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 4 I P a g e s 4 1 1 -5 5 0 I D e c e m b e r 2 0 2 3 december 2023 vol.57 no.4 Radiol Oncol 2023; 57(4): A. December 2023 Vol. 57 No. 4 Pages 411-550 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(4): 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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57(4): C. review 411 Imaging microvascular changes in nonocular oncological clinical applications by optical coherence tomography angiography: a literature review Rok Hren, Gregor Sersa, Urban Simoncic, Matija Milanic 419 Locoregional therapy combined with systemic therapy (LRT + ST) for unresectable and metastatic intrahepatic cholangiocarcinoma: a systematic review and meta-analysis Mengqi Zhang, Weiwei Qi, Xiaofei Qiu, Chunpeng Yu, Wensheng Qiu, Song Wang, Zhenkang Qiu 430 Retropharyngeal calcific tendinitis in the neurological emergency unit, report of three cases and review of the literature Tatjana Filipovic, Jernej Avsenik nuclear medicine 436 Quantitative SSTR-PET/CT for predicting response and survival outcomes in patients with nancreatic neuroendocrine tumors receiving CAPTEM Maria Ingenerf, Homeira Karim, Christoph Auernhammer, Matthias Zacherl, Vera Wenter, Michael Winkelmann, Jens Ricke, Frank Berger, Christine Schmid-Tannwald radiology 446 Breast-lesion assessment using amide proton transfer-weighted imaging and dynamic contrast-enhanced MR imaging Lulu Zhuang, Chun Lian, Zehao Wang, Ximin Zhang, Zhigang Wu, Rong Huang 455 Apparent diffusion coefficient measurements of bone marrow infiltration patterns in multiple myeloma for the assessment of tumor burden − a feasibility study Xing Xiong, Yuzhu Ma, Yao Dai, Chunhong Hu, Yu Zhang 465 Assessment of short-term effect of platelet-rich plasma treatment of tendinosis using texture analysis of ultrasound images Karlo Pintaric, Vladka Salapura, Ziga Snoj, Andrej Vovk, Mojca Bozic Mijovski, Jernej Vidmar clinical oncology 473 The association of genetic factors with serum calretinin levels in asbestos-related diseases Cita Zupanc, Alenka Franko, Danijela Strbac, Viljem Kovac, Vita Dolzan, Katja Goricar contents contents Radiol Oncol 2023; 57(4): D. 487 Correlation of t(14;18) translocation breakpoint site with clinical characteristics in follicular lymphoma Matej Panjan, Lucka Boltezar, Srdjan Novakovic, Ira Kokovic, Barbara Jezersek Novakovic 493 The prognostic significance of tumor-immune microenvironment in ascites of patients with high-grade serous carcinoma Simona Miceska, Erik Skof, Simon Bucek, Cvetka Grasic Kuhar, Gorana Gasljevic, Spela Smrkolj, Veronika Kloboves Prevodnik 507 Management of tumor volume changes during preoperative radiotherapy for extremity soft tissue sarcoma: a new strategy of adaptive radiotherapy Marion Geneau De Lamarliere, Amélie Lusque, Justine Attal Khalifa, Vincent Esteyrie, Christine Chevreau, Thibaud Valentin, Dimitri Gangloff, Thomas Meresse, Louis Courtot, Philippe Rochaix, Bérénice Boulet, Eliane Graulieres, Anne Ducassou 516 Dosimetric comparison of postoperative interstitial high-dose-rate brachytherapy and modern external beam radiotherapy modalities in tongue and floor of the mouth tumours in terms of doses to critical organs Örs Ferenczi, Tibor Major, Georgina Fröhlich, Dalma Béla, Szabolcs Tódor, Csaba Polgár, Akiyama Hironori, Botond Bukovszky, Zoltán Takácsi-Nagy 524 Phase angle as a prognostic indicator of surgical outcomes in patients with gastrointestinal cancer Jana Gulin, Ester Ipavic, Denis Mlakar Mastnak, Erik Brecelj, Ibrahim Edhemović, Nada Rotovnik Kozjek medical physics 530 Correlation between maximum heart distance and thoracic diameter changes and diaphragmatic descent in left-sided breast cancer patients during deep inspiration breath-hold (DIBH) He-Gou Wu, Guang-Wei Zhang, Jian-Feng Liu, Jun-Guo Yang, Xiao-Hui Su study protocol 538 Post-radiation xerostomia therapy with allogeneic mesenchymal stromal stem cells in patients with head and neck cancer: study protocol for phase I clinical trial Primoz Strojan, Gaber Plavc, Marko Kokalj, Goran Mitrovic, Olga Blatnik, Luka Lezaic, Aljaz Socan, Aljosa Bavec, Natasa Tesic, Katrina Hartman, Urban Svajger erratum 550 Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses Angelika Vizintin, Stefan Markovic, Janez Scancar, Jerneja Kladnik, Iztok Turel, Damijan Miklavcic I slovenian abstracts contents Radiol Oncol 2023; 57(4): 411-418. doi: 10.2478/raon-2023-0057 411 review Imaging microvascular changes in nonocular oncological clinical applications by optical coherence tomography angiography: a literature review Rok Hren1,2,3, Gregor Sersa4, Urban Simoncic1,5, Matija Milanic1,5 1 Faculty of Mathematics and Physics, Ljubljana, Slovenia 2 Institute of Mathematics, Physics, and Mechanics, Ljubljana, Slovenia 3 Syreon Research Institute, Budapest, Hungary 4 Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Jozef Stefan Institute, Ljubljana, Slovenia Radiol Oncol 2023; 57(4): 411-418. Received 15 September 2023 Accepted 3 October 2023 Correspondence to: Matija Milanic, Ph.D., Faculty of Mathematics and Physics, University of Ljubljana, Jadranska ulica 19, SI-1000 Ljubljana, Slovenia. E-mail: matija.milanic@fmf.