovàcs A. Percutaneous electrochemotherapy in primary and secondary liver malignancies - local tumor control and impact on overall survival. Radiol Oncol 2022; 56: 102-10. doi: 10.2478/raon-2022-0003 11. Schipilliti FM, Onorato M, Arrivi G, Panebianco M, Lerinò D, Milano A, et al. Electrochemotherapy for solid tumors: literature review and presentation of a novel endoscopic approach. Radiol Oncol 2022; 56: 285-91. doi: 10.2478/ raon-2022-0022 12. Bosnjak M, Jesenko T, Markelc B, Cerovsek A, Sersa G, Cemazar M. Sunitinib potentiates the cytotoxic effect of electrochemotherapy in pancreatic car- cinoma cells. Radiol Oncol 2022; 56: 164-72. doi: 10.2478/raon-2022-0009 13. Perrone AM, Galuppi A, Pirovano C, Borghese G, Covarelli P, De Terlizi F, et al. Palliative electrochemotherapy in vulvar carcinoma: preliminary results of the ELECHTRA (Electrochemotherapy Vulvar Cancer) multicenter study. Cancers 2019; 11: E657. doi: 10.3390/cancers11050657 14. Tranoulis A, Georgiou D, Founta C, Mehra G, Sayasneh A, Nath R. Use of electrochemotherapy in women with vulvar cancer to improve quality-of- life in the palliative setting: a meta-analysis. Int J Gynecol Cancer 2020; 30: 107-14. doi: 10.1136/ijgc-2019-000868 15. Merlo S, Vivod G, Bebar S, Bosnjak M, Cemazar M, Srsa G, et al. Literature review and our experience with bleomycin-based electrochemotherapy for cutaneous vulvar metastases from endometrial cancer. Technol Cancer Res Treat 2021; 20: 15330338211010134. doi: 10.1177/15330338211010134 16. Corrado G, Cutillo G, Fragomeni SM, Bruno V, Tagliaferri L, Mancini E, et al. Palliative eletrochemotherapy in primary or recurrent vulvar cancer. Int J Gynecol Cancer 2020; 30: 927-31. doi: 10.1136/ijgc-2019-001178 17. Perrone AM, Galuppi A, Borghese G, Corti B, Ferioli M, Della Gatta AN, et al. Electrochemotherapy pre-treatment in primary squamous vulvar cancer. Our preliminary experience. J Surg Oncol 2018; 117: 1813-7. doi: 10.1002/ jso.25072 18. Pellegrino A, Damiani GR, Mangioni C, Stripoli D, Loverro G, Cappello A, et al. Outcomes of bleomycin-based electrochemotherapy in patients with repeated loco-regional recurrences of vulvar cancer. Acta Oncol 2016; 55: 619-24. doi: 10.3109/0284186X.2015.1117134 19. Vivod G, Kovacevic N, Čemažar M, et al. Electrochemotherapy as an alternative treatment option to pelvic exenteration for recurrent vulvar cancer of the perineum region. Technol Cancer Res Treat 2022; 21: 15330338221116488. doi: 10.1177/15330338221116489 20. Perrone AM, Galuppi A, Cima S, Pozatti F, Arcelli A, Cortesi A, et al. Electrochemotherapy can be used as palliative treatment in patients with repeated loco-regional recurrence of squamous vulvar cancer: a preliminary study. Gynecol Oncol 2013; 130: 550-3. doi: 10.1016/j.ygyno.2013.06.028 21. Perrone AM, Cima S, Pozzati F, Fraculli R, Cammelli S, Tesei M, et al. Palliative electro-chemotherapy in elderly patients with vulvar cancer: a phase II trial: Electro-chemotherapy in vulvar cancer. J Surg Oncol 2015; 112: 529-32. doi: 10.1002/jso.24036 22. Clover AJP, de Terlizzi F, Bertino G, Curatolo O, Odili J, Campana LG, et al. Electrochemotherapy in the treatment of cutaneous malignancy: Outcomes and subgroup analysis from the cumulative results from the pan-European International Network for Sharing Practice in Electrochemotherapy data- base for 2482 lesions in 987 patients (2008-2019). Eur J Cancer 2020; 138: 30-40. doi: 10.1016/j.ejca.2020.06.020 23. Groselj A, Kranjc S, Bosnjak M, Krzan M, Kosjek T, Prevc A, et al. Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy. Basic Clin Pharmacol Toxicol 2018; 123: 247-56. doi: 10.1111/bcpt.13012 24. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sergent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45: 228-47. doi: 10.1016/j.ejca.2008.10.026 25. Zmuc J, Gasljevic G, Sersa G, Edhemovic I, Boc N, Seliskar A, et al. Large liver blood vessels and bile ducts are not damaged by electrochemotherapy with bleomycin in pigs. Sci Rep 2019; 9: 1-11. doi: 10.1038/s41598-019-40395-y 26. Wei J, Hu M, Du H. Improving cancer immunotherapy: exploring and target- ing metabolism in hypoxia microenvironment. Front Immunol 2022; 13: 845923. doi: 10.3389/fimmu.2022.845923 27. Tannock IF, Gordon Steel G. Cell proliferation, drug distribution and thera- peutic effects in relation to the vascular system of solid tumours. Br J Cancer 2022 Dec 23. [Ahead of print]. doi: 10.1038/s41416-022-02109-6 28. Calvet CY, Mir LM. The promising alliance of anti-cancer electrochemo- therapy with immunotherapy. Cancer Metastasis Rev 2016; 35: 165-77. doi: 10.1007/s10555-016-9615-3 29. Ursic K, Kos S, Kamensek U, Cemazar M, Miceska S, Markelc B, et al. Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune sta- tus. J Control Release 2021; 332: 623-35. doi: 10.1016/j.jconrel.2021.03.009 Radiol Oncol 2023; 57(1): 127-139. doi: 10.2478/raon-2023-0012 127 research article CT-guided 125I brachytherapy for hepatocellular carcinoma in high-risk locations after transarterial chemoembolization combined with microwave ablation: a propensity score-matched study Zixiong Chen1, Xiaobo Fu1, Zhenkang Qiu2, Maoyuan Mu1, Weiwei Jiang1, Guisong Wang1, Zhihui Zhong1, Han Qi1, Fei Gao1 1 Department of Minimally Invasive & Interventional Radiology, Sun Yat-sen University Cancer Center and Sun Yat-sen University State Key Laboratory of Oncology in South China, and Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, China 2 Interventional Medical Center, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China Radiol Oncol 2023; 57(1): 127-139. Received 29 December 2022 Accepted 30 January 2023 Correspondence to: Han Qi, M.D., Ph.D., Department of Minimally Invasive & Interventional Radiology, Sun Yat-sen University Cancer Center and Sun Yat-sen University State Key Laboratory of Oncology in South China, and Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng East Road, Guangzhou 510060, Guangdong Province, China. E-mail: qihan@sysucc.org.cn and Fei Gao, M.D., Ph.D., Department of Minimally Invasive & Interventional Radiology, Sun Yat-sen University Cancer Center and Sun Yat-sen University State Key Laboratory of Oncology in South China, and Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng East Road, Guangzhou 510060, Guangdong Province, China. E-mail: gaof@sysucc.org.cn Disclosure: No potential conflicts of interest were disclosed. Zixiong Chen and Xiaobo Fu contributed equally to this work. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. This study aimed to evaluate the safety and efficacy of 125I brachytherapy combined with transarterial chemoembolization (TACE) and microwave ablation (MWA) for unresectable hepatocellular carcinoma (HCC) in high-risk locations. Patients and methods. After 1:2 propensity score matching (PSM), this retrospectively study analyzed 49 patients who underwent TACE +MWA+125I brachytherapy (group A) and 98 patients who only received TACE +MWA (group B). The evaluated outcomes were progression-free survival (PFS), overall survival (OS), and treatment complications. Cox proportional hazards regression analysis survival was used to compare the two groups. Results. The patients in group A showed a longer PFS than group B (7.9 vs. 3.3 months, P = 0.007). No significant differences were observed in median OS between the two groups (P = 0.928). The objective response rate (ORR), disease control rate of tumors in high-risk locations, and the ORR of intrahepatic tumors were 67.3%, 93.9%, and 51.0%, respectively, in group A, and 38.8%, 79.6% and 29.6%, respectively, in group B (P < 0.001, P = 0.025 and P = 0.011, respectively). TACE-MWA-125I (HR = 0.479, P < 0.001) was a significant favorable prognostic factor that affected PFS. The present of portal vein tumor thrombosis was an independent prognostic factor for PFS (HR = 1.625, P = 0.040). The Barcelona clinic liver cancer (BCLC) stage (BCLC C vs. B) was an independent factor affecting OS (HR = 1.941, P = 0.038). The incidence of complications was similar between the two groups, except that the incidence of abdominal pain was reduced in the group A (P = 0.007). Conclusions. TACE-MWA-125I resulted in longer PFS and better tumor control than did TACE-MWA in patients with unresectable hepatocellular carcinoma in high-risk locations. Key words: hepatocellular carcinoma; high-risk location; transarterial chemoembolization; microwave ablation; 125I brachytherapy Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma128 Introduction Hepatocellular carcinoma (HCC) ranks the third leading cause of cancer-related death worldwide.1,2 Up to 60% of patients with HCC are diagnosed at the intermediate to the advanced stages and po- tentially curative treatments are unapplicable.3,4 Previous studies have demonstrated the benefits of combination therapies in patients with unre- sectable HCC.5-8 The combined treatments of tran- scatheter arterial chemoembolization (TACE) and thermal ablation could induce extensive tumor necrosis and result in a significant survival ben- efit.5,9-14 Prior studies of combined therapies have primarily focused on the Barcelona clinic liver cancer (BCLC) 0/A stage patients as a radical treat- ment.4 For unresectable HCCs, a meta-analysis demonstrated that microwave ablation (MWA) outperformed radiofrequency ablation (RFA) in large neoplasms.15 Tumor location is an important factor for the procedural success of ablative techniques and TACE. For thermal ablation, firstly, tumors close to vital structures may increase the risk of bleed- ing and injuring of the adjacent organs.16 Secondly, the blood flow can dissipate the heat away from the ablated lesion due to the heat sink effect.17 Lesions adjacent to large intrahepatic vessels, he- patic capsules, or extrahepatic organs may lead to insufficient ablation. MWA exhibited better tumor control than RFA for subcapsular HCC within the Milan criteria18 and the small single periportal HCC.19 However, most patients with intermediate- advanced HCC require a large ablation volume and more ablation time.12 For TACE, adjacent to organs, or previous treatment was reported to pro- mote the formation of extrahepatic collateral arter- ies, leading to incomplete embolization.20 Overall, the efficacy of combined therapies and complica- tions related to the high-risk locations still need to be carefully considered. Brachytherapy with iodine-125 (125I) seeds im- plantation has been accepted as a useful method to achieve local control with low complication rates in prostate cancer, HCC, and some other solid tu- mors.21-24 In previous studies, the combination of 125I and RFA, as well as 125I and TACE was reported to be effective for the treatment of HCC in high- risk locations.25-28 Thus, we have conducted this retrospective study to assess the efficacy, safety, and prognostic factors of computer tomography3 guided 125I brachytherapy combined with TACE and MWA for HCC in high-risk locations. To re- duce the influence of confounding bias, propensi- ty score matching (PSM) was performed to assess survival outcomes.29 Patients and methods Patients This retrospective study was approved by the in- stitutional review board of Sun Yat-sen University Cancer Center that waived the need for written in- formed consent. The data of 1577 primary HCC pa- tients who had received TACE plus MWA as first- line therapeutic options between February 2015 to March 2021 at our center were retrospectively iden- tified. A total of 287 patients were identified by the following eligibility criteria: (a) Diagnosed with HCC according to the Guidelines of the European Association for the Study of the Liver or American Association for the Study of Liver Diseases3,30; (b) BCLC stage B or C patients who were not eligible for surgical resection or liver transplantation4; (c) presenting with HCC in high-risk locations; (d) TACE combined with sequential MWA was per- formed as the first-line therapy; (e) Patients had not previously undergone liver resection or liver transplantation. Totally 106 patients were excluded based on the following exclusion criteria: (a) HCC complicated with other malignancies; (b) MWA or 125I brachytherapy was not used to treat the tumors in high-risk locations; (c) Incomplete preoperative and postoperative clinical and radiographic data; (d) Child-Pugh class C or D disease and Eastern Cooperative Oncology Group (ECOG) perfor- mance status≥2 (Figure 1). Definition of high-risk locations Referring to the previous studies31,32, The high-risk locations were defined as: (1) Type 1: less than 5 mm from the large vessels (the first- or second- grade branches of the portal veins, hepatic veins, and bile ducts); (2) Type 2: less than 5mm from the hepatic capsule or extrahepatic organs (such as heart, thoracic/abdominal wall, diaphragm, gas- tric, intestinal, and gall bladder). If the tumor was closed to both structures, the nearest one was cho- sen to define a high-risk location. TACE MWA treatment protocol Before TACE-MWA treatment, all patients re- ceived a standardized pretreatment evaluation in- cluding history, laboratory, and imaging. All TACE and MWA were performed by five interventional Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma 129 radiologists with experience of more than 5 years. For TACE, a selective 5-F catheter (Yashiro type; Terumo Corporation) was introduced, and hepatic arterial angiography was performed to identify the tumor-feeding arteries. Then the tumor-feed- ing arteries were super-selective catheterized with a 2.7-F microcatheter (Renegade Hi Flo; Boston Scientific Corporation). The embolization emulsion was a mixture of 50 mg/m2 of lobaplatin (Hainan Changan International Pharmaceutical Co., Ltd.), 10–40 mg of pirarubicin (Shenzhen Main Luck Pharmaceuticals Inc.) diluted in iodized oil (Lipoid ultra-fluid, Guerbet). MWA was performed within two weeks after TACE. MWA was performed with a commercially available system (ECO-100; ECO Microwave Electronic Institute) under CT guid- ance. A suitable route for puncture and ablation was designed. According to the size, number and anatomic location of the tumors, physicians chose the number of needles, the power (40–80W), and corresponding time (5–20 min) of ablation as well as the adjustable position of needles to eliminate the residual tumor. All ablations were conducted under intravenous moderate sedation and local anesthesia. 125I seed implantation protocol After MWA, CT scanning was conducted imme- diately to assessed the ablation area and residual tumor adjacent to high-risk sites. Then, interstitial needles were inserted into the target zone under CT guidance. The number and distribution of particles were determined by the treatment plan- ning system (TPS) (BT-RSI, Beijing Atom and High Technique Inc.) to achieve a satisfactory dose dis- tribution. After finishing implantation, a repeated CT scan was performed to check for complications and transmitted to TPS for dose verification. A to- tal of 493 125I seeds (Yunke Pharmaceutical Limited Liability Company) were implanted by three ra- diologists with at least 5 years’ experience in 49 patients with an average of 12.1 ± 15.1 seeds per patient. Follow-up protocol and study outcomes In both groups, patients generally underwent con- trast-enhanced CT or MRI 4–6 weeks postopera- tively to evaluate initial tumor response. Patients were reviewed every 3 months during the first year and every 6 months thereafter. Repeated TACE, MWA, or 125I seeds implantation was performed according to the location and proportion of residu- al tumor. Intrahepatic tumor response was graded according to the modified Response Evaluation Criteria in Solid Tumors (mRECIST) by two radi- ologists independently and discrepancies of as- sessment results was resolved by discussions.33 Tumor response in the high-risk sites was assessed between baseline and best response according to a modified standard: the product of maximum per- pendicular diameters of the tumors in the high-risk sites were calculated and compared to the initial value.34 The primary study endpoint was progres- sion-free survival (PFS). The secondary endpoints included overall survival (OS), objective response rate [ORR], disease control rate (DCR) and safety. PFS was calculated from the date of the first ses- sion of MWA or MWA-125I for the tumor in high- risk site to the date of tumor progression, death, or last follow-up; OS was calculated from the same treatment to the date of death due to any cause or last follow-up. The ORR was defined as the sum of complete and partial response, whereas DCR was defined as ORR plus stable disease rate. The best overall response was categorized as the final re- sponse during the treatment. Adverse events were graded according to the Common Terminology Criteria for Adverse Events (Version 5.0). Propensity score matching analysis To decrease the selection bias between the two groups, propensity scores were computed by lo- gistic regression model for each patient using the following covariates: age (y), BCLC stage (B/C), maximum tumor diameter35, tumor number (1- 2/≥3), high-risk site (Type 1/2), portal vein tumor thrombosis (PVTT) (Y/N). A 1:2 propensity score FIGURE 1. Patient flow diagram. TACE = transarterial chemoembolization; MWA = microwave ablation Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma130 TABLE 1. Baseline patient characteristics before and after propensity score-matched Parameter Total (N = 181) Total cohort PSM cohort Group A Group B P value Group A Group B P valueN = 49 N = 132 N = 49 N = 98 Sex 0.37 0.751 Male 162 (89.5%) 46 (93.9%) 116 (87.9%) 46 (93.9%) 89 (90.8%) Female 19 (10.5%) 3 (6.12%) 16 (12.1%) 3 (6.12%) 9 (9.18%) Age (y) 56.0 [47.0;64.0] 56.0 [47.0;68.0] 55.5 [48.0;62.0] 0.425 56.0 [47.0;68.0] 55.0 [48.0;62.0] 0.468 BCLC 0.041* 1 B 72 (39.8%) 13 (26.5%) 59 (44.7%) 13 (26.5%) 26 (26.5%) C 109 (60.2%) 36 (73.5%) 73 (55.3%) 36 (73.5%) 72 (73.5%) Maximum tumor diameter, median (IQR), mm 29.0 [18.0;49.0] 36.0 [22.0;53.0] 26.5 [18.0;46.0] 0.039* 36.0 [22.0;53.0] 31.0 [18.0;57.8] 0.579 Tumor number 0.927 0.66 1-2 60 (33.1%) 17 (34.7%) 43 (32.6%) 17 (34.7%) 29 (29.6%) ≥ 3 121 (66.9%) 32 (65.3%) 89 (67.4%) 32 (65.3%) 69 (70.4%) PVTT 0.202 1 None 108 (59.7%) 25 (51.0%) 83 (62.9%) 25 (51.0%) 49 (50.0%) Yes 73 (40.3%) 24 (49.0%) 49 (37.1%) 24 (49.0%) 49 (50.0%) Distant metastasis 0.171 0.953 None 109 (60.2%) 25 (51.0%) 84 (63.6%) 25 (51.0%) 52 (53.1%) Yes 72 (39.8%) 24 (49.0%) 48 (36.4%) 24 (49.0%) 46 (46.9%) High risk location 0.045* 0.76 1 98 (54.2%) 33 (67.3%) 65 (49.2%) 33 (67.3%) 62 (63.3%) 2 83 (45.8%) 16 (32.7%) 67 (50.8%) 16 (32.7%) 36 (36.7%) TACE sessions 2.00 [1.00;3.00] 2.00 [1.00;2.00] 2.00 [1.00;3.00] 0.442 2.00 [1.00;2.00] 1.00 [1.00;3.00] 0.464 MWA sessions 1.00 [0.00;2.00] 1.00 [0.00;2.00] 1.00 [0.00;2.00] 0.162 1.00 [0.00;2.00] 1.00 [0.00;2.00] 0.31 Cause of liver disease: 0.09 0.181 Continued HCV/HBV 169 (93.4%) 43 (87.8%) 126 (95.5%) 43 (87.8%) 93 (94.9%) Other 12 (6.6%) 6 (12.2%) 6 (4.55%) 6 (12.2%) 5 (5.10%) ECOG score 0.189 0.669 0 126 (69.6%) 30 (61.2%) 96 (72.7%) 30 (61.2%) 65 (66.3%) 1 55 (30.4%) 19 (38.8%) 36 (27.3%) 19 (38.8%) 33 (33.7%) Child-pugh score 0.563 0.386 A 82 (45.3%) 46 (93.9%) 36 (27.3%) 46 (93.9%) 86 (87.8%) B 99 (54.7%) 3 (6.12%) 96 (72.7%) 3 (6.12%) 12 (12.2%) AFP, ng/mL 0.811 0.576 < 400 125 (69.1%) 35 (71.4%) 90 (68.2%) 35 (71.4%) 64 (65.3%) ≥ 400 56 (30.9%) 14 (28.6%) 42 (31.8%) 14 (28.6%) 34 (34.7%) PT (s) 12.2 [11.5;13.1] 12.0 [11.5;12.8] 12.3 [11.6;13.1] 0.167 12.0 [11.5;12.8] 12.2 [11.5;13.1] 0.331 ALB (g/L) 40.35 [36.2;43.9] 40.8 [36.7;44.1] 40.2 [36.0;43.9] 0.329 40.8 [36.7;44.1] 40.3 [36.1;44.1] 0.518 TBIL (mg/dL) 13.55 [10.1;19.32] 12.3 [8.73;18.2] 14.6 [10.4;20.4] 0.167 12.3 [8.73;18.2] 14.6 [10.1;20.2] 0.227 AFP = alpha fetoprotein; ALB = albumin; BCLC = Barcelona Clinic Liver Cancer; ECOG = Eastern Cooperative Oncology Group; IQR = interquartile range; MWA = microwave ablation; PSM = propensity score-matched; PVTT = portal vein tumor thrombosis; PT = prothrombin time; TACE = transcatheter arterial chemoembolization; TBIL = total bilirubin SI conversion factors: To convert albumin to grams per liter, multiply by 10.0; to convert bilirubin to micromoles per liter, multiply by 17.104; and to convert α-fetoprotein to micrograms per liter, multiply by 1.0. *p value ≤ 0.05 was considered to indicate statistical significance. Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma 131 matching was performed using the nearest-neigh- bor matching algorithm with an optimal caliper of 0.1 without replacement. Statistical analysis Categorical data are reported as counts and per- centages, and continuous data are reported as medians or interquartile ranges. The Chi-square test or Fisher’s exact test were used to analyze the differences in categorical variables, and the Mann- Whitney test was applied to the continuous vari- ables. PFS and OS curves were constructed by the Kaplan-Meier (KM) method and estimated by the log-rank test. Univariate was used to analyze prog- nostic factors for PFS and OS using a Cox propor- tional hazards model. Significant univariate factors were included in the multivariate models. All tests were two-tailed and P values less than 0.05 were considered to indicate a statistically significant dif- ference. All statistical analyses were performed by R statistical package version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Results Baseline characteristics Between February 2015 to March 2021, a total of 181 HCC patients with tumors in high-risk locations were enrolled in this study. Before PSM, there were significant differences between the two groups in the BCLC stage, maximum tumor diameter, and high-risk location. In the TACE-MWA group (group B), 59 (44.7%) and 73 (55.3%) patients were classified as BCLC stage B and C, respectively. The maximum tumor diameter was significantly smaller than that in the TACE +MWA+125I group (group A) (26.5 vs. 36.0 mm, P = 0.039). And there were more tumors in type 2 high-risk sites (50.8%) in group B. After PSM, all characteristics were balanced between the two groups. The baseline characteristics of the unweighted and weighted cohorts are outlined in Table 1. During the follow-up, patients in group A required significantly fewer sessions of TACE and MWA than that in group B (0.73 vs. 1.27, P = 0.048; 0.88 vs. 1.74, P = 0.013, respectively). Local tumor control In the PSM cohort, Tumor responses of the intra- hepatic tumor and high-risk locations tumor are shown in Table 2. For the tumor in high-risk loca- tions, the ORR and DCR were significantly higher in the group A (67.3% vs. 38.8%, P < 0.001; 93.9% vs. 79.6%, P = 0.025, respectively). Likewise, the ORR of intrahepatic tumors in the group A was higher than that in the TACE-MWA group (51.0% vs. 29.6%, P = 0.011). The DCR in the group A was slightly higher than that in the group B without a statistical difference (85.7% vs. 71.4%, P = 0.055) Progression-free survival and overall survival In the unweighted cohort, the median PFS time was 7.9 months (95CI%: 10.9–18.5 months) for pa- tients in the group A, and 3.7 months in the group B (95CI%: 7.1–11.4 months) (P = 0.021) (Figure 2A). In the weighted cohort, the median follow-up for the study population was 16.2 months (range, 1.0–80.4 months). At the time of censoring, median TABLE 2. Intrahepatic and high-risk locations tumor responses in the two groups after propensity score matching (PSM) Tumor response Intrahepatic Tumor Tumor in high-risk locations Group A (n = 49) Group B (n = 98) P value Group A (n = 49) Group B (n = 98) P value Complete response (CR) 4 6 5 9 Partial response (PR) 21 23 28 29 Stable disease (SD) 17 41 13 40 Progressive disease (PD) 7 28 3 20 Objective response rate (ORR) (%) 51.0% 29.6% 0.011†* 67.3% 38.8% < 0.001†* Disease control rate (DCR) (%) 85.7% 71.4% 0.055† 93.9% 79.6% 0.025†* † Pearson χ2 test was used * p value ≤ 0.05 was considered to indicate statistical significance. Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma132 PFS was 3.3 months (95%CI: 6.1–10.5 months) for patients in the group B, and 7.9 months (95%CI: 10.9–18.5 months) in the group A with significant difference (P = 0.007) (Figure 2B). Univariable anal- yses revealed that treatment method, maximum tumor diameter, high-risk location, PVTT, and AFP level were significantly correlated with PFS (P < 0.1). Based on these results, the multivariate analysis including all factors of univariate analysis was performed and it identified treatment method and PVTT as independent prognostic factors for PFS (Table 3) (P < 0.05) (Figure 3A). There was no significant difference in OS be- tween the two groups in both unweighted and weighted cohorts (Figure 2C, 2D). As shown in Supplementary Table 1, univariate analysis results showed that sex, BCLC stage, high-risk location, PVTT, total bilirubin, and Child-pugh class were significant (P < 0.1). Because of assumed collinear- ity between PVTT and BCLC stage, as well as the total bilirubin and Child-pugh class, PVTT and total bilirubin were deleted from the multivariate model. Results of the multivariate analysis demon- strated that sex and BCLC stage were prognostic A B C D FIGURE 2. Kaplan-Meier curves comparing progression-free survival (PFS) and overall survival overall survival (OS) from different groups. (A) Cumulative PFS between the unmatched A and B groups. (B) Cumulative PFS between the matched A and B groups. (C) Cumulative OS between the unmatched A and B groups. (D) Cumulative OS between the matched A and B groups. Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma 133 TABLE 3. Multivariate analyses of predictors of progression-free survival after treatment Risk Factor Univariate Multivariate HR 95%CI P value HR 95%CI P value Sex Female 1 Male 0.755 0.393–1.451 0.399 Age (y) < 60 1 ≥ 60 1.093 0.761–1.569 0.63 Maximum tumor diameter (mm) 1.006 1.000–1.013 0.071* 1.003 0.996-1.010 0.458 Tumor number 1-2 1 ≥ 3 1.104 0.915–1.332 0.302 BCLC stage B 1 C 1.395 0.929–2.095 0.109 High-risk location 1 1 2 0.665 0.456–0.970 0.034** 0.846 0.550-1.302 0.447 PVTT None Yes 1.615 1.124–2.319 0.010** 1.625 1.015-2.546 0.040** Distant metastasis None Yes 1.099 0.772–1.565 0.602 TACE sessions 1.062 0.964–1.170 0.223 MWA sessions 1.006 0.914–1.106 0.905 Treatment TACE+MWA 1 TACE+MWA+ 125I 0.527 0.357–0.778 0.002** 0.479 0.328-0.733 <0.001** Cause of liver disease Other 1 HBV/HCV 1.36 0.662–2.791 0.402 ECOG score 0 1 1 1.269 0.876–1.837 0.208 AFP ≤ 400 ng/mL 1 Continued > 400 ng/mL 1.72 1.164–2.542 0.006** 1.403 0.948-2.129 0.090 Prothrombin time (s) 0.942 0.827–1.073 0.367 Albumin (g/L) 0.987 0.951–1.024 0.477 Continued Total Bilirubin (μmol/L) 1.001 0.995–1.008 0.649 Child-pugh class A 1 B 1.216 0.654–2.263 0.536 AFP = alpha fetoprotein; ALB = albumin; BCLC = Barcelona Clinic Liver Cancer; ECOG = Eastern Cooperative Oncology Group; HR = hazard ratio; MWA = microwave ablation; PVTT = portal vein tumor thrombosis; PT = prothrombin time; TACE = transarterial chemoembolization; TBIL = total bilirubin *P value ≤ 0.1 in uni,variate were included in multivariate analysis, **P value ≤ 0.05 was considered to indicate statistical significance in multivariate analysis Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma134 factors for OS (P < 0.05). However, no significant difference was detected between male and female groups (P = 0.057). KM analysis showed signifi- cant difference between the BCLC B/C groups (P = 0.012) (Figure 3B, 3C). Subgroup analyses The patients without PVTT tended to get a better PFS in group B (P < 0.05) (Figure 4A). No significant difference was detected in group A between PVTT and non-PVTT patients (Figure 4B). Moreover, we observed significant differences between the two groups concerning PFS in both PVTT and non- PVTT patients (Figure 4C, 4D). For the 95 patients with tumors located in the high-risk site 1 (less than 5 mm from the large vessels or bile ducts), the mean PFS time was 11.4 months (95%CI: 7.9–14.9 months) in group A and 7.1 months (95%CI: 4.6–9.5 months) in group B (P < 0.01) (Figure 4e). Similarly, for the 52 patients with tumors located in the high- risk site 2 (less than 5 mm from the hepatic cap- sule or extrahepatic organs), the PFS time in group A was longer than that in group B (20.3 vs. 10.1 months, P = 0.02) (Figure 4F). Complications Adverse events for both treatment groups were listed in Table 4. During follow-up, there were no treatment-related deaths in either group and all of these patients were relieved after symptomatic treatment. The most common complication after treatment was abdominal pain in both groups and it was more frequent in the group B (P = 0.007). A slightly higher puncture hemorrhage rate was found in the group B (8.2%), without a significant difference (P = 0.274). One patient developed liver abscess after MWA in the group B. No displace- ment of 125I seeds or radiation-induced liver dis- ease was noted. All of these patients recovered with conservative treatment during the hospital stay. FIGURE 3. Kaplan-Meier curves comparing progression-free survival (PFS) and overall survival (OS) according to the statistically significant prognostic factors in the multivariate analysis after propensity score-match. (A) Cumulative PFS between portal vein tumor thrombosis (PVTT) and None-PVTT patients. (B). Cumulative OS between male and female. (C) Cumulative OS between Barcelona clinic liver cancer (BCLC) B and C stages patients. A B C Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma 135 FIGURE 4. Subgroup analyses revealed by Kaplan-Meier curves comparing progression-free survival (PFS). (A) Comparison of the PFS between portal vein tumor thrombosis (PVTT) and None-PVTT patients in group B. (B) Comparison of the PFS between PVTT and None-PVTT patients in group A. (C) Comparison of the PFS between group A and B in patients with PVTT. (D) Comparison of the PFS between group A and B in none-PVTT patients. (E) Comparison of the PFS between group A and B in high-risk location 1. (F) Comparison of the PFS between group A and B in high-risk location 2. A B C D E F Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma136 Discussion Our study aims to compare the effectiveness and safety outcome of TACE-MWA-125I with that of TACE-MWA in patients with tumors in high-risk locations. As far as we know, this is the first study addressing this topic. A 1:2 PSM was performed to adjust for a variety of covariates and potential confounders between the two groups. A peri-hepatic-vein location was a risk factor for the regional recurrence and a peri-portal-vein loca- tion was a potential high-risk factor for incomplete RFA in small HCCs.36 Based on that, the results of our study demonstrated that treatment of high- risk sites contributes to the local tumor control and PFS. The initial intrahepatic tumor DCR and ORR of 85.7% and 51.0%, respectively, in the group A, are slightly higher than the published outcomes of Peng et al., who demonstrated that patients with advanced recurrent HCC treated with TACE-RFA combined with sorafenib received DCR and ORR of 84% and 40.6%, respectively.7 The PFS yielded higher PFS than the previously TACE-MWA out- comes of Zhang et al. (median PFS 4.2 months).37 The results of the group B in our study were slight- ly lower than the results presented by Zhang et al. which may in part be due to the more advanced stage of HCC (BCLC C) patients included in our study. Considering the theoretical advantages of MWA in the controlling of high-risk site tumors, these results indicated the strengths of TACE- MWA-125I in the local tumor control. As a low-dose rate brachytherapy, X-rays and γ-rays emit from the 125I seeds could suppress the proliferation and in- duce apoptosis in tumor cells.38,39 Numerous stud- ies have demonstrated the value of brachytherapy in the locoregional therapies.21-24 Furthermore, the sy nergy between brachytherapy and TACE-MWA might further increase the therapeutic effect.40 The tumors neighboring large vessels, hepatic capsule, or extrahepatic organs, may increase the risk of sublethal temperatures and reversible injury due to the heat sink effect or the limited margin of ab- lation. But the increased vasodilation and vascu- lar permeability due to the hyperthermia would improve the oxygenation in the high-risk tumors. The cytotoxicity of 125I seeds radiation is primarily oxygen dependent, which might explain the syn- ergy and survival advantages.39 The previous studies for the high-risk location related to the thermal ablation have mainly fo- cused on the RFA in the small HCCs. The studies by Kang et al. suggested that neither perivascular nor subcapsular was a statistically significant risk factor for the OS outcomes.41,42 Since the 125I seeds implantation was only targeted at high-risk sites and extrahepatic metastasis occurred in 47.6% (n = 70) patients, there was no significant improvement in OS in our study. Interestingly, previous studies indicated that low dose irradiation could lead to an increase in CD8 + T cells and promote antitumor immunity.43,44 These findings suggest that further combination of systemic therapies such as immu- notherapy and targeted therapy may yield better survival benefit. From the Cox proportional hazards regression, we found that the presence of PVTT was an inde- pendent prognostic factor for PFS (P = 0.04). In the TABLE 4. Complications related to the procedure Adverse events Group A (N = 49) AE Grade Group B (N = 98) AE Grade P value 1 2 3-4 1 2 3-4 Fever 3 3 0 0 3 9 10 0 0.608‡ Nausea 5 3 2 0 5 9 11 0 0.852† Diarrhea 3 2 1 0 3 5 5 0 1‡ Pain required treatment 13 10 3 0 13 39 42 0 0.007†* Liver abscess 0 0 0 0 0 1 1 0 1‡ Puncture hemorrhage 1 1 0 0 1 5 8 0 0.274‡ Displacement of seeds 0 0 0 0 0 - - - - RILD 0 0 0 0 0 - - - - AE = adverse effects; RILD = radiation-induced liver disease † Pearson χ2 test was used; ‡ Continuity correction was used *p value ≤ 0.05 was considered to indicate statistical significance. Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma 137 subgroup analysis, we were delighted to find that the difference in PFS between the PVTT and non- PVTT patients in the group B was not detected in the group A, and the group A achieved better PFS in both PVTT and non-PVTT patients. 125I seeds implanted around the portal vein might play a role in the treatment of PVTT. Results from previ- ous studies have indicated the effectiveness of 125I seeds implantation for PVTT which might account for the survival advantages.45,46 Besides, the TACE- MWA-125I therapy achieved superior PFS in both high-risk location 1 and 2 patients, suggesting its potential broad applicability. In terms of safety, there were no treatment-re- lated deaths. Most adverse events were considered mild or moderate and easily managed. Our results showed that the combination with 125I did not in- crease the risk of complications, and the incidence of abdominal pain and Puncture hemorrhage was decreased during the follow-up period (P < 0.01, P = 0.274). This might be due to the 125I seeds implan- tation providing a sufficiently safe distance from the ablation boundary to the high-risk location, which could reduce the difficulty and risk of abla- tion. No brachytherapy-related complications such as displacement of seeds and radiation-induced liver disease were detected during follow-up. In addition, as a less invasive treatment, the utility of stereotactic body radiation therapy (SBRT) for the treatment of HCC in high-risk site compensates for the limitations of thermal ablation and the clini- cal effectiveness of SBRT plus TACE and AMW is worth further investigation.47 Nevertheless, there were several limitations in our study. Firstly, the present study was an obser- vational, retrospective, single-center study with inherent limitations. Secondly, the classification of high-risk locations was more meticulous and com- prehensive in previous studies.36,41,42 Considering the complex anatomical structure of inter- to advance-stage HCC included in this study, the classification stratified by peri-hepatic-vein, peri- portal-vein and subcapsular was hard to apply. We can only make a relatively rough classification by referring to the studies by Teratani et al. and Lin et al.31,32 Further comparison based on a more comprehensive classification might reveal more valuable information. Third, the gender distribu- tion in this study was unbalanced (Female 12/147), suggesting the impact of sex on OS might be am- plified in the multivariate analysis. larger prospec- tive randomized clinical trials are still needed. 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Hepatocellular carcinoma with portal vein tumor thrombus treated with transarterial chemoembo- lization and sorafenib vs. (125)Iodine implantation. Front Oncol 2021; 11: 806907. doi: 10.3389/fonc.2021.806907 Radiol Oncol 2023; 57(1): 127-139. Chen Z et al. / TACE-MWA-125I of unresectable hepatocellular carcinoma 139 46. Yang M, Fang Z, Yan Z, Luo J, Liu L, Zhang W, et al. Transarterial chemoembo- lisation (TACE) combined with endovascular implantation of an iodine-125 seed strand for the treatment of hepatocellular carcinoma with portal vein tumour thrombosis versus TACE alone: a two-arm, randomised clinical trial. J Cancer Res Clin Oncol 2014; 140: 211-9. doi: 10.1007/s00432-013-1568-0 47. Lee J, Shin IS, Yoon WS, Koom WS, Rim CH. Comparisons between radiofre- quency ablation and stereotactic body radiotherapy for liver malignancies: meta-analyses and a systematic review. Radiother Oncol 2020; 145: 63-70. doi: 10.1016/j.radonc.2019.12.004 Radiol Oncol 2023; 57(1): 140. doi: 10.2478/raon-2023-0011 140 erratum Similar complication rates for irreversible electroporation and thermal ablation in patients with hepatocellular tumors Niklas Verloh1, Isabel Jensch1, Lukas Lürken1, Michael Haimerl1, Marco Dollinger1, Philipp Renner2, Philipp Wiggermann3, Jens Martin Werner4, Florian Zeman5, Christian Stroszczynski1, Lukas Philipp Beyer1 1 Department of Radiology, University Hospital Regensburg, Germany 2 Department of Surgery, Robert-Bosch-Hospital, Stuttgart, Germany 3 Department of Radiology, Hospital Braunschweig, Germany 4 Department of Surgery, University Hospital Regensburg, Regensburg, Germany 5 Center for Clinical Trials, University Hospital Regensburg, Regensburg, Germany Radiol Oncol 2019; 53(1): 116-122. Received 2 November 2018 Accepted 22 January 2019 Correspondence to: Lukas Philipp Beyer, M.D., Department of Radiology, University Hospital Regensburg, 93053 Regensburg, Germany. Phone: +49 941 944 17464; E-mail: lukas@lukasbeyer.com Disclosure: No potential conflicts of interest were disclosed. doi: 10.2478/raon-2019-0011 Unfortunately co-author surename is spelled incorrectly (Lukas Lürken), the correct spelling is “Lukas Luerken”. Niklas Verloh1, Isabel Jensch1, Lukas Luerken1, Michael Haimerl1, Marco Dollinger1, Philipp Renner2, Philipp Wiggermann3, Jens Martin Werner4, Florian Zeman5, Christian Stroszczynski1, Lukas Philipp Beyer1 Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. I Radiol Oncol 2023; 57(1): 1-11. doi: 10.2478/raon-2023-0015 Verukozni karcinom ustne votline Krištofelc N, Zidar N, Strojan P Izhodišča. Verukozni karcinom je oblika ploščatoceličnega karcinoma s specifičnimi morfološkimi, cito- kinetičnimi in kliničnimi lastnostmi. Kljub majhni mitotski aktivnosti in počasni rasti se lahko širi v okolna tkiva, vendar ne metastazira v regionalne bezgavke in oddaljene organe. Najpogosteje vznikne v ustni votlini. Predstavljamo etiologijo, klinično sliko, diagnostiko in metode zdravljenja verukoznega karcinoma ustne votline ter izpostavljamo z njim povezane dileme. Zaključki. Verukozni karcinom ustne votline moramo zaradi manj agresivne narave in boljše napovedi poteka bolezni razlikovati od običajne oblike ploščatoceličnega karcinoma. Za pravilno diagnosticiranje je ključnega pomena tesno sodelovanje med zdravnikom klinikom in patologom. Priporočila o optimal- nem načinu zdravljenja temeljijo na opisih posameznih primerov in majhnih retrospektivnih raziskavah ter posledično niso enotna. Načrtovane multicentrične prospektivne raziskave bi lahko omogočile boljšo obravnavo bolnikov z verukoznim karcinomom ustne votline. Radiol Oncol 2023; 57(1): 12-19. doi: 10.2478/raon-2023-0013 Molekularno profiliranje redkega timoma z uporabo sekvenciranja naslednje generacije. Metaanaliza Kostić Perić J, Ćirković A, Srzentić Dražilov S, Samardžić N, Skodrić Trifunović V, Jovanović D, Pavlović S Izhodišča. Timomi spadajo med redke tumorje, ki vzniknejo iz epitelnega tkiva timusa. Razlikujemo več oblik timoma: A, AB, B1, B2, B3, timični karcinom in timični nevroendokrini timom. V metaanalizni raziskavi smo se osredotočili na timom z uporabo člankov, ki temeljijo na genomskem profiliranju bolezni z metodo sekvenciranja naslednje generacije (NGS). Materiali in metode. Izvedli smo sistematičen pregled in metaanalizo razširjenosti raziskav, ki so odkrile gene in različice, ki se pojavljajo v manj agresivnih oblikah timičnih epitelijskih tumorjev. Raziskave, ob- javljene pred 12. decembrom 2022, smo našli v zbirkah podatkov PubMed, Web of Science in SCOPUS. Dva pregledovalca sta preiskala baze podatkov in izbrala članke za končno analizo na podlagi natanč- no opredeljenih kriterijev za izključitev in vključitev. Rezultati. V kvalitativno in kvantitativno analizo smo na koncu vključili 12 objav. Trije geni, GTF2I, TP53 in HRAS, so se v opazovanih raziskavah pokazali kot pomembne za bolezen. Razmerja obetov (angl. odds ratio, OR) za vse tri ekstrahirane gene so bila: GTF2I (OR = 1,58; interval zaupanja [angl. confi- dence interval, CI 1,51, 1,66]; p < 0,00001), TP53 (OR = 1,36; CI [1,12, 1,65]; p < 0,002) in HRAS (OR = 1,02; CI [1,00, 1,04]; p < 0,001). Zaključki. Glede na rezultate, smo videli, da ima gen GTF2I pomembno prevalenco v kohorti opazo- vanih bolnikov s timomom. Prav tako smo pri analizi objavljenih člankov videli, da NGS kaže, da so geni GTF2I, TP53 in HRAS najpogosteje mutirani geni v timomu. Ti imajo patogene enonukleotidne različice in insercije/delecije, ki prispevajo k razvoju in napredovanju bolezni. Te različice bi lahko bile dragoceni biološki označevalci in tarčna mesta za zdravila, specifična za timom. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. II Radiol Oncol 2023; 57(1): 20-34. doi: 10.2478/raon-2023-0008 Multimodalno računalniškotomografsko slikanje prispeva k izboljšanju diagnostične natančnosti solitarnih pljučnih nodulov. Večinstitucionalna, prospektivna raziskava Yan G, Li H, Fan X, Deng J, Yan J, Qiao F, Yan G, Liu T, Chen J, Wang L, Yang Y, Li Y, Zhao L, Bhetuwal A, McClure MA, Li N, Peng C Izhodišča. Solitarni pljučni noduli so klinično ena najpogostejših nenormalnih najdb pri računalniški tomografiji (CT) prsnega koša. Namen večinstitucionalne prospektivne raziskave je bil raziskati vrednost preiskave CT brez kontrasta, CT s kontrastom, perfuzijskega CT-ja in preiskave CT-ja z dvojno energijo, ki jo uporabljamo za razlikovanje benignih in malignih solitarni pljučni nodulov. Bolniki in metode. Bolnike z 285 solitarnimi pljučnimi noduli smo pregledali s preiskavami: CT bez kon- trasta, CT s kotrastom, perfuzijski CT in CT z dvojno energijo. Razlike med benignimi in malignimi noduli smo ocenjevali s posamičnio preiskavo CT ter z različnimi kombinacijami CT-ja brez kontrasta in ostalimi tremi preiskavami. Kombinacije smo ozačili kot metode A, B in C in preiskave kombinirali tudi med seboj (metoda A + B, A + C , B + C in A + B + C). Ugotovitve smo primerjali z analizo karakteristik krivulje spreje- mnika (angl. receiver operating characteristic curve analysis). Rezultati. Multimodalno CT slikanje je pokazalo boljše rezultate (občutljivosti od 92,81 % do 97,60 %, specifičnosti od 74,58 % do 88,14 % in natančnost od 86,32 % do 93,68 %) kot tiste pri enomodalnem CT slikanju (občutljivosti od 83,23 % do 85,63 %, specifičnosti 63,56 % do 67,80 % in natančnosti od 75,09 % do 78,25 %, vse p < 0,05). Zaključki. Solitarni pljučni noduli, ovrednoteni z multimodalnim slikanjem CT, prispevajo k izboljšanju di- agnostične natančnosti razlikovanja med njihovo benignostjo in malignostjo. CT brez kontrasta pomaga poiskati in oceniti morfološke značilnosti nodulov; CT s kontrastom pomaga oceniti njihovo vaskularnost. Dodatno sta v pomoč pri izboljšanju diagnostične učinkovitosti perfuzijski CT z uporabo parametra pre- pustne površine in CT z dvojno energijo ter uporabo parametra normalizirane koncentracije joda v venski fazi. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. III Radiol Oncol 2023; 57(1): 35-41. doi: 10.2478/raon-2023-0005 Ultrazvočno diagnosticiranje tumorjev perifernih živcev. Skupina primerov Podnar S Izhodišča. Tumorji perifernih živcev so redki, a predstavljajo pomemben vzrok okvare perifernih živcev. Namen raziskave je bil predstaviti skupino zaporednih bolnikov s tumorji perifernih živcev, obravnavanih v avtorjevi ultrazvočni praksi. Bolniki in metode. Retrospektivno smo pregledali elektronske zdravstvene kartoteke bolnikov s tumorji perifernih živcev, ki smo jih izvedli v ultrazvočnem laboratoriju Inštituta za klinično nevrofiziologijo od febru- arja 2013 do maja 2020. Pri vseh bolnikih smo zbrali podatke o spolu, starosti, kliničnih značilnostih, lokaciji tumorjev perifernih živcev, elektrodiagnostiki in o ultrazvočnih ugotovitvah. Rezultati. V analiziranem obdobju smo pregledali 2845 pacientov. Med temi smo prepoznal 15 bolnikov (0,5 %) s tumorji perifernih živcev. Štirje izmed njih (3 s potrjeno nevrofibromatozo) so imeli več tumorjev perifernih živcev. Polovica bolnikov (53 %) je imela znake okvare perifernega živca, ostali pa tipno maso ali pa so navajali bolečine. Najpogosteje je bil prizadet ulnarni živec (36 %). Površine preseka tumorjev perifernih živcev so bile od 24 mm2 do 1250 mm2 (srednja vrednost 61 mm2). Na podlagi histološkega izvida smo pri 5 bolnikih in na podlagi ultrazvočnih preiskav pri preostalih bolnikih diagnosticirali švanom, skupaj pri 40 % bolnikov, nevrofibrom pri 27 % in perineuriom pri 27 % bolnikov. Zaključki. Kot v prejšnjih objavah so se tumorji perifernih živcev kazali z nevrološkimi simptomi, kot tipna masa ali z bolečino. V nasprotju z drugimi žariščnimi nevropatijami so zlasti živci s švanomom, kljub izraziti zadebelitvi, pogosto ohranjali dobro funkcijo. Dodajanje ultrazvoka v klinično prakso nam je omogočilo diagnosticiranje teh redkih lezij perifernih živcev, ki smo jih pred tem praviloma spregledali. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. IV Radiol Oncol 2023; 57(1): 42-50. doi: 10.2478/raon-2023-0007 Učinki dinamične ojačitve s kontrastnim sredstvom ob slikanju prostate zaradi raka v tranzicijski coni pri poročanju v slikovnem in podatkovnem sistemu diagnostike prostate (PI- RADS), različica 2.1. Zhang J, Xu L, Zhang G, Zhang X, Bai X, Sun H, Jin Z Izhodišča. Namen raziskave je bil analizirati učinke dinamične ojačitve s kontrastnim sredstvom v tranzi- cijski coni prostate pri raku prostate in klinično pomembnem raku prostate v sistemu PI-RADS, različica 2.1. Bolniki in metode. Diagnostično učinkovitost različnih kombinacij preiskav (T2 poudarjeno slikanje [T2WI] + difuzijsko poudarjeno slikanje [DWI]; T2WI + dinamično ojačano s kontrastnim sredstvom slikanje [DCE]; in T2WI + DWI + DCE) pri raku prostate v tranzicijski coni ter pri klinično pomembnem raku prostate v tranzicijski coni smo primerjali z biopsijo prostate, ki je pomenila referenčni standard. Uporabili smo oceno ≥ 4, ki je predstavljala pozitivni prag. Rezultati. V raziskavi smo ovrednotili 425 vzorcev. 203 vzorcev je sodilo v skupino rak v tranzicijski co- ni prostate in 146 vzorcev v skupino klinično pomembni rak prostate v tranzicijski coni. Kombinacija 3 sekvenc je imela podobna področja pod krivuljo pri diagnosticiranju obeh rakov prostate (P > 0,05). Senzitivnost T2WI + DCE in T2WI + DWI + DCE (84,7 % in 85,7 % za rak prostate v tranzicijski coni; 88,4 % in 89,7 % za klinično pomemben rak v tranzicijski coni) pri diagnosticiranju obeh vrst rakov je bila značilno večja kot senzitivnost pri T2WI + DWI (79,3 % za rak prostate v tranzicijski coni; 82,9 % za klinično pomem- ben rak v isti coni). Specifičnost T2WI + DWI (86,5 % za rak v tranzicijski coni; 74,9 % za klinično pomemben rak v tranzicijski coni prostate) je bila značilno večja kot specifičnost pri T2WI + DCE in T2WI + DWI + DCE (68,0 % in 68,5 % za rak v tranzicijski coni; 59.1 % in 59.5 % za klinično pomemben rak v tranzicijski coni.), (vsi P < 0,05). Diagnostična učinkovitost T2WI + DCE in T2WI + DWI + DCE ni vsebovala značilnih razlik (P > 0.05). Zaključki. DCE lahko izboljša senzitivnost diagnoze za rak prostate v tranzicijski coni in klinično pomemb- ni rak prostate v tranzicijski coni ter je uporabna pri zaznavi majhnih rakavih lezij. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. V Radiol Oncol 2023; 57(1): 51-58. doi: 10.2478/raon-2023-0006 Presaditev otočkov trebušne slinavke v ekstracelularni matriks po ireverzibilni elektroporaciji jeter Zhang Y, Lv Y, Wang Y, Chang TT, Rubinsky B Izhodišča. Presaditev otočkov trebušne slinavke z infuzijo skozi portalno veno je postala uveljavljen kli- nični način zdravljenja bolnikov s sladkorno boleznijo tipa 1. Ker je učinkovitost presaditve majhna, iščejo nove pristope za vsaditev pankreatičnih otočkov. Cilj pričujoče raziskave je bil raziskati možnost, da bi decelularizirani matriks v jetrih, ki smo ga ustvarili z netermično ireverzibilno elektroporacijo (NTIRE), lahko uporabili kot mesto za presaditev pankreatičnih otočkov. Materiali in metode. Pankreatične otočke ali kontrolne vzorce s fiziološko raztopino smo 16 ur po obdelavi jeter z NTIRE injicirali na ista mesta v jetrih pri 7 podganah. Sedem dni po zdravljenju z NTIRE smo ocenili delovanje otočnih presadkov z zaznavanjem insulina in glukagona v jetrih, kar smo naredili z imunohistokemično preiskavo. Rezultati. Otočki trebušne slinavke, implantirani v volumen jeter, ki smo ga predhodno obdelali z NTIRE, so se vključili v jetrni parenhim ter proizvajali inzulin in glukagon v 2 od 7 jeter podgan. Možni razlogi za to, da pri preostalih 5/7 podgan nismo zaznali pankreatičnih otočkov, so lahko bila lokalna vnetna reakcija, zavrnitev presadka, majhno število začetnih otočkov ali pa čas implantacije. Zaključki. Raziskava kaže, da lahko otočke trebušne slinavke vgradimo in da delujejo v prostoru zu- najceličnega matriksa, ustvarjenega z NTIRE, čeprav je stopnja uspešnosti nizka. Nadaljnji razvoj na tem področju bi lahko dosegli z boljšim razumevanjem mehanizmov neuspeha in bi tako lahko razvili načine, kako zaobiti ali premagati te mehanizme. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. VI Radiol Oncol 2023; 57(1): 59-69. doi: 10.2478/raon-2023-0002 Ocena izpostavljenosti nizkofrekvenčnim magnetnim poljem v bližini visokonapetostnih daljnovodov in oceana tveganja za nastanek raka pri slovenskih otrocih in mladostnikih Žagar T, Valič B, Kotnik T, Korat S, Tomšič S, Zadnik V, Gajšek P Izhodišča. Posamične predhodne raziskave so pokazale, da bi povprečna dnevna izpostavljenost magnetnim poljem z izjemno nizko frekvenco (MP ENF) vrednosti nad 0,3 ali 0,4 μT lahko potencialno povečala tveganje za otroško levkemijo. Metode. Da bi omogočili izračune MP ENF okoli visokonapetostnih daljnovodov za celotno Slovenijo, smo razvili novo tri-dimenzionalno metodo za izračun dolgoročnega povprečja MP ENF, ki vključuje natančne podatke o reliefu terena. Iz populacijskega Registra raka smo pridobili podatke o populaciji slovenskih otrok in mladostnikov ter bolnikov z otroškim rakom (0–14 let), levkemijo (0–19 let) ter možgan- skimi tumorji (0–29 let) za 12-letno obdobje 2005–2016. Rezultati. V Sloveniji je le 0,5 % otrok in mladostnikov mlajših od 19 let živelo na območju z gostoto MP ENF nad 0,1 μT v bližini visokonapetostnih daljnovodov. Tveganje za raka pri otrocih in mladostnikih, ki so živeli na območjih z višjo MP ENF, se bistveno ni razlikovalo od tveganja njihovih vrstnikov. Zaključki. Nova metoda omogoča razmeroma hiter izračun vrednosti nizkofrekvenčnih magnetnih polj za poljubne obremenitve elektrodistribucijskega omrežja, saj vrednost posameznega vira za poljubno obremenitev izračunamo s skaliranjem vrednosti za nazivno obremenitev. To omogoča tudi bistveno hitrejše prilagajanje spremembam v elektrodistribucijskem omrežju. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. VII Radiol Oncol 2023; 57(1): 70-79. doi: 10.2478/raon-2023-0001 Primerjava mikrokroglic CalliSpheres®, ki sproščajo zdravilo, in običajne transarterijske kemoembolizacije pri bolnikih s primarnim rakom jeter. Randomizirana kontrolirana raziskava Shi Z, Wang D, Kang T, Yi R, Cui L, Jiang H Izhodišča. Namen raziskave je bil primerjati rezultate transarterijske kemoembolizacije z mikrokroglicami CalliSpheres®, ki sproščajo zdravilo (angl. drug-eluting beads transarterial chemoembolization, DEB- TACE) in običajne transarterijske kemoembolizacije (angl. conventional transarterial chemoemboliza- tion, cTACE) pri zdravljenju bolnikov z neoperabilnim hepatocelularnim rakom jeter. Bolniki in metode. Skupno 90 bolnikov smo razdelili v skupino DEB-TACE (n = 45) in skupino cTACE (n = 45). Obe skupini smo primerjali glede na odgovor na zdravljenje, celokupno preživetje, preživetje brez napredovanja bolezni in varnost zdravljenja. Rezultati. Objektivni odgovor na zdravljenje v skupini DEB-TACE je bil po 1, 3 in 6 mesecih spremljanja značilno večji kot v skupini cTACE (P = 0,031, P = 0,003, P = 0,002). Prav tako je bil popolni odgovor v skupini DEB-TACE po 3 mesecih pomembno večji kot v skupini cTACE (P = 0,036). Analiza preživetja je pokazala, da so bolniki v skupini DEB-TACE imeli boljše preživetje kot v skupini cTACE (srednja vrednost ce- lokupnega preživetja: 534 proti 367 dni, P = 0,027; srednja vrednost preživetje brez napredovanja bolezni: 352 proti 278 dni, P = 0,004). Stopnja okvare delovanja jeter je bila v skupini DEB-TACE višja v prvem tednu, vendar je bila v obeh skupinah podobna v prvem mesecu. DEB-TACE z mikrokroglicami CalliSpheres® je povzročila pogosteje povišane temperature in hude bolečine v trebuhu (P = 0,031, P = 0,037). Zaključki. Bolniki, ki smo jih zdravili z DEB-TACE z mikrokroglicami CalliSpheres® so imeli boljši odgovor na zdravljenje in boljše preživetje kot bolniki v skupini cTACE. Čeprav so se v skupini DEB-TACE pogosteje pojavili prehodne resne poškodbe jeter, povišane telesne temperature in hude bolečine v trebuhu, jih je bilo mogoče obvladati s simptomatskim zdravljenjem. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. VIII Radiol Oncol 2023; 57(1): 80-85. doi: 10.2478/raon-2022-0026 Ali sočasna ginekološka operacija vpliva na stopnjo okužb po mastektomiji in rekonstrukciji z vsadki? Pišlar N, Perić B, Ahčan U, Cencelj-Arnež R, Žgajnar J, Perhavec A Izhodišča. Pri bolnicah z rakom dojk, ki potrebujejo operacijo, je pogosto indicirana tudi ginekološka operacija. Namen pričujoče raziskave je bil primerjati stopnjo infekcijskih zapletov po mastektomiji in rekonstrukciji z vsadki pri bolnicah s sočasno ginekološko operacijo in brez nje. Bolnice in metode. Opravili smo retrospektivno analizo medicinske dokumentacije 159 zaporedno operiranih bolnic, pri katerih smo napravili mastektomijo in rekonstrukcijo z vsadki. Bolnice smo razdelili v dve skupini: 102 bolnici brez (1. skupina) in s sočasno ginekološko operacijo (2. skupina). Primerjali smo stopnjo okužb med skupinama s testom χ2. Za ugotavljanje povezanosti različnih dejavnikov s stopnjo okužb smo napravili logistično regresijo. Rezultati. Napravili smo 240 rekonstruktivnih operacij z vsadki. Srednja vrednost časa sledenja bolnicam je bila 297 dni (10–1061 dni). Srednja starost bolnic je bila 47,2 let (95 % interval zaupanja [CI] 32,8–65,9); 48,2 let (95 % CI 46,1–50,3) za 1. skupino in 45,8 let (95 % CI 43,2–48,3) za 2. skupino; p = 0,002). Stopnja okužb je bila 17,6 % (17,6 % proti 17,5 %, p = 0,987), odstranitev vsadka je bila potrebna v 5,7 % (4,9 % proti 7,0 %; p = 0.58). Debelost (indeks telesne mase > 30 kg/m2), starost in predhodna operacija dojke z obsevanjem so bili dejavniki tveganja za okužbo v univariatni analizi. Debelost (prilagojeno razmerje obetov [aOR] 3,319; 95 % CI 1,085–10,157; p = 0,036) in predhodna operacija dojke z obsevanjem (aOR 7,481; 95 % CI 2,230–25,101; p = 0,001) sta bila neodvisno povezana z okužbami v multivariatnem modelu. Zaključki. Sočasna ginekološka operacija pri bolnicah po mastektomiji in rekonstrukciji z vsadki ni po- večala tveganja za okužbo. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. IX Radiol Oncol 2023; 57(1): 86-94. doi: 10.2478/raon-2022-0048 Verifikacija lege z računalniško tomografijo s stožčastim snopom ter poravnavo na karino in hrbtenico kot nadomestek poravnave na tarčo pri obsevanju lokalno napredovalega pljučnega raka But-Hadžić J, Strljič K, Žager Marciuš V Izhodišča. Namen raziskave je bil oceniti natančnost volumetrične slikovno vodene radioterapije pljuč- nega raka s poravnavo na hrbtenico ali karino kot nadomestka poravnave na tumor, ker najboljši pristop še ni znan. Bolniki in metode. Slike računalniške tomografije s stožčastim snopom (angl. cone beam computed tomography, CBCT) iz 1., 10., 15. in 20. frakcije obsevanja pri 40 radikalno obsevanih bolnikih s pljučnim rakom smo retrospektivno poravnali s simulatorsko CT sliko in uporabili tri pristope. Analizirali in primerjali smo odstopanja nastavitev poravnave na hrbtenico in karino od referenčne nastavitve na tarčo (na tu- mor in na bezgavke) v lateralni, longitudinalni in vertikalni smeri. Preverili smo morebitni vpliv lege tumorja in stadija bezgavk na natančnost poravnave. Rezultati. Povprečno odstopanje nastavitev na hrbtenico in karino od referenčne nastavitve je bilo naj- večje v longitudialni smeri, z najboljšim ujemanjem v vertikalni in lateralni smeri. Obe strategiji poravnave sta bili natančnejši pri centralno ležečih tumorjih, ob tem je bila nastavitev na karino natančnejša v 50 % v lateralni smeri in 66 % v longitudinalni smeri povprečnih odstopanj. Pri vseh meritvah pri vseh bolnikih je pri- merjava nastavitev na karino in hrbtenico pokazala večjo natančnost karine v lateralni in longitudinalni smeri. V primerjalni analizi podskupin je bila karina v primerjavi s hrbtenico v lateralni in longitudinalni smeri boljša pri centralno ležečih tumorjih, N2 in N3. Obe strategiji sta bili primerljivi pri perifernih tumorjih in N0. Zaključki. Poravnava CBCT in simulatorskega CT na karino kaže večjo natančnost v primerjavi s hrb- tenico v lateralni in longitudialni smeri ter je superiorna pri centralno ležečih tumorjih ter stadiju N2 in N3. Hrbtenica in karina kot nadomestek poravnave na tarčo sta enako natančni za periferno ležeče tumorje in za stadij N0. Predlagamo uporabo karine kot standardnega nadomestka za tarčo pri poravnavi CBCT slikovno vodenega obsevanja pri lokalno napredovalem pljučnem raku. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. X Radiol Oncol 2023; 57(1): 95-102. doi: 10.2478/raon-2023-0004 Učinki implantacije zlatih fiducijskih označevalcev na kontrolo tumorja in toksičnost pri teleradioterapiji raka prostate Moll M, Weiß M, Stanisav V, Zaharie A, Goldner G Izhodišča. Utemeljitve, ki govorijo o vplivih fiducijskih označevalcev pri slikovno vodeni radioterapiji (angl. image-guided radiotherapy, IGRT) na kontrolo tumorja ter akutno in pozno toksičnost so redke. Bolniki in metode. V retrospektivno raziskavo smo vključili bolnike s primarnim rakom prostate z nizko in srednjo stopnjo tveganja, ki smo jih zdravili med leti 2010 in 2015. 40 bolnikov smo pripravili na obsevanje z in 21 bolnikov brez zlatih fiducijskih označevalcev. Pri odločitvi za ali proti implantaciji označevalcev smo upoštevali anesteziološko oceno in bolnikovo izbiro. IGRT smo izvedli z uporabo elektronskih portal- nih slikovnih naprav. Predpisana doza je bila 78 Gy, 2 Gy na frakcijo. Biokemična ponovitev bolezni brez morfološko dokazane bolezni smo opredelili s Phoenixovimi merili. Akutno in pozno gastrointestinalno in genitourinalno toksičnost smo ocenili z merili Onkološke skupine za radioterapijo (angl. Radiation Therapy Oncology Group). Rezultati. Zaradi kontraindikacij za anestezijo ni prejelo fiducijskih označevalcev 60 % bolnikov in zaradi osebne izbire 25 % bolnikov. Ko smo ocenjevali kontrolo tumorja, nismo ugotovili pomembnih razlik glede biokemične ponovitve bolezni ter celokupnega in za bolezen specifičnega preživetja (p = 0.61, p = 0.56 in p > 0.9999). Prav tako nismo ugotovili pomembnih razlik pri ocenjevanju akutnih in poznih gastrointesti- nalnih (p = 0,16 in 0,64) in genitourinalnih toksičnosti (p = 0,58 in 0,80). Zaključki. Nismo ugotovili statistično značilne prednosti po implantaciji zlatih fiducijskih označevalcev tako ne pri biokemični ponovitvi bolezni kot ne pri zgodnjih ali poznih gastrointestinalnih in genitourinalnih stranskih učinkih. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. XI Radiol Oncol 2023; 57(1): 103-110. doi: 10.2478/raon-2023-0014 Petletni rezultati analize KRAS, NRAS in BRAF ter vzorci zdravljenja v vsakdanji klinični praksi v Sloveniji pri zdravljenju prvega reda bolnikov z metastatskim rakom debelega črevesa nemutiranega tipa RAS. Podatki iz vsakodnevne klinične prakse Mesti T, Reberšek M, Ocvirk J Izhodišča. Izvedli smo neintervencijsko raziskavo IV. faze za oceno statusa KRAS, NRAS in BRAF pri bolni- kih z metastatskim rakom debelega črevesa in danke, ki so bili primerni za sistemsko zdravljenje prvega reda. Analizirali smo odločitve za zdravljenje prvega reda pri bolnikih z nemutiranim tipom RAS. Bolniki in metode. V raziskavo smo vključili bolnike s histološko potrjenim metastatskim rakom debele- ga črevesa in danke, ki so bili primerni za sistemsko zdravljenje prvega reda in so izpolnjevali vsa vključi- tvena merila. Analizo KRAS, NRAS in BRAF smo naredili iz vzorcev tkiva primarnega tumorja ali metastaz. Vsi vključeni bolniki so pisno privolili v sodelovanje v raziskavi. Rezultati. Od aprila 2013 do marca 2018 smo na Onkološkem inštitutu Ljubljana v raziskavo vključili 650 bolnikov. Med njimi je 637 bolnikov prejelo sistemsko zdravljenjem prvega reda glede na status RAS in BRAF. Porazdelitev bolnikov s tumorsko mutacijo KRAS in brez te mutacije je bila skoraj enaka (48,8 % oziroma 47,9 %), nadalje 89 % bolnikov je imelo nemutirane tumorje NRAS in 86,1 % nemutirane tumorje BRAF. Najpogosteje smo predpisali zdravljenje z bevacizumabom (53,1 %), bodisi v kombinaciji z dvema kemoterapevtikoma ali z enim. Zaviralca EGFR cetuksimab in panitumumab smo predpisali bolnikom, ki so imeli tumorje z nemutiranim RAS (30,9 %). Zaključki. Petletna analiza naše vsakodnevne klinične prakse v terciarni ustanovi, je pokazala, da je porazdelitev med tumorji nemutiranega in mutiranega tipa pri bolnikih z metastatskim rakom debelega črevesa in danke približno enaka kot v svetu. Slovenska populacija z metastatskim rakom debelega črevesa in danke ima tudi enako razmerje porazdelitve nemutiranih in mutiranih genov KRAS, NRAS in BRAF. Ugotovili smo, da dvotedenska čakalna doba za določitev tumorskih označevalcev ni vplivala na odločitev o načinu zdravljenja prvega reda. Tako so bile odločitve za zdravljenje v skladu s svetovnimi smernicami zdravljenja, ki temeljijo na z dokazi podprti medicini. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. XII Radiol Oncol 2023; 57(1): 111-120. doi: 10.2478/raon-2023-0003 Vpliv genetske variabilnosti OPRM1, MIR23B in MIR107 na akutno in kronično bolečino ter neželene učinke zdravljenja s tramadolom in paracetamolom po operaciji raka dojk Vidic Z, Goričar K, Stražišar B, Bešić N, Dolžan V Izhodišča. Tramadol je opioidni analgetik, ki ga pogosto uporabljamo za lajšanje bolečin po operaciji raka dojke. Protibolečinski učinek posreduje preko aktivacije opioidnega receptorja mu, ki ga kodira gen OPRM1. V raziskavi smo želeli preveriti povezavo med genetskimi spremembami gena OPRM1 in genov za njegove regulatorne molekule miRNA ter izidom zdravljenja s tramadolom po operaciji raka dojke z odstranitvijo pazdušnih bezgavk. Bolnice in metode. Raziskava je vključevala 113 bolnic po operaciji raka dojke z odstranitvijo paz- dušnih bezgavk, ki so v randomizirani klinični študiji KCT 04/2015-DORETAonko/si Onkološkega inštituta Ljubljana prejemale 75/650 mg ali 37,5/325 mg tramadola s paracetamoloma za lajšanje pooperativne bolečine. Z genotipizacijo s kompetitivno alelno specifično verižno reakcijo s polimerazo smo pri preisko- vankah preverili prisotnost polimorfizmov OPRM1 rs1799971, OPRM1 rs677830, MIR23B rs1011784 in MIR107 rs2296616 ter z logistično regresijo, Fisherjevim testom in Mann-Whitneyevim testom preverili povezavo teh genetskih sprememb z akutno in kronično bolečino ter neželenimi učinki zdravljenja s tramadolom. Rezultati. Pri nobenem izmed preiskovanih polimorfizmov nismo potrdili vpliva na stopnjo akutne bole- čine, ocenjene z analogno vizualno skalo (VAS), v prvih štirih tednih po operaciji (P > 0,05). Nosilke vsaj enega polimorfnega alela OPRM1 rs1799971 so imele v prvih štirih tednih po operaciji večje tveganje za pojav zaprtosti v primerjavi z nosilkami dveh normalnih alelov (razmerje obetov [RO] = 4,5; 95 % interval zaupanja [IZ] = 1,6–12,64; P = 0,004). Nosilke vsaj enega polimorfnega alela OPRM1 rs677830 so imele po treh tednih zdravljenja s tramadolom večje tveganje za pojav zaprtosti (RO = 3,11; 95 % IZ = 1,08–8,89; P = 0,035). Nosilke dveh polimorfnih alelov MIR23B rs1011784 so imele povečano tveganje za pojav sla- bosti po 28 dneh zdravljenja s tramadolom (RO = 7,35, 95 % IZ = 1,27–42,6, P = 0,026), medtem ko so imeli heterozigoti za MIR107 rs2296616 nižje tveganje za pojav slabosti po 21 dneh zdravljenja s tramadolom (RO = 0,21, 95 % IZ = 0,05–0,87, P = 0,031). Nosilke dveh polimorfnih alelov MIR107 rs2296616 so eno leto po operaciji pogosteje navajale kronične bolečine kot nosilke dveh normalnih alelov (P = 0,004). Nosilke vsaj enega polimorfnega alela MIR23B rs1011784 so ob prilagoditvi za višino odmerka pogosteje poročale o nevropatski bolečini (RO = 2,85, 95 % IZ = 1,07–7,59, P = 0,036), medtem ko so nosilke vsaj enega poli- morfnega alela OPRM1 rs677830 redkeje poročale o nevropatski bolečini v primerjavi z nosilkami dveh normalnih alelov (RO = 0,38, 95 % IZ = 0,15–0,99, P = 0,047). Zaključki. Genetske spremembe OPRM1 in genov, ki kodirajo miRNA s potencialnim vplivom na izraža- nje OPRM1, bi lahko bile povezane tako s pojavom neželenih učinkov zdravljenja s tramadolom/para- cetamolom kot tudi prisotnostjo kronične in nevropatske bolečine po operaciji raka dojke z odstranitvijo pazdušnih bezgavk. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. XIII Radiol Oncol 2023; 57(1): 121-126. doi: 10.2478/raon-2023-0010 Zdravljenje ponovitve raka zunanjega spolovila z elektrokemoterapijo. Podrobna analiza možnih vzrokov neučinkovitega zdravljenja Vivod G, Jesenko T, Gašljević G, Kovačevič N, Bošnjak M, Serša G, Merlo S, Čemažar M Izhodišča. Zdravljenje z elektrokemoterapijo je lokalno učinkovito pri bolnicah z rakom zunanjega spolovila. Dosedanje raziskave opisujejo varno in učinkovito uporabo elektrokemoterapije pri obravnavi ginekoloških rakov v paliativne namene, večinoma pri ženskah s ploščatoceličnim rakom zunanjega spolovila. V posameznih primerih pa je zdravljenje z elektrokemoterapijo neučinkovito. Biološke lastnosti, ki povzročajo neučinkovitost, še niso opredeljene. Bolniki in metode. Elektrokemoterapijo z intravenskim apliciranjem bleomicina smo uporabili pri po- novitvi ploščatoceličnega raka v predelu zunanjega spolovila. Zdravljenje smo izvedli po standardnih operativnih postopkih s heksagonalno elektrodo. Zanimalo nas je, katere značilke so določale neučinko- vitost elektrokemoterapije. Rezultati. Na podlagi predstavljenega primera neučinkovitega zdravljenja ponovitve raka zunanjega spolovila z elektrokemoterapijo domnevamo, da lahko analiza ožiljenosti tumorja pred zdravljenjem na- pove učinkovitost zdravljenja z elektrokemoterapijo. Histološka analiza je pokazala minimalno prisotnost krvnih žil v predelu tumorja. Slabša perfuzija zmanjša količino zdravila v tumorju, kar vodi v zmanjšano učinkovitost elektrokemoterapije. V predstavljenem primeru zdravljenje z elektrokemoterapijo ni povzro- čilo željenega imunskega odziva v tumorju. Zaključki. V predstavljenem primeru smo analizirali možne dejavnike, ki bi lahko napovedali neučin- kovitost zdravljenja ponovitve raka zunanjega spolovila z elektrokemoterapijo. Na podlagi histološke analize smo ugotovili slabšo ožiljenost tumorja, kar je zmanjšalo vnos in porazdelitev zdravila v tumorju ter povzročilo neučinkovitost elektrokemoterapije. Slovenian abstracts Radiol Oncol 2023; 57(1): I-XIV. XIV Radiol Oncol 2023; 57(1): 127-139. doi: 10.2478/raon-2023-0012 CT-vodena brahiterapija z 125J pri hepatocelularnem raku na mestih z visokim tveganjem, po transkarterijski kemoembolizaciji in v kombinaciji z mikrovalovno ablacijo. Primerjava dveh metod zdravljenja Chen Z, Fu X, Qiu Z, Mu M, Jiang W, Wang G, Zhong Z, Qi H, Gao F Izhodišča. Namen raziskave je bil oceniti varnost in učinkovitost brahiterapije z 125J v kombinaciji s transarterialno kemoembolizacijo (angl. transarterial chemoembolization, TACE) in mikrovalovno abla- cijo (angl. microwave ablation, MWA) pri neresektabilnem hepatocelularnem raku na mestih z visokim tveganjem. Bolniki in metode. Po primerjavi 1 : 2 smo v retrospektivni raziskavi analizirali 49 bolnikov, ki so prejeli TACE + MWA + brahiterapijo z 125J (skupina A) in 98 bolnikov, ki so prejeli samo TACE +MWA (skupina B). Ocenjevali smo preživetje brez napredovanja bolezni, celokupno preživetje in zaplete zdravljenja. Obe skupini smo primerjali s Coxovo regresijsko analizo proporcionalnih tveganj. Rezultati. Bolniki v skupini A so imeli daljše preživetje brez napredovanja bolezni kot v skupini B (7,9 proti 3,3 meseca, P = 0,007). Med obema skupinama nismo videli pomembnih razlik v srednjem celokupnem preživetju (P = 0,928). Stopnje objektivnega odgovora kontrole bolezni v primerih tumorjev na mestih z visokim tveganjem in stopnje objektivnega odgovora intrahepatičnih tumorjev so bile v skupini A 67,3 %, 93,9 % oziroma 51,0 %, v skupini B pa 38,8 %, 79,6 % in 29,6 % (P < 0,001, P = 0,025 oziroma P = 0,011). Kombinacija TACE-MWA-125J (razmerje obetov [angl. hazard ratio, HR] = 0,479, P < 0,001) je bila pomem- ben in ugoden prognostični dejavnik, ki je vplival na preživetje brez napredovanja bolezni. Tumorska tromboza v portalni veni je bila neodvisen prognostični dejavnik za preživetje brez napredovanja bolezni (HR = 1,625, P = 0,040). Barcelonski stadij jetrnega raka ([angl. Barcelona clinic liver cancer, BCLC] C proti B) je bil neodvisen dejavnik, ki je vplival na celokupno preživetje (HR = 1,941, P = 0,038). Incidenca zapletov je bila med obema skupinama podobna, le da je bila incidenca bolečine v trebuhu manjša v skupini A (P = 0,007). Zaključki. Kombinacija TACE-MWA-125J pri bolnikih z neoperabilnim hepatocelularnim rakom na mestih z visokim tveganjem je omogočila daljše preživetje brez napredovanja bolezni in boljši nadzor tumorja kot TACE-MWA. 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 predpi- sovanjem 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. 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-0166, 21.10.2022, 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 domnevnem neželenem učinku zdravila. Glejte poglavje 4.8, kako poročati o neželenih učinkih. IME ZDRAVILA: Verzenios 50 mg/100 mg/150 mg filmsko obložene tablete. KAKOVOSTNA IN KOLIČINSKA SESTAVA: Ena filmsko obložena tableta vsebuje 50 mg/100 mg/150 mg abemacikliba. Ena filmsko obložena tableta vsebuje 14 mg/28 mg/42 mg laktoze (v obliki monohidrata). Terapevtske indikacije: 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 faktorja 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 kombi- nirati z agonistom gonadoliberina (LHRH – Luteinizing Hormone–Releasing Hormone). Napredovali ali metastatski 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 fulvestrantom 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 zdravlje- nje 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 naslednji odmerek vzame ob predvi- denem 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 abe- macikliba nadaljevati ob natančnem spremljanju znakov toksičnosti. Alternativno je mogoče odmerek abemacikliba znižati na 50 mg enkrat na dan ali prekiniti dajanje abemacikliba. Če je uporaba zaviralca CYP3A4 prekinjena, je treba odmerek abemacikliba povečati na odmerek, kakršen je bil pred uvedbo zaviralca CYP3A4 (po 3–5 razpolovnih časih zaviralca CYP3A4). Prilagajanje odmerka glede na starost in pri bolnicah z blago ali zmerno ledvično okvaro ter z blago (Child Pugh A) ali zmerno (Child Pugh B) jetrno okvaro ni potrebno. Pri dajanju abemacikliba bolnicam s hudo ledvično okvaro sta potrebna previdnost in skrbno spremljanje 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 utemeljeno. Glede na stopnjo VTE bo morda treba spremeniti odmerek abemacikliba. Glede na povečanje vrednosti ALT ali AST je mogoče potrebna prilagoditev odmerka. Driska je najpogostejši neželeni učinek. Bolnice je treba ob prvem znaku tekočega blata začeti zdraviti z antidiaroiki, kot je lopera- mid, povečati vnos peroralnih tekočin in obvestiti zdravnika. Sočasni uporabi induktorjev CYP3A4 se je treba izogibati zaradi tveganja za zmanjšano učinkovitost abemacikliba. Bolnice z redkimi dednimi mot- njami, 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/pnevmo- nitis, 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: karbama- zepin, 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, levkopenija, 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-aminotransferaze. Pogosti: povečano solzenje, venska trombembolija, ILD/pnevmonitis, dispepsija, spremembe na nohtih, suha koža, mišična šibkost. Občasni: febrilna nevtropenija. Rok uporabnosti 3 leta. Posebna navodila za shranjevanje Za shranjevanje zdravila niso potrebna posebna navodila. Imetnik dovoljenja za promet z zdravilom: Eli Lilly Neder- land B.V., Papendorpseweg 83, 3528BJ, Utrecht, Nizozemska. Datum prve odobritve dovoljenja za promet: 27. september 2018 Datum zadnje revizije besedila: 1.4.2022 Režim izdaje: Rp/Spec - Predpisova- nje 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 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 SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA TAGRISSO 40 mg filmsko obložene tablete / TAGRISSO 80 mg filmsko obložene tablete SESTAVA: Ena filmsko obložena tableta vsebuje 40 ali 80 mg osimertiniba. INDIKACIJE: Zdravilo Tagrisso je kot monoterapija indicirano za: adjuvantno zdravljenje po popolni resekciji tumorja pri odraslih bolnikih z nedrobnoceličnim rakom pljuč v stadiju IB-IIIA (NSCLC - non-small cell lung cancer), pri katerem ima tumorski receptor za epidermalni rastni faktor (EGFR - epidermal growth factor receptor) delecije eksona 19 ali substitucije eksona 21 (L858R). • prvo linijo zdravljenja odraslih bolnikov z lokalno napredovalim ali metastatskim nedrobnoceličnim rakom pljuč (NSCLC – “non-small-cell lung cancer”), katerega receptor za epidermalni rastni faktor (EGFR – “epidermal growth factor receptor”) ima aktivirajoče mutacije. • zdravljenje odraslih bolnikov z lokalno napredovalim ali metastatskim NSCLC, pozitivnim za mutacijo T790M EGFR. ODMERJANJE IN NAČIN UPORABE: Zdravljenje z zdravilom Tagrisso mora uvesti zdravnik, ki ima izkušnje z zdravili za zdravljenje raka. Pri odločanju o uporabi zdravila Tagrisso je treba določiti stanje mutacije EGFR (v vzorcih tumorja pri adjuvantnem zdravljenju in v vzorcih tumorja ali plazme pri lokalno napredovalem ali metastatskem raku) z uporabo validirane testne metode. Odmerjanje: Priporočeni odmerek je 80 mg osimertiniba enkrat na dan. Bolniki na adjuvantnem zdravljenju morajo zdravilo jemati do ponovitve bolezni ali nesprejemljivih toksičnih učinkov. Zdravljenja, daljšega od 3 let, niso preučili. Bolniki z lokalno napredovalim ali metastatskim rakom pljuč morajo zdravilo jemati do napredovanja bolezni ali nesprejemljivih toksičnih učinkov. Če bolnik izpusti odmerek zdravila Tagrisso, ga mora vzeti, razen če je do naslednjega odmerka že manj kot 12 ur. Zdravilo Tagrisso je mogoče vzeti s hrano ali brez nje, vsak dan ob istem času. Prilagoditve odmerka: Glede na varnost in prenašanje pri posameznem bolniku je lahko potrebna prekinitev odmerjanja in/ali zmanjšanje odmerka. V primeru potrebe po zmanjšanju odmerka je treba odmerek zmanjšati na 40 mg enkrat na dan. Smernice za zmanjšanje odmerka v primeru neželenih učinkov/toksičnosti so navedene v preglednici v Povzetku glavnih značilnosti zdravila.To zdravilo je namenjeno za peroralno uporabo. Tableto je treba zaužiti celo z vodo in se je ne sme drobiti, lomiti ali gristi. KONTRAINDIKACIJE: Preobčutljivost na učinkovino ali katerokoli pomožno snov. Šentjanževke se ne sme uporabljati skupaj z zdravilom Tagrisso. OPOZORILA IN PREVIDNOSTNI UKREPI: Pri odločanju o uporabi zdravila TAGRISSO za adjuvantno zdravljenje bolnikov z NSCLC po popolni resekciji tumorja je pomembno določiti prisotnost mutacije EGFR (delecije eksona 19 (Ex19del) ali substitucije eksona 21 (L858R)). V kliničnem laboratoriju je treba opraviti validirano preiskavo tumorske DNK iz vzorca tkiva, pridobljenega z biopsijo ali kirurškim posegom. Pri odločanju o uporabi zdravila Tagrisso za zdravljenje lokalno napredovalega ali metastatskega NSCLC je pomembno določiti stanje mutacije T790M EGFR. Opraviti je treba validirano preiskavo tumorske DNK, dobljene iz vzorca tkiva, ali tumorske DNK v obtoku (ctDNA – circulating tumor DNA), dobljene iz vzorca plazme. Določitev prisotne mutacije EGFR (aktivirajoče mutacije EGFR pri prvi liniji zdravljenja ali mutacije T790M po napredovanju bolezni med zdravljenjem ali po zdravljenju z zaviralcem tirozin kinaze receptorja za epidermalni rastni faktor) v vzorcu tkiva ali plazme pomeni, da je bolnik primeren za zdravljenje z zdravilom TAGRISSO. A če je uporabljena preiskava za ctDNA iz plazme in je izvid negativen, je priporočljivo opraviti še preiskavo tkivnega vzorca, če je le mogoče. Pri preiskavah vzorcev plazme namreč obstaja možnost lažno negativnih rezultatov. Uporabiti se sme le robustne, zanesljive in občutljive preiskave z dokazano upo- rabnostjo za določanje stanja mutacije EGFR v tumorski DNK (iz vzorca tkiva ali plazme). O intersticijski bolezni pljuč (IBP) ali neželenih učinkih, podobnih IBP, so poročali pri 3,7 % od 1479 bolnikov, ki so v študijah ADAURA, FLAURA in AURA prejemali zdravilo Tagrisso. Pri zdravljenju lokalno napredovalega ali metastats- kega raka so poročali o petih smrtnih primerih. Pri adjuvantnem zdravljenju niso poročali o smrtnih primerih. Pojavnost IBP je bila pri bolnikih japonske etnične pripadnosti 10,49 %, pri bolnikih azijske etnične pripadnosti 1,6 % in pri neazijskih bolnikih 2,5 %. Vse bolnike z akutnim nastankom in/ali nepojasnjenim po- slabšanjem pljučnih simptomov (dispneja, kašelj, zvišana telesna temperatura) je treba skrbno pregledati, da bi izključili IBP. V času preiskovanja teh simptomov je treba zdravljenje s tem zdravilom prekiniti. Če je diagnosticirana IBP, je treba ukiniti zdravljenje z zdravilom Tagrisso in uvesti ustrezno zdravljenje, kot je potrebno. Ponovna uvedba zdravila Tagrisso pride v poštev le po skrbnem pretehtanju koristi in tveganj pri posameznem bolniku. Stevens-Johnsonov sindrom (SJS): V povezavi z zdravljenjem z zdravilom TAGRISSO so poročali o redkih primerih SJS. Pred uvedbo zdravljenja je treba bolnike seznaniti z znaki in simptomi SJS. Če se pojavijo znaki ali simptomi, ki nakazujejo SJS, je treba zdravljenje z zdravilom TAGRISSO nemudoma prekiniti ali končati. Podaljšanje intervala QTc: Bolnikom, zdravljenim z zdravilom Tagrisso, se pojavi podaljšanje intervala QTc. Takšno podaljšanje lahko poveča tveganje za ventrikularne tahiaritmije (npr. torsade de pointes) ali nenadno smrt. V študijah ADAURA, FLAURA ali AURA niso poročali o motnjah srčnega ritma. Uporabi osimertiniba se je treba pri bolnikih s prirojenim sindromom dolgega intervala QT izogniti, če je le mogoče. O rednih kontrolah elektrokardiograma (EKG) in elektrolitov je treba razmisliti pri bolnikih s kongestivnim srčnim popuščanjem, elektrolitskimi motnjami in prejemnikih zdravil, za katera je znano, da podaljšajo interval QTc. Prekinite uporabo pri bolnikih, ki se jim interval QTc podaljša preko 500 msec na vsaj 2 ločenih posnetkih EKG, in ga ne uporabljajte, dokler ni interval QTc manj kot 481 msec oziroma do njegove vrnitve na izhodiščno vrednost, če je izhodiščni interval QTc 481 msec ali več. Potem začnite zdravilo Tagrisso znova uporabljati v manjšem odmerku. Trajno ukinite zdravljenje z osimertinibom, če se bolniku pojavi podaljšanje intervala QTc v kombinaciji s čimer koli od naslednjega: torsade de pointes, polimorf- na ventrikularna tahikardija, znaki/simptomi resne motnje srčnega ritma. Spremembe v krčljivosti srca: V kliničnih preskušanjih se je zmanjšanje iztisnega deleža levega prekata (LVEF “left ventricular ejection fraction”) za ≥ 10 odstotnih točk ali na manj kot 50 % pojavilo pri 3,2 % (40/1233) bolnikov, zdravljenih z zdravilom TAGRISSO, ki so imeli LVEF izmerjen izhodiščno in vsaj še enkrat med obdobjem spremljanja. Pri bolnikih s srčnimi dejavniki tveganja in bolnikih s stanji, ki prizadenejo LVEF, je treba razmisliti o nadziranju delovanja srca, vključno z ocenjevanjem LVEF izhodiščno in med zdravljenjem. Pri bolnikih, ki se jim med zdravljenjem pojavijo pomembni srčni znaki ali simptomi, je treba razmisliti o nadziranju delovanja srca, vključno z ocenjevanjem LVEF. V s placebom nadzorovanem preskušanju adjuvantnega zdravljenja (ADAURA) se je zmanjšanje LVEF za ≥ 10 odstotnih točk ali na manj kot 50 % pojavilo pri 1,6 % (5/312) bolnikov, zdravljenih z zdravilom TAGRISSO, in 1,5 % (5/331) bolnikov, zdravljenih s placebom. Keratitis: O keratitisu so poročali pri 0,7 % bolnikov, zdravljenih z zdravilom Tagrisso v študijah ADAURA, FLAURA in AURA. Bolnike z znaki in simptomi, ki nakazujejo keratitis (na primer vnetje očesa, solzenje, občutljivost na svetlobo, zamegljen vid, bolečine v očesu in/ali pordelost očesa), je treba nemudoma napotiti k specialistu oftalmologu. Aplastična anemija: V povezavi z zdravljenjem z osimertinibom so poročali o redkih primerih aplastične anemije, vključno s smrtnimi primeri. Pred uvedbo zdravljenja je treba bolnike seznaniti z znaki in simptomi aplastične anemije, ki vključujejo, a niso omejeni le na dolgotrajno zvišano telesno temperaturo, podplutbe, krvavitve, bledico, okužbe in utrujenost. Če se pojavijo znaki in simptomi, ki kažejo na aplastično anemijo, je treba razmisliti o natančnem nadziranju bolnika ter o prekinitvi ali prenehanju zdravljenja z osimertinibom. Pri bolnikih s potrjeno aplastično anemijo je treba zdravljenje z osimertinibom ukiniti. Starost in telesna masa: Pri bolnikih starih nad 65 let ali bolnikih z telesno maso pod 50 kg je lahko prisotno povečano tveganje za pojav neželenih učinkov 3. ali višje stopnje. Pri teh bolnikih je priporočeno skrbno spremljanje. MEDSEBOJNO DELOVANJE Z DRUGIMI ZDRAVILI: Močni induktorji CYP3A lahko zmanjšajo izpostavljenost osimertinibu. Osimertinib lahko poveča izpostavljenost substratom BCRP in P-glikoproteina (P-gp). Študije in vitro so pokazale, da poteka presnova I. faze osimertiniba pretežno s CYP3A4 in CYP3A5. Podatki iz klinične farmakokinetične študije so pokazali, da ni verjetno, da bi zaviralci CYP3A4 vplivali na izpostavljenost osimertinibu. Dodatnih katalizacijskih encimov niso odkrili. Podatki klinične farmakokinetične študije o sočasni uporabi z rifampicinom kažejo, da se je sočasni uporabi močnih induktorjev CYP3A (npr. fenitoina, rifampicina, karbamazepina) in zdravila Tagrisso priporočljivo izogniti. Izpostavljenost osimertinibu lahko zmanjšajo tudi zmerni induktorji CYP3A4 (npr. bosentan, efavirenz, etravirin, modafinil), zato jih je treba uporabljati previdno oziroma se jim je treba izogniti, če je mogoče. Kliničnih podatkov, ki bi omogočali priporočilo za prilagoditev odmerka zdravila Tagrisso, ni na voljo. Sočasna uporaba šentjanževke je kontraindicirana. Glede na podatke klinične farmakokinetične študije je pri sočasni uporabi zdravila Tagrisso in rosuvastatina ter ostalih zdravil, katerih odstranjevanje je odvisno od BCRP in imajo ozek tera- pevtski indeks, treba bolnike skrbno spremljati glede znakov spremenjenega prenašanja zaradi večje izpostavljenosti sočasnemu zdravilu med prejemanjem zdravila Tagrisso. Tveganja za manjšo izpostavljenost hormonskim kontraceptivom ni mogoče izključiti. Bolnike, ki sočasno jemljejo zdravila, katerih odstranje- vanje je odvisno od P-gp in imajo ozek terapevtski indeks (npr. digoksin, dabigatran, in aliskiren), je treba skrbno spremljati glede znakov spremenjenega prenašanja zaradi večje izpostavljenosti sočasnemu zdravilu v času prejemanja zdravila Tagrisso. NEŽELENI UČINKI: Podatki iz treh randomiziranih študij lll. faze (ADAURA – adjuvantno zdravljenje, FLAURA – prva linija in AURA3 – le druga linija) in iz dveh študij z eno samo skupino (AURAex in AURA2 – druga linija ali več) in eni študiji l. faze (AURA1 – prva linija ali več) povzemajo izpostavljenost zdravilu Tagrisso pri 1479 bolnikih z nedrobnoceličnim rakom pljuč in pozitivno mutacijo EGFR. Večina neželenih učinkov je bila glede na resnost 1. ali 2. stopnje. Najpogostejši neželeni učinki zdravila so bili driska (47 %), izpuščaj (45 %), paronihija (33 %), suha koža (32 %) in stomatitis (24 %). V vseh študijah skupaj je bilo neželenih učinkov 3. stopnje 10 % in 4. stopnje 0,1 %. Med bolniki, ki so prejemali zdravilo Tagrisso 80 mg enkrat na dan, so zaradi neželenih učinkov odmerek zmanjšali 3,4 % bolnikom. Ukinitev uporabe zdravila zaradi neželenih učinkov je bilo 4,8 %. Zelo pogosti neželeni učinki: zmanjšan apetit, driska, stomatitis, izpuščaj, suha koža, paronihija, srbenje ter zmanjšano število levkocitov, limfocitov trombocitov in nevtrofilcev. Pogosti neželeni učniki: epistaksa, intersticijska bolezen pljuč, alopecija, urtikarija, sindrom palmarno-plantarne eritrodizestezije, zvišanje ravni kreatinina v krvi, zvišanje ravni kreatin fosfokinaze v krvi, zmanjšan iztisni delež levega prekata. VRSTA IN VSEBINA OVOJNINE: Al/Al perforirani pretisni omoti za enkratni odmerek. Škatle z 28 x 1 tableto (4 pretisni omoti). NAČIN IZDAJANJA ZDRAVILA: samo na recept DATUM REVIZIJE BESEDILA: 6.10.2022 (SI-2632) IMETNIK DOVOLJENJA ZA PROMET: AstraZeneca AB, S-151 85, Sodertalje, Švedska Zdravilo Tagrisso v Sloveniji še ni razvrščeno na listo zdravil za adjuvantno zdravljenje po popolni resekciji tumorja pri odraslih bolnikih z nedrobnoceličnim rakom pljuč v stadiju IB-IIIA (NSCLC - non-small cell lung cancer), pri katerem ima tumorski receptor za epidermalni rastni faktor (EGFR - epidermal growth factor receptor) delecije eksona 19 ali substitucije eksona 21 (L858R). 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, telefon +386 1 51 35 600. Reference: 1. Tsuboi M, Wu YL, Grohe C, et al. Osimertinib as adjuvant therapy in patients with resected EGFRm stage IB-IIIA NSCLC: updated results from ADAURA. Presented at: ESMO Congress 2022; September 9-13, 2022; Paris, France. Tagrisso Za adjuvantno zdravljenje po popolni resekciji tumorja pri odraslih bolnikih z EGFRm NSCLC Sa m o za st ro ko vn o ja vn os t. In fo rm ac ija p rip ra vl je na n ov em br a 20 22 . SI -2 47 2 Zdravilo Tagrisso je v adjuvantnem zdravljenju bolnikov v stadijih IB-IIIA NSCLC doseglo mediano preživetje brez ponovitve bolezni 65,8 mesecev (DFS)1 73% ROg - 0,27 (95% IZ: 0,21, 0,34) 1.0 0 0.8 0.6 0.4 0.2 0.0 6 12 18 726660544842363024 25 33 Meseci 343 339 288 316 73 48 139 84 0 0 5 4 201 115 249 137 270 162 278 181 289 205 307 230 TAGRISSO Placebo 90% 85% 73% 55% 44% 38% V er je tn os t pr ež iv et ja b re z po no vi tv e bo le zn i Odstotek bolnikov živih in brez ponovitve bolezni po 48 mesecih 48 - mesecev 80% VS 59% Stad IB: ROg=0,41 74% VS 42% Stad II: ROg=0,34 65% VS 14% Stad IIIA: ROg=0,20 Število bolnikov izpostavljenih tveganju TAGRISSO Placebo zmanjšano tveganje za smrt ali ponovitev bolezni * 65,8 mesecev pri bolnikih zdravljenih z zdravilom Tagrisso (95% IZ: 61,7.ND) v primerjavi z 28,1 mese- cev pri bolnikih zdravljenih s placebom (95% IZ: 22,1- 35,0). Zrelost podatkov je bila v času analize 45%. *Primarni izid učinkovitosti je bilo preživetje brez ponovitve bolezni (DFS - disease-free survival) po oceni raziskovalca v populaciji s stadijem II-IIIA. DFS po oceni raziskovalca v populaciji s stadijem IB-IIIA (celotna populacija) je bil sekundarni izid učinkovitosti. * DFS... preživetje brez ponovitve bolezni * DFS pri bolnikih v stadijih IB-IIIA Končna analiza DFS* Oglas TAGRISSO Adauara 48mes_210x280_JAN 2023.indd 1 13/02/2023 10:56 POSTAVLJA NOVE STANDARDE ZDRAVLJENJA RAZSEJANEGA HER2+ RAKA DOJK2 NEPRIMERLJIVO PREŽIVETJE* SEDAJ ODOBRENO PO VSAJ ENI PREDHODNI TERAPIJI NA PODLAGI ANTI-HER21 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: februar 2023. Samo za strokovno javnost. SI-2807 ENHERTU® je registrirana blagovna znamka družbe Daiichi Sankyo Company, Limited. © 2023 Daiichi Sankyo Company, Ltd. in AstraZeneca Ltd. 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 fl uorescenčne 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 nevtrofi lcev 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: S tarejši: Pri bolnikih, starih 65 let ali starejših, prilagajanje odmerka zdravila Enhertu ni potrebno. Podatki pri bolnikih, starih ≥ 75 let, so omejeni. Okvara ledvic: P rilagajanje odmerka pri bolnikih z blago (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: P ri 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 tomografi jo (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: P ri 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 (ehokardiografi ja 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 me- secev 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. P LODNOST, NOSEČNOST IN DOJENJE: N osečnost: D ajanje 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 po- tencial 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. P lodnost: 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 me- seca). 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, fl atulenca, 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, limfopenija, 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: 0,28; 95 % IZ: 0,22-0,37; p<0,000001, ključni opazovani dogodek raziskave: PFS glede na BICR;)1,2 ***po oceni raziskovalca je mediani PFS znašal 25,1 mesecev pri bolnikih, ki so prejemali ENHERTU, v primerjavi s 7,2 mesecev pri bolnikih zdravljenih s T-DM1 (HR: 0,26; 95 % IZ: 0,20, 0,35; sekundarni opazovani dogodek;)2 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 Zdravilo Enhertu v Sloveniji še ni razvrščeno na listo zdravil. 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 NEDROBNOCELIČNI RAK PLJUČ DROBNOCELIČNI RAK PLJUČ TROJNO NEGATIVNI RAK DOJK UROTELIJSKI KARCINOM HEPATOCELULARNI KARCINOM ZDRAVILO TECENTRIQ JE INDICIRANO ZA ZDRAVLJENJE RAZLIČNIH VRST RAKA: Skrajšan povzetek glavnih značilnosti zdravila Tecentriq M- SI- 00 00 07 83 (v1 .0) Da tu m pr ipr av e i nf orm ac ije : f eb ru ar 20 23 Ime zdravila: Tecentriq 840 mg/1200 mg koncentrat za raztopino za infundiranje. Kakovostna in količinska sestava: 840 mg: ena 14-ml viala s koncentratom vsebuje 840 mg atezolizumaba.1200 mg: ena 20-ml viala s koncentratom vsebuje 1200 mg atezolizumaba. Po redčenju je končna koncentracija razredčene raztopine med 3,2 mg/ml in 16,8 mg/ml. Atezolizumab je humanizirano monoklonsko protitelo IgG1 z inženirsko obdelano domeno Fc, ki je pridobljeno iz celic jajčnika kitajskega hrčka s tehnologijo rekombinantne DNA in deluje na ligand za programirano celično smrt 1 (PD-L1). Terapevtske indikacije: Urotelijski karcinom (UC): Zdravilo Tecentriq je kot monoterapija indicirano za zdravljenje odraslih bolnikov z lokalno napredovalim ali razsejanim UC, ki: so bili predhodno zdravljeni s kemoterapijo na osnovi platine ali niso primerni za zdravljenje s cisplatinom in katerih tumorji izražajo PD-L1 v ≥ 5 %. Zgodnji stadij nedrobnoceličnega raka pljuč (NDRP): Zdravilo Tecentriq je kot monoterapija indicirano za adjuvantno zdravljenje po popolni resekciji in kemoterapiji na osnovi platine za odrasle bolnike z NDRP in velikim tveganjem za ponovitev, katerih tumorji izražajo PD-L1 na ≥ 50 % tumorskih celic (TC) in nimajo EGFR mutiranega ali ALK pozitivnega NDRP. Razsejani NDRP: Zdravilo Tecentriq je v kombinaciji z bevacizumabom, paklitakselom in karboplatinom indicirano v prvi liniji zdravljenja odraslih bolnikov z razsejanim neploščatoceličnim NDRP. Pri bolnikih z EGFR mutiranim ali ALK pozitivnim NDRP je zdravilo Tecentriq v kombinaciji z bevacizumabom, paklitakselom in karboplatinom indicirano le, ko so izčrpana ustrezna tarčna zdravljenja. Zdravilo Tecentriq je v kombinaciji z nab-paklitakselom in karboplatinom indicirano kot prva linija zdravljenja odraslih bolnikov z razsejanim neploščatoceličnim NDRP, ki ni EGFR mutiran ali ALK pozitiven. Zdravilo Tecentriq je kot monoterapija indicirano v prvi liniji zdravljenja odraslih bolnikov z razsejanim NDRP, pri katerih je PD-L1 izražen na ≥ 50 % TC ali ≥ 10 % imunskih celic (IC), ki infiltrirajo tumor, ter nimajo EGFR mutiranega ali ALK pozitivnega NDRP. Zdravilo Tecentriq je kot monoterapija indicirano za zdravljenje odraslih bolnikov z lokalno napredovalim ali razsejanim NDRP, ki so bili predhodno zdravljeni s kemoterapijo. Bolniki z EGFR mutiranim ali ALK pozitivnim NDRP morajo pred uvedbo zdravila Tecentriq prejeti tudi tarčna zdravljenja. Drobnocelični rak pljuč (DRP): Zdravilo Tecentriq je v kombinaciji s karboplatinom in etopozidom indicirano kot prva linija zdravljenja odraslih bolnikov z razsejanim DRP. Trojno negativni rak dojk (TNRD): Zdravilo Tecentriq je v kombinaciji z nab-paklitakselom indicirano za zdravljenje odraslih bolnikov z inoperabilnim lokalno napredovalim ali razsejanim TNRD, katerih tumorji izražajo PD-L1 v ≥ 1 % in predhodno še niso prejemali kemoterapije zaradi razsejane bolezni. Hepatocelularni karcinom (HCC): Zdravilo Tecentriq je v kombinaciji z bevacizumabom indicirano za zdravljenje odraslih bolnikov z napredovalim ali neresektabilnim HCC, ki predhodno še niso prejemali sistemskega zdravljenja. Odmerjanje in način uporabe: Zdravilo Tecentriq morajo uvesti in nadzorovati zdravniki z izkušnjami pri zdravljenju raka. Odmerjanje: priporočeni odmerek zdravila Tecentriq je 840 mg, danega intravensko na dva tedna, ali 1200 mg, danega intravensko na tri tedne, ali 1680 mg, danega intravensko na štiri tedne, kot je navedeno v celotnem Povzetku glavnih značilnosti zdravila Tecentriq. Kadar zdravilo Tecentriq dajete v kombinaciji, glejte tudi celotne informacije za predpisovanje zdravil, ki se uporabljajo v kombinaciji. Prilagoditev odmerka med zdravljenjem: odmerkov zdravila Tecentriq ni priporočljivo zmanjševati. Zapoznitev odmerka ali prenehanje uporabe glede na neželeni učinek je opisano v SmPC. Način uporabe: zdravilo Tecentriq je namenjeno za intravensko uporabo. Infuzij se ne sme dajati kot hiter intravenski odmerek ali bolus. Začetni odmerek zdravila Tecentriq je treba dati v 60 minutah. Če bolnik prvo infuzijo dobro prenese, je mogoče vse nadaljnje infuzije dati v 30 minutah. Kontraindikacije: Preobčutljivost na atezolizumab ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Sledljivost: Za izboljšanje sledljivosti bioloških zdravil je treba lastniško ime in številko serije uporabljenega zdravila jasno zabeležiti v bolnikovi dokumentaciji. Imunsko pogojeni neželeni učinki: Večina imunsko pogojenih neželenih učinkov, ki so se pojavili med zdravljenjem z atezolizumabom, je bila po prekinitvi atezolizumaba in uvedbi kortikosteroidov in/ali podpornega zdravljenja reverzibilna. Opazili so imunsko pogojene neželene učinke, ki vplivajo na več kot en organski sistem. Imunsko pogojeni neželeni učinki, povezani z atezolizumabom, se lahko pojavijo po zadnjem odmerku atezolizumaba. Pri sumu na imunsko pogojene neželene učinke je treba opraviti temeljito oceno za potrditev etiologije oziroma izključitev drugih vzrokov. Glede na izrazitost neželenega učinka je treba uporabo atezolizumaba odložiti in uvesti kortikosteroide. Atezolizumab je treba trajno prenehati uporabljati pri vseh imunsko pogojenih neželenih učinkih 3. stopnje, ki se ponovijo, in pri vseh imunsko pogojenih neželenih učinkih 4. stopnje, z izjemo endokrinopatij, ki jih je mogoče nadzorovati z nadomestnimi hormoni. Bolnike je treba spremljati glede znakov in simptomov pnevmonitisa ter izključiti druge možne vzroke, razen imunsko pogojenega pnevmonitisa. Bolnike je treba spremljati glede znakov in simptomov hepatitisa. Vrednosti AST, ALT in bilirubina je treba spremljati pred začetkom zdravljenja z atezolizumabom, redno med zdravljenjem in kot je potrebno glede na klinično oceno. Bolnike je treba spremljati glede znakov in simptomov kolitisa in endokrinopatij, meningitisa ali encefalitisa. V primeru meningitisa ali encefalitisa je treba zdravljenje z atezolizumabom trajno ukiniti ne glede na njuno stopnjo. Bolnike je treba spremljati glede znakov in simptomov motorične in senzorične nevropatije. V primeru miastenijskega sindroma/miastenije gravis ali Guillain- Barréjevega sindroma je treba zdravljenje z atezolizumabom trajno prekiniti ne glede na njihovo stopnjo. Bolnike je treba nadzorovati glede znakov in simptomov, ki kažejo na akutni pankreatitis. Bolnike je treba nadzorovati glede znakov in simptomov, ki kažejo na miokarditis. Imunsko pogojeni nefritis: Bolnike je treba nadzorovati glede sprememb v delovanju ledvic. Bolnike je treba nadzorovati glede znakov in simptomov, ki kažejo na miozitis. Z infundiranjem povezane reakcije: pri zdravljenju z atezolizumabom so opažali z infundiranjem povezane reakcije. Pri bolnikih, ki imajo z infundiranjem povezane reakcije 1. ali 2. stopnje, je treba hitrost infundiranja zmanjšati ali zdravljenje prekiniti. Pri bolnikih, ki imajo z infundiranjem povezane reakcije 3. ali 4. stopnje, je treba zdravljenje z atezolizumabom trajno ukiniti. Bolniki, ki imajo z infundiranjem povezane reakcije 1. ali 2. stopnje, lahko še naprej prejemajo atezolizumab pod natančnim nadzorom; v poštev pride premedikacija z antipiretikom in antihistaminikom. Pri bolnikih, ki so prejemali atezolizumab, so poročali o imunsko pogojenih hudih kožnih neželenih učinkih, vključno s primeri Stevens-Johnsonovega sindroma (SJS) in toksične epidermalne nekrolize (TEN). Bolnike je treba spremljati glede sumov na hude kožne neželene učinke in izključiti druge vzroke. V primeru suma na hude kožne neželene učinke je treba bolnike napotiti k specialistu po nadaljnjo diagnozo in zdravljenje. Uporabo atezolizumaba je treba odložiti pri bolnikih s sumom na SJS ali TEN. Pri potrjenem SJS ali TEN je treba trajno prenehati z uporabo atezolizumaba. Kartica za bolnika: Zdravnik, ki predpiše zdravilo, se mora z bolnikom pogovoriti o tveganjih zdravljenja z zdravilom Tecentriq. Bolniku je treba dati kartico za bolnika in mu naročiti, naj jo ima vedno pri sebi. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Formalnih farmakokinetičnih študij medsebojnega delovanja z atezolizumabom niso izvedli. Ker se atezolizumab odstrani iz obtoka s katabolizmom, ni pričakovati presnovnih medsebojnih delovanj med zdravili. Uporabi sistemskih kortikosteroidov ali imunosupresivov se je pred uvedbo atezolizumaba treba izogibati, ker lahko vplivajo na farmakodinamično aktivnost in učinkovitost atezolizumaba. Vendar pa se sistemske kortikosteroide ali druge imunosupresive lahko uporabi po začetku zdravljenja z atezolizumabom za zdravljenje imunsko pogojenih neželenih učinkov. Neželeni učinki: Informacije o varnosti atezolizumaba v monoterapiji: najpogostejši neželeni učinki (> 10 %) so bili utrujenost, zmanjšan apetit, navzea, izpuščaj, zvišana telesna temperatura, kašelj, diareja, dispneja, artralgija, astenija, bolečina v hrbtu, bruhanje, okužba sečil in glavobol. Varnost atezolizumaba v kombinaciji z drugimi učinkovinami: najpogostejši neželeni učinki (≥ 20 %) so bili anemija, nevtropenija, navzea, utrujenost, alopecija, izpuščaj, diareja, trombocitopenija, zaprtost, zmanjšan apetit in periferna nevropatija. Poročanje o domnevnih neželenih učinkih: Poročanje o domnevnih neželenih učinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoča namreč stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da poročajo o katerem koli domnevnem neželenem učinku zdravila na: Javna agencija Republike Slovenije za zdravila in medicinske pripomočke, Sektor za farmakovigilanco, Nacionalni center za farmakovigilanco, Slovenčeva ulica 22, SI-1000 Ljubljana, Tel: +386 (0)8 2000 500, Faks: +386 (0)8 2000 510, e-pošta: h-farmakovigilanca@jazmp.si, spletna stran: www.jazmp.si. Za zagotavljanje sledljivosti zdravila je pomembno, da pri izpolnjevanju obrazca o domnevnih neželenih učinkih zdravila navedete številko serije biološkega zdravila. Režim izdaje zdravila: H. Imetnik dovoljenja za promet: Roche Registration GmbH, Emil-Barell-Strasse 1, 79639 Grenzach-Wyhlen, Nemčija. Verzija: 3.0/22 DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o., Stegne 13G, 1000 Ljubljana Samo za strokovno javnost. Vir: 1. Povzetek glavnih značilnosti zdravila Tecentriq je dosegljiv na povezavi: https://www.ema.europa.eu/en/documents/product-information/tecentriq-epar-product-information_sl.pdf Za adjuvantno zdravljenje po radikalni resekciji in kemoterapiji na osnovi platine za odrasle bolnike z NDRP in velikim tveganjem za ponovitev, katerih tumorji izražajo PD-L1 na ≥ 50 % tumorskih celic (TC) in nimajo EGFR mutiranega ali ALK pozitivnega NDRP. CILJ JE OZDRAVITEV. NDRP = nedrobnocelični rak pljuč 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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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 SOOČITE ALK+ mNSCLC Z ZDRAVILOM LORVIQUA Pri bolnikih z metastazami v CŽS ali brez njih BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA Lorviqua 25 mg, 100 mg fi lmsko obložene tablete Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o kateremkoli domnevnem neželenem učinku zdravila. Glejte poglavje 4.8 povzetka glavnih značilnosti zdravila, kako poročati o neželenih učinkih. Sestava in oblika zdravila: Ena fi lmsko obložena tableta vsebuje 25 mg ali 100 mg lorlatiniba in 1,58 mg oz. 4,20 mg laktoze monohidrata. Indikacije: Zdravljenje odraslih bolnikov z napredovalim nedrobnoceličnim rakom pljuč (NSCLC – Non-Small Cell Lung Cancer), ki je ALK (anaplastična limfomska kinaza) pozitiven in se predhodno niso zdravili z zaviralcem ALK, ter pri bolnikih, pri katerih je bolezen napredovala po: zdravljenju z alektinibom ali ceritinibom kot prvim ALK zaviralcem tirozin kinaze (TKI – Tyrosine Kinase Inhibitor) ali zdravljenju s krizotinibom in vsaj še 1 drugim ALK TKI. Odmerjanje in način uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. Odkrivanje ALK-pozitivnega NSCLC je potrebno pri izbiri bolnikov, saj so to edini bolniki, pri katerih so dokazali korist. Priporočeni odmerek je 100 mg peroralno enkrat na dan. Zdravljenje je treba nadaljevati do napredovanja bolezni ali nesprejemljive toksičnosti. Če bolnik izpusti odmerek, ga mora vzeti takoj, ko se spomni, razen če do naslednjega odmerka manjka manj kot 4 ure. Bolniki ne smejo vzeti 2 odmerkov hkrati, da bi nadomestili izpuščeni odmerek. Prilagajanje odmerkov: Ravni zmanjšanja odmerka: prvo zmanjšanje odmerka: 75 mg peroralno enkrat na dan; drugo zmanjšanje odmerka: 50 mg peroralno enkrat na dan. Zdravljenje je treba trajno prekiniti, če bolnik ne prenaša odmerka 50 mg peroralno enkrat na dan. Za prilagajanje odmerkov zaradi neželenih učinkov glejte preglednico 1 v SmPC-ju. Posebne populacije: Starejši bolniki (≥ 65 let): Zaradi omejenih podatkov priporočil o odmerjanju ni mogoče dati. Okvara ledvic: Prilagajanje odmerkov pri bolnikih z normalnim delovanjem in blago ali zmerno okvaro [absolutna ocena hitrosti glomerulne fi ltracije (eGFR – estimated Glomerular Filtration Rate): ≥  30  ml/min] ni potrebno. Pri bolnikih s hudo okvaro ledvic (absolutna vrednost eGFR  <  30  ml/min) je priporočljiv zmanjšan odmerek lorlatiniba, npr. začetni odmerek 75 mg peroralno enkrat na dan. Podatkov pri bolnikih na ledvični dializi ni na voljo. Okvara jeter: Pri bolnikih z blago okvaro ni potrebno prilagajanje odmerkov. Podatkov o uporabi pri zmerni ali hudi okvari ni, zato uporaba ni priporočljiva. Pediatrična populacija: Varnost in učinkovitost pri otrocih in mladostnikih, starih <  18  let, nista bili dokazani. Način uporabe: Peroralna uporaba, vsak dan ob približno istem času, s hrano ali brez nje. Tablete je treba pogoltniti cele. Kontraindikacije: Preobčutljivost na učinkovino ali katerokoli pomožno snov. Uporaba močnih induktorjev CYP3A4/5. Posebna opozorila in previdnostni ukrepi: Hiperlipidemija: Uporaba je povezana z zvečanji vrednosti holesterola in trigliceridov v serumu – morda bo treba uvesti ali povečati odmerek zdravil za zniževanje ravni lipidov. Učinki na osrednje živčevje: Opazili so učinke na osrednje živčevje, vključno s psihotičnimi učinki in spremembami v kognitivni funkciji, razpoloženju, duševnem stanju ali govoru – morda bo treba prilagoditi odmerek ali prekiniti zdravljenje. Atrioventrikularni blok: Pri bolnikih, ki so prejemali lorlatinib, so poročali o podaljšanju intervala PR in AV-bloku. Potrebno je spremljanje EKG in morda bo treba prilagoditi odmerek. Zmanjšanje iztisnega deleža levega prekata: Pri bolnikih, ki so prejemali lorlatinib in pri katerih so opravili izhodiščno in še vsaj eno nadaljnjo oceno iztisnega deleža levega prekata (LVEF – Left Ventricular Ejection Fraction), so poročali o zmanjšanju LVEF. Če imajo bolniki dejavnike tveganja za srce ali stanja, ki vplivajo na LVEF, ali se jim med zdravljenjem pojavijo pomembni srčni znaki/simptomi, je treba razmisliti o spremljanju srca, vključno z oceno LVEF. Zvečanje vrednosti lipaze in amilaze: Pri bolnikih, ki so prejemali lorlatinib, se je pojavilo zvečanje vrednosti lipaze in/ali amilaze. Zaradi sočasne hipertrigliceridemije in/ali morebitnega intrinzičnega mehanizma je treba upoštevati tveganje za pankreatitis. Bolnike je treba spremljati glede zvečanja vrednosti lipaze in amilaze. Intersticijska bolezen pljuč (ILD – Interstitial Lung Disease)/pnevmonitis: Pri uporabi lorlatiniba so se pojavili hudi ali življenjsko ogrožajoči pljučni neželeni učinki, skladni z ILD/pnevmonitisom. V se bolnike, pri katerih pride do poslabšanja respiratornih simptomov, ki kažejo na ILD/pnevmonitis, je treba takoj pregledati glede ILD/pnevmonitisa. Hipertenzija: Pri bolnikih, ki so prejemali loratinib, so poročali o hipertenziji. Pred uvedbo lorlatiniba mora biti krvni tlak pod nadzorom. Med zdravljenjem je treba krvni tlak preveriti po 2 tednih in nato najmanj enkrat na mesec ter glede na stopnjo resnosti zdravljenje prekiniti in nato nadaljevati z zmanjšanim odmerkom ali trajno prekiniti. Hiperglikemija: Pri bolnikih, ki so prejemali loratinib, se je pojavila hiperglikemija. Pred uvedbo je treba oceniti koncentracijo glukoze v serumu na tešče in jo nato redno spremljati v skladu z nacionalnimi smernicami ter glede na stopnjo resnosti zdravljenje prekiniti in nato nadaljevati z zmanjšanim odmerkom ali trajno prekiniti. 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: Učinek zdravil na lorlatinib: Induktorji CYP3A4/5: Sočasna uporaba močnih induktorjev CYP3A4/5 (npr. rifampicin, karbamazepin, enzalutamid, mitotan, fenitoin in šentjanževka) je kontraindicirana. Zaviralci CYP3A4/5: Sočasni uporabi močnih zaviralcev CYP3A4/5 (npr. boceprevir, kobicistat, itrakonazol, ketokonazol, posakonazol, troleandomicin, vorikonazol, ritonavir, paritaprevir v kombinaciji z ritonavirom in ombitasvirom in/ali dasabuvirom ter ritonavir v kombinaciji z elvitegravirom, indinavirom, lopinavirom ali tipranavirom in grenivka ali grenivkin sok), se je treba izogibati, saj lahko pride do zvečanja koncentracij lorlatiniba v plazmi (če je sočasna uporaba nujna, je priporočljivo zmanjšati odmerek lorlatiniba). Učinek lorlatiniba na druga zdravila: Substrati CYP3A4/5: Izogibati se je treba sočasnemu dajanju lorlatiniba in substratov CYP3A4/5 z ozkimi terapevtskimi indeksi (npr. alfentanil, ciklosporin, dihidroergotamin, ergotamin, fentanil, hormonski kontraceptivi, pimozid, kinidin, sirolimus in takrolimus), saj lahko lorlatinib zmanjša koncentracije teh zdravil. Substrati P-glikoproteina: Substrate P-gp, ki imajo ozke terapevtske indekse (npr. digoksin, dabigatraneteksilat), je treba v kombinaciji z lorlatinibom uporabljati previdno, saj obstaja verjetnost, da se koncentracija teh substratov v plazmi zmanjša. Študije in vitro s prenašalci zdravil, ki niso P-gp: Lorlatinib je treba v kombinaciji s substrati BCRP, OATP1B1, OATP1B3, OCT1, MATE1 in OAT3 uporabljati previdno, saj klinično pomembnih sprememb v plazemski izpostavljenosti teh substratov ni mogoče izključiti. Plodnost, nosečnost in dojenje: Ženskam v rodni dobi je treba svetovati, naj se med zdravljenjem z lorlatinibom izogibajo zanositvi in naj med zdravljenjem uporabljajo visoko učinkovito nehormonsko metodo kontracepcije, saj lahko lorlatinib povzroči, da hormonski kontraceptivi postanejo neučinkoviti. Učinkovito kontracepcijo je treba uporabljati še vsaj 35 dni po zaključku zdravljenja. Med zdravljenjem in še vsaj 14 tednov po zadnjem odmerku morajo bolniki, ki imajo partnerice v rodni dobi, uporabljati učinkovito kontracepcijo. Nosečnost: Študije na živalih so pokazale embriofetalno toksičnost, zato uporaba med nosečnostjo ali pri ženskah v rodni dobi, ki ne uporabljajo kontracepcije, ni priporočljiva. Dojenje: Med zdravljenjem in še 7  dni po zadnjem odmerku je treba prenehati z dojenjem. Plodnost: Zdravljenje lahko ogrozi plodnost pri moških. Vpliv na sposobnost vožnje in upravljanja strojev: Ima zmeren vpliv na sposobnost vožnje in upravljanja strojev. Potrebna je previdnost, saj se pri bolnikih lahko pojavijo učinki na osrednje živčevje. Neželeni učinki: Zelo pogosti: anemija, hiperholesterolemija, hipertrigliceridemija, učinki na razpoloženje, učinki na kognitivne funkcije, periferna nevropatija, glavobol, motnja vida, hipertenzija, diareja, navzea, zaprtje, izpuščaj, artralgija, mialgija, edem, utrujenost, zvečanje telesne mase, zvečanje vrednosti lipaze, zvečanje vrednosti amilaze. 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: Pfi zer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. Datum zadnje revizije besedila: 04.04.2022 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. ALK=¯anaplastična¯limfomska¯kinaza, CŽS = centralni živčni sistem, mNSCLC = (Metastatic Non-Small Cell Lung Cancer) metastatski nedrobnocelični rak pljuč, NSCLC = (Non-Small Cell Lung Cancer) nedrobnocelični rak pljuč,¯¯TKI=(Tyrosine¯Kinase¯Inhibitor) zaviralec¯tirozin¯kinaze. Pfi zer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J.F. Kennedy, L – 1855, Pfi zer, podružnica Ljubljana, Letališka cesta 29a, 1000 Ljubljana PP-LOR-SVN-0005 Datum priprave: april 2022. Samo za strokovno javnost. Literatura: 1. Povzetek glavnih značilnosti zdravila Lorviqua, 4.4.2022. Zdravilo LORVIQUA v monoterapiji je indicirano za zdravljenje odraslih bolnikov z napredovalim nedrobnoceličnim rakom pljuč (NSCLC), ki je ALK pozitiven, in se predhodno niso zdravili z zaviralcem ALK.1 Zdravilo LORVIQUA v monoterapiji je indicirano za zdravljenje odraslih bolnikov z napredovalim NSCLC, ki je ALK-pozitiven, pri katerih je bolezen napredovala po: • zdravljenju z alektinibom ali ceritinibom kot prvim ALK zaviralcem tirozin kinaze (TKI); ali • zdravljenju s krizotinibom in vsaj še 1 drugim ALK TKI.1 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 1 I P a g e s 1 -1 4 0 I M a r c h 2 0 2 3 march 2023 vol.57 no.1