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. Optical coherence tomography angiography (OCTA) is an emerging imaging modality that enables noninvasive visualization and analysis of tumor vasculature. OCTA has been particularly useful in clinical ocular on- cology, while in this article, we evaluated OCTA in assessing microvascular changes in clinical nonocular oncology through a systematic review of the literature. Method. The inclusion criterion for the literature search in PubMed, Web of Science and Scopus electronic data- bases was the use of OCTA in nonocular clinical oncology, meaning that all ocular clinical studies and all ocular and nonocular animal, phantom, ex vivo, experimental, research and development, and purely methodological studies were excluded. Results. Eleven articles met the inclusion criteria. The anatomic locations of the neoplasms in the selected articles were the gastrointestinal tract (2 articles), head and neck (1 article) and skin (8 articles). Conclusions. While OCTA has shown great advancements in ophthalmology, its translation to the nonocular clinical oncology setting presents several limitations, with a lack of standardized protocols and interpretation guidelines pos- ing the most significant challenge. Key words: optical coherence tomography angiography (OCTA); oncology; endoscopy; skin carcinoma Introduction It was demonstrated that angiogenesis is closely associated with tumor growth, as the develop- ment of vasculature has the capacity to supply oxygen and nutrients to dividing tumor cells.1 Microvascular alterations are therefore typical sig- natures of early tumor development and progres- sion. Conventional techniques for assessing micro- vascular changes are narrow band imaging (NBI)23 and confocal laser endomicroscopy (CLE)45 in en- doscopy and confocal laser microscopy (CLM)6 and dermoscopy7 in skin diagnosis. However, NBI has limited resolution, and CLE utilizes exog- enous tracers, while CLM and dermoscopy cannot visualize deeper vascular changes due to a limited Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology412 penetration depth, with blood vessels also often being hidden in pigmented lesions. To address these shortcomings, various emerging imaging techniques have been explored for microvascular imaging. Optical coherence tomography (OCT) is a ma- ture imaging technique that uses low-coherence light to capture high-resolution, cross-sectional images of biological tissues in real time. It has been applied in various fields of medicine8 due to its noninvasiveness, high resolution, and ability to visualize microstructure and has become the gold standard for diagnosis in ophthalmology.9 It is thus not surprising that OCT has found its way into oncological applications as well.10 To enable further functional assessment of tumors, optical coherence tomography angiography (OCTA) is an impending valuable extension of OCT in on- cological research and clinical practice. OCTA is a modification of OCT and works by comparing the light waves that are reflected from stationary tissue with the light waves that are reflected from moving red blood cells (RBCs), and this informa- tion is then used to create a detailed map of the blood vessels (Figure 1). The distinct advantage of OCTA is that it is a noncontact, nonionizing, and noninvasive modality and does not require a con- trast agent. OCTA has proven highly valuable in helping to better understand and manage a range of nononcological ocular pathologies11,12,13, while in oncological ocular clinical applications, OCTA has potential for use in the diagnosis and monitoring of chorioretinal pathologies, such as neovasculari- zation and macular edema.14 How valuable OCTA could be in quantifying microvascular changes in nonocular clinical on- cology remains unclear, and to that end, we decid- ed to systematically review the literature with the intention of exclusively focusing only on studies in which OCTA was performed on patients in the clinical oncology setting. Materials and methods Two authors (R.H. and M.M.) conducted jointly— to preclude potential bias—a comprehensive litera- ture search on August 3, 2023, through PubMed, Web of Science and Scopus electronic databases using the following search terms: “optical coher- FIGURE 1. Optical coherence angiography (OCTA) scanning protocol. (A) A raster scanning protocol for blood vessel visualization, with the x-axis sampling density determined by A-scans per B-scan sets and the y-axis determined by B scans per volume sets. (B) OCTA B-scans created by four repeated B-scans at one y-location, repeated for varying positions along the y-axis, impacting sampling density; ΔT represents interscan time and Ta denotes acquisition time. (C) Maximum intensity projection (MIP) applied to the OCTA B-scan within the depth range of interest (where vessels are located) to generate one line of the en face OCTA image. (D) Illustration depicting the equal distribution of sampling points for smaller and larger imaging areas. Taken from Sampson et al.12 and reprinted with permission from the publisher by the Creative Commons license. To view a copy of Creative Commons license, visit http://creativecommons.org/licenses/by/4.0/. A B C D Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology 413 ence tomography angiography tumors” and “dy- namic optical coherence tomography tumors”. No restrictions on publication date or language were imposed. The inclusion criterion was the nonocu- lar application of OCTA in the oncological clinical setting, meaning that all ocular oncological clini- cal studies and all ocular and nonocular animal and phantom, ex vivo, experimental, research and TABLE 1. Included articles reporting the use of optical coherence tomography angiography (OCTA) to quantify microvascular changes in nonocular clinical applications in oncology Reference Year of publication Number of patients Oncologic setting GI tract Tsai et al.15 2014 1 Nondysplastic Barrett’s esophagus Lee et al.16 2017 52 Nondysplastic Barrett’s esophagus surveillance or endoscopic eradication therapies for low-grade/high-grade dysplasia Head and neck Maslennikova et al.17 2017 25 Radiotherapy of oropharyngeal and nasopharyngeal cancer Skin De Carvalho et al.18 2016 1 Naevus to melanoma transition Themstrup et al.19 2017 47 Actinic keratosis, Bowen’s disease and squamous cell carcinoma Themstrup et al.20 2018 81 Basal cell carcinoma Meiburger et al.21 2019 7 Basal cell carcinoma Gubarkova et al.22 2019 27 Basal cell carcinoma De Carvalho et al.23 2018 127 Melanoma Welzel et al.24 2021 159 Melanoma Perwein et al.25 2023 130 Nevi GI = gastrointestinal FIGURE 2. Images obtained through optical coherence tomography angiography (OCTA) for (TOP ROW) nondysplastic Barrett’s esophagus (NDBE) and (BOTTOM ROW) low-grade/high-grade dysplasia (LGD/HGD) (bottom left LGD; bottom center and bottom right HGD). NDBE images show a regular honeycomb microvascular pattern (arrows, top row), while abnormal vascular features, such as abnormal vessel branching (arrows, bottom left), heterogeneous vessel size (arrows, bottom center) or both (bottom right), are observed in LGD/HGD. Motion artifacts are denoted by asterisks. OCTA images can assist in distinguishing the boundary between abnormal microvasculature and neighboring nondysplastic regions (dashed line, bottom left and bottom center). Taken from Lee et al.16 and reprinted with permission from the publisher. A B C D E F Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology414 development, and purely methodological studies were excluded. Special care was taken that dupli- cations were removed, both across databases and across studies; for example, if the study was first published in proceedings and later in the journal, then proceedings article was considered a nonpri- mary publication and therefore excluded. Studies were categorized with respect to the anatomical location of the tumors. Results In total, 3977 articles were found to be of interest in the PubMed, Web of Science and Scopus data- bases; it is noteworthy that 3855 articles (96.9% of total) were linked to ocular oncological studies. After excluding duplicates and applying the exclu- sion criteria, first considering the title and abstract and then, if necessary, reading the entire article, 11 articles were identified for further analysis. The anatomical locations of tumors in the selected arti- cles were the gastrointestinal (GI) tract (2 articles), head and neck (1 article) and skin (8 articles). GI tract A pioneering effort in assessing microvascula- ture by means of OCTA in clinical oncology was the work of Tsai et al.15 They applied a modality to image subsurface vascular patterns in a patient with nondysplastic Barrett’s esophagus (NDBE) and demonstrated that in this way, the diagnostic capability of endoscopic OCT was enhanced. Lee et al.16 continued their work and collected 97 data- sets from 52 patients with NDBE and low-grade/ high-grade dysplasia (LGD/HGD). Their goal was to differentiate NDBE patients from LGD/HGD pa- tients; however, due to insufficient image quality, 43 datasets (44%) in 20 patients were not used for analysis; OCTA images were also not generated in real time due to the high computational burden. The findings of the study revealed distinct differ- ences in microvascular OCTA features of abnor- mal vessel branching and heterogeneous vessel size between the NDBE and LGD/HGD groups, as shown in Figure 2. Further research with a larger patient population is required to validate these findings and establish the clinical utility of endo- scopic OCTA in routine practice. Head and neck In the study by Maslennikova et al.17, clinicians aimed to investigate the use of OCTA for imag- ing microvascular changes in the oral mucosa of cancer patients undergoing radiotherapy (RT). Authors conducted a longitudinal study involving 25 patients with oropharyngeal and nasopharyn- geal cancer undergoing RT. OCTA was employed to visualize and analyze the microvascular network within the oral mucosa over time, and imaging was performed before the treatment and at regular inter- vals during and after RT. OCTA images were gener- ated in real time by the acquisition system shown in Figure 3. The findings of the study demonstrated significant alterations in the microvascular mor- phology and density in the irradiated oral mucosa over the course of RT, with the microvascular net- work showing an increase in the vascular density and total length of capillary-like vessels compared to the baseline measurements. These changes were found to be more prominent when grade two and three mucositis developed. The study demonstrat- ed the potential of OCTA as a valuable tool for lon- gitudinal monitoring of microvascular changes in radiation-induced oral mucosal damage. However, it is important to note that this study has several limitations, as the sample size was relatively small, and the results may not be generalizable to the broader population; additionally, the prognostic significance of the observed microvascular changes needs further investigation. FIGURE 3. Optical coherence tomography angiography (OCTA) acquisition system. OCTA images were acquired in real time. Taken from Maslennikova et al.17 and reprinted with permission from the publisher by the Creative Commons license. To view a copy of Creative Commons license, visit http:// creativecommons.org/licenses/by/4.0/. Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology 415 Skin De Carvalho et al.18 published a case report in which they showed an increased vasculature in the mel- anoma region compared to the nevus. Following this, Themstrup et al.19 conducted a study to dis- tinguish subtypes within the keratinocyte skin cancer spectrum enrolling 18 patients with actinic keratosis (AK), 12 patients with Bowen’s disease (BD) and 24 patients with squamous cell carcino- ma (SCC). In this exploratory clinical study, they identified two vascular features that showed sig- nificant differences across the lesion types. One of these vascular features, referred to as “blobs”, i.e., small, isolated points with a simple round appear- ance, was more frequently present in BD cases but either absent or only slightly present in AK and SCC lesions. The other feature, called “curves”, i.e., narrow, curved, continuous structures of varying length, was predominantly present in AK lesions. These findings are illustrated in Figure 4. FIGURE 4. Illustration of two distinct vascular features observed through dermoscopy and optical coherence tomography angiography (OCTA). The first feature, referred to as “blobs”, is small, isolated points with a simple round appearance; the second feature, called “curves”, is narrow, curved, continuous structures of varying length. Panel (A) displays a dermoscopic image of a Bowen’s disease (BD) lesion, and panel (B) shows the corresponding OCTA image. The asterisk in both panels points to the same vessel. The thin arrows in panel (B) indicate examples of vascular blobs. Similarly, panels (C) and (D) display a dermoscopic image of AK and the corresponding OCTA image, respectively. The thick arrows in panel (D) indicate examples of vascular curves. Taken from Themstrup et al.19 and reprinted with permission from the publisher. In a subsequent study20, the same group contin- ued with the differentiation of common basal cell carcinoma (BCC) subtypes by scanning 81 patients with 98 BCC lesions, of which 27 were superficial BCC (sBCC), 55 were nodular BCC (nBCC) and 16 were infiltrative BCC (iBCC). In this study, they found various structural and microvascular fea- tures that would aid in identifying nBCC, iBCC and sBCC subtypes. For example, it was shown that the presence of so-called “serpiginous” ves- sels, i.e., wavy structures of varying length, indi- cated an increased risk of nBCC and a reduced risk of sBCC. Meiburger et al.21 applied OCTA to a patient with nBCC and six patients with sBCC and developed an algorithm for automatically determining skin lesion area using vascular density. While authors were hopeful in their conclusion that proposed method could facilitate diagnosis and treatment of BCC, no further study was published. Gubarkova et al.22 examined 27 patients with BCC who received photodynamic therapy (PDT). They utilized OCTA imaging before and imme- diately after PDT and during follow-up visits to monitor vascular changes. Analysis of the OCTA images allowed for quantification of parameters such as blood vessel density and uniformity, aid- ing in distinguishing among BCC subtypes. The study demonstrated that OCTA offers real-time information on vascular changes in response to PDT. The researchers observed a decrease in blood vessel density at 24 hours after PDT, with OCTA images having 97% predictive value for differen- tiation between complete and partial responders. De Carvalho et al.23 conducted a systematic anal- ysis of melanoma lesions in 127 patients and found a significant link between specific microvascular features and Breslow’s thickness. In a more re- cent study, Welzel et al.24 assessed 159 melanomas from 156 consecutive patients and found that ir- regular vascular shapes, including blobs, curves and serpiginious vessels, were more common in high-risk and metastatic melanomas than in low- risk lesions. Most recently, the same group25 pro- spectively examined a total of 167 nevi, including dysplastic ones, in 130 participants and compared these microvascular features to those found ear- lier in 159 melanomas.24 They found that increased blood vessel density and diameter and irregular tissue architecture were associated with melano- mas, while nevi showed more regular structures and lower blood vessel density and diameter, indi- cating their benign nature (Figure 5). Researchers also found excellent predictive diagnostic value of B C D A Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology416 microvascular features (e.g., blobs, serpiginious vessels) for nevi (88.2% to 91%) and melanoma (95.5% to 96.8%) and concluded that OCTA “may be a valuable addition to the current clinical-der- moscopic gold standard”.25 Discussion Based on this literature review, the inference could be made that OCTA is still finding its place in on- cological clinical applications. It appears that the translation of OCTA from ocular applications to the nonocular clinical oncology setting faces cer- tain limitations that could potentially hinder its widespread adoption. Limited penetration depth One of the obvious limitations of OCTA in nonoc- ular clinical oncology settings is its restricted pen- etration depth. OCTA relies on detecting motion contrast generated by moving RBCs, which limits its applicability to superficial structures. Tumors FIGURE 5. Microvascularization in skin lesions (nevi, dysplastic nevi, and melanomas) through optical coherence tomography angiography (OCTA) scans (denoted as D-OCT). (A) Compound nevus on the scapula, displaying a globular appearance with recent changes. (B) Dysplastic nevus, flat lesion on the scapula, exhibiting a complex appearance, atypical network, irregular pigmentation, and dots/globules. (C) Melanoma, a lesion on the scapula, measuring 3.35 mm in tumor thickness, classified as pT3aN1bM0S2, stage IIIB. Taken from Perwein et al.25 and reprinted with permission from the publisher by the Creative Commons license. To view a copy of Creative Commons license, visit http://creativecommons.org/licenses/by/4.0/. B C A Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology 417 and lesions in deeper anatomical locations, such as within organs or soft tissues, may not be adequate- ly visualized using OCTA due to limited tissue penetration. This constraint hampers its potential for comprehensive evaluation and monitoring of oncological conditions. However, this limitation can be overcome by us- ing endoscopic techniques bringing the instrument closer to the tissue of interest. As demonstrated in the GI tract studies by Tsai et al.15 and Lee et al.16, OCTA can be used endoscopically. Namely, com- mon OCT has been developed as endoscopic probes of different types and used to obtain microscopy images of entire luminal organs, solid tumors, or vessels.26 Since OCTA is an extension of OCT, the same already developed technology can be used to bring the system closer to the tissue of interest. Another possibility to increase the OCTA pen- etration depth is to use OCTA systems with longer wavelengths. In the studies presented in this ar- ticle, the OCTA systems utilized 1.3 µm wave- lengths, which is a typical wavelength also used for skin imaging; in ophthalmology, a shorter wavelength of 0.8 µm is typically used, result- ing in an approximately 60% lower penetration depth. In a recent publication by Nishizawa and Yamanaka27, it was shown that by using a 1.7 µm wavelength, the penetration depth increases by approximately 40% compared to a 1.3 µm wave- length. Therefore, by developing OCTA systems with even longer wavelengths, larger penetration depths could be obtained. Inability to differentiate vessel types OCTA provides detailed structural information about blood vessels but lacks the ability to differ- entiate between different vessel types. In the field of oncology, the distinction between arterial and venous vasculatures is crucial, as tumor angio- genesis is primarily associated with the growth of new abnormal blood vessels. Accurate differentia- tion between these types of vessels aids in assess- ing tumor progression and treatment response. Unfortunately, OCTA’s current capabilities fail to provide this level of vessel characterization, limit- ing its effectiveness in nonocular oncological set- tings. In ophthalmology, recent articles report the possibility of differentiating between arteries and veins utilizing various OCTA image parameters, including vascular diameters and shape and per- fusion intensity density.28 However, the current methods for artery-vein classification in OCTA employ complex algorithms, thereby making it dif- ficult for clinical applications. To alleviate this hin- drance, deep learning algorithms were developed to reduce the complexity and automate artery-vein classification.29 Similar algorithms should also be developed for other OCTA modalities. Motion artifacts Movement, including patient motion during OCTA acquisition, can introduce motion artifacts, leading to image distortions and reduced image quality. Unlike ophthalmology, where patients can fixate on a target, patients in nonocular oncology settings often have limited control over motion, making motion artifacts more challenging to miti- gate. This limitation can compromise the accuracy and reliability of OCTA in nonocular clinical on- cology, demanding the need for advanced post- processing algorithms to improve image quality. Since motion artifacts are well-known sources of artifacts in OCT imaging, they have been ex- tensively researched. One possibility is to detect and compensate for the axial motion artifacts pixelwise by comparing the topology of different layers in tissue, and the motion artifacts are then compensated by shifting the pixel numbers with the value detected.29 Another possibility is to re- move the affected scans in the software and to use only the nonaffected scans for vasculature image reconstruction.30 However, this approach may in- crease the duration of imaging sessions; therefore, it would be better to use an approach without the need for rescanning. As a solution, it was demon- strated that the motion contribution to the OCT signal can be reasonably estimated by consider- ing statistics of the measured flow signal across all voxels.30 By implementing motion artifact compen- sation strategies, the translation of OCTA to clini- cal workflow would become more feasible. Lack of standardized protocols and interpretation The lack of standardized protocols and interpreta- tion guidelines is a significant limitation of OCTA in nonocular clinical oncology. Unlike ophthal- mology, where standardized imaging protocols and interpretation criteria exist, the application of OCTA in oncology lacks such standardization. As a result, different centers may use varying ac- quisition settings, image processing algorithms, or interpretation approaches, leading to inconsistent and noncomparable results. Establishing stand- Radiol Oncol 2023; 57(4): 411-418. Hren R et al. / Optical coherence tomography angiography in nonocular oncology418 ardized protocols and guidelines specific to non- ocular oncology would enhance the accuracy and reproducibility of OCTA findings. While OCTA has shown great promise in oph- thalmology, its translation to the nonocular clini- cal oncology setting faces limitations. In particu- lar, the lack of standardized protocols and inter- pretation guidelines poses a significant challenge. Addressing these limitations through advance- ments in technology, algorithm development, and a larger number of clinical sites initiating clinical trials is essential for realizing the full potential of OCTA in nonocular clinical oncology. Acknowledgment This work was financially supported by the state budget by the Slovenian Research Agency, re- search grant no. J3-3083 and research programs no. P3-0003, P3-0307, and P1-0389. References 1. Folkman J. Role of angiogenesis in tumor growth and metastasis. Sem Oncol 2002; 29(6 Suppl 16): 15-8. doi: 10.1053/sonc.2002.37263 2. Yang Q, Liu Z, Sun H, Jiao F, Zhang B, Chen J. A narrative review: narrow-band imaging endoscopic classifications. Quant Imaging Med Surg 2023; 13: 1138-63. doi: 10.21037/qims-22-728 3. Goda K, Takeuchi M, Ishihara R, Fujisaki J, Takahashi A, Takaki Y, et al. Diagnostic utility of a novel magnifying endoscopic classification system for superficial Barrett’s esophagus-related neoplasms: a nationwide multicent- er study. Esophagus 2021; 18: 713-23. doi: 10.1007/s10388-021-00841-1 4. Wallace M, Lauwers G, Chen Y, Dekker E, Fockens P, Sharma P, et al. Miami classification for probe-based confocal laser endomicroscopy. Endoscopy 2011; 43: 882-91. doi: 10.1055/s-0030-1256632 5. Canakis A. The diagnostic performance of probe-based confocal laser endomicroscopy in the detection of gastric cancer: a systematic review and meta-analysis. Ann Gastroenterol 2022; 35: 496-502. doi: 10.20524/ aog.2022.0741 6. Gerger A, Hofmann-Wellenhof R, Samonigg H, Smolle J. In vivo confocal laser scanning microscopy in the diagnosis of melanocytic skin tumours. Br J Dermatol 2009; 160: 475-81. doi: 10.1111/j.1365-2133.2008.08995.x 7. Zalaudek I, Kreusch J, Giacomel J, Ferrara G, Catricalà C, Argenziano G. How to diagnose nonpigmented skin tumors: A review of vascular struc- tures seen with dermoscopy. J Am Acad Dermatol 2010; 63: 361-74. doi: 10.1016/j.jaad.2009.11.698 8. Wan B, Ganier C, Du-Harpur X, Harun N, Watt FM, Patalay R, et al. Applications and future directions for optical coherence tomography in dermatology. Br J Dermatol 2021; 184: 1014-22. doi: 10.1111/bjd.19553 9. Fujimoto J, Swanson E. The development, commercialization, and impact of optical coherence tomography. Invest Ophthalmol Vis Sci 2016; 57: OCT1. doi: 10.1167/iovs.16-19963 10. Suppa M, Palmisano G, Tognetti L, Lenoir C, Cappilli S, Fontaine M, et al. Line-field confocal optical coherence tomography in melanocytic and non- melanocytic skin tumors. Ital J Dermatol Venereol 2023; 158: 180-9. doi: 10.23736/S2784-8671.23.07639-9 11. Chen CL, Wang RK. Optical coherence tomography based angiography [Invited]. Biomed Opt Express 2017; 8: 1056. doi: 10.1364/BOE.8.001056 12. Sampson DM, Dubis AM, Chen FK, Zawadzki RJ, Sampson DD. Towards standardizing retinal optical coherence tomography angiography: a review. Light Sci Appl 2022; 11: 63. doi: 10.1038/s41377-022-00740-9 13. Courtie E, Gilani A, Veenith T, Blanch RJ. Optical coherence tomography angiography as a surrogate marker for end-organ resuscitation in sepsis: A review. Front Med 2022; 9: 1023062. doi: 10.3389/fmed.2022.1023062 14. Pellegrini M, Staurenghi G, Preziosa C. Clinical Applications of optical coher- ence tomography angiography in ocular oncology: Pearls and pitfalls. Ocul Oncol Pathol 2022; 8: 79-87. doi: 10.1159/000520951 15. Tsai TH, Ahsen OO, Lee HC, Liang K, Figueiredo M, Tao YK, et al. Endoscopic optical coherence angiography enables 3-dimensional visualization of subsurface microvasculature. Gastroenterology 2014; 147: 1219-21. doi: 10.1053/j.gastro.2014.08.034 16. Lee HC, Ahsen OO, Liang K, Wang Z, Figueiredo M, Giacomelli MG, et al. Endoscopic optical coherence tomography angiography microvas- cular features associated with dysplasia in Barrett’s esophagus (with video). Gastrointestinal Endoscopy 2017; 86: 476-84.e3. doi: 10.1016/j. gie.2017.01.034 17. Maslennikova AV, Sirotkina MA, Moiseev AA, Finagina ES, Ksenofontov SY, Gelikonov GV, et al. In vivo longitudinal imaging of microvascular changes in irradiated oral mucosa of radiotherapy cancer patients using optical coher- ence tomography. Sci Rep 2017; 7: 16505. doi: 10.1038/s41598-017-16823- 2 18. De Carvalho N, Ciardo S, Cesinaro AM, Jemec GBE, Ulrich M, Welzel J, et al. In vivo micro-angiography by means of speckle-variance optical coherence tomography (SV-OCT) is able to detect microscopic vascular changes in nae- vus to melanoma transition. J Eur Acad Dermatol Venereol 2016; 30: e67-8. doi: 10.1111/jdv.13311 19. Themstrup L, Pellacani G, Welzel J, Holmes J, Jemec GBE, Ulrich M. In vivo microvascular imaging of cutaneous actinic keratosis, Bowen’s disease and squamous cell carcinoma using dynamic optical coherence tomography. J Eur Acad Dermatol Venereol 2017; 31: 1655-62. doi: 10.1111/jdv.14335 20. Themstrup L, De Carvalho N, Nielsen SM, Olsen J, Ciardo S, Schuh S, et al. In vivo differentiation of common basal cell carcinoma subtypes by microvas- cular and structural imaging using dynamic optical coherence tomography. Exp Dermatol 2018; 27: 156-65. doi: 10.1111/exd.13479 21. Meiburger KM, Chen Z, Sinz C, Hoover E, Minneman M, Ensher J, et al. Automatic skin lesion area determination of basal cell carcinoma using optical coherence tomography angiography and a skeletonization approach: Preliminary results. J Biophotonics 2019; 12: e201900131 doi: 10.1002/ jbio.201900131 22. Gubarkova EV, Feldchtein FI, Zagaynova EV, Gamayunov SV, Sirotkina MA, Sedova ES, et al. Optical coherence angiography for pre-treatment assess- ment and treatment monitoring following photodynamic therapy: a basal cell carcinoma patient study. Sci Rep 2019; 9: 18670. doi: 10.1038/s41598- 019-55215-6 23. De Carvalho N, Welzel J, Schuh S, Themstrup L, Ulrich M, Jemec GBE, et al. The vascular morphology of melanoma is related to Breslow index: An in vivo study with dynamic optical coherence tomography. Exp Dermatol 2018; 27: 1280-86. doi: 10.1111/exd.13783 24. Welzel J, Schuh S, De Carvalho N, Themstrup L, Ulrich M, Jemec GBE, et al. Dynamic optical coherence tomography shows characteristic alterations of blood vessels in malignant melanoma. J Eur Acad Dermatol Venereol 2021; 35: 1087-93. doi: 10.1111/jdv.17080 25. Perwein MKE, Welzel J, De Carvalho N, Pellacani G, Schuh S. Dynamic optical coherence tomography: A non-invasive imaging tool for the distinction of nevi and melanomas. Cancers 2022; 15: 20. doi: 10.3390/cancers15010020 26. Gora MJ, Suter MJ, Tearney GJ, Li X. 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Quant Imaging Med Surg 2020; 11: 1120-33. doi: 10.21037/qims-20-730 Radiol Oncol 2023; 57(4): 419-429. doi: 10.2478/raon-2023-0059 419 review Locoregional therapy combined with systemic therapy (LRT + ST) for unresectable and metastatic intrahepatic cholangiocarcinoma: a systematic review and meta-analysis Mengqi Zhang1, Weiwei Qi1, Xiaofei Qiu3, Chunpeng Yu2, Wensheng Qiu1, Song Wang2, Zhenkang Qiu2 1 Department of Oncology, Key Laboratory of Cancer Molecular and Translational Research, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China 2 Interventional Medical Center, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China 3 Qingdao Municipal Center for Disease Control & Prevention, Qingdao Institute of Preventive Medicine, Qingdao, Shandong, China Radiol Oncol 2023; 57(4): 419-429. Received 1 June 2023 Accepted 18 September 2023 Correspondence to: Zhenkang Qiu, Ph.D., M.D., Interventional Medical Center, The Affiliated Hospital of Qingdao, University, 16 Jiangsu Road, Qingdao 266003, Shandong Province, China; E-mail: dr_qiuzk@126.com Mengqi Zhang and Weiwei Qi have made equal contributions as co-authors to this article. 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 outcome of systemic therapy (ST) for unresectable and metastatic intrahepatic cholangiocarci- noma (iCCA) is poor. This study aims to further evaluate the efficacy and safety of locoregional therapy combined with systemic therapy (LRT + ST) compared with only ST in unresectable and metastatic iCCA by performing a system- atic literature review and meta-analysis. Methods. A comprehensive search was performed in PubMed, Web of Science, EMBASE, and the Cochrane Library up to November 3, 2022. The primary outcome was overall survival (OS), and the secondary outcomes were progres- sion-free survival (PFS), objective response rate (ORR), and adverse events (AEs). Results. Ten retrospective cohort studies with 3,791 unresectable or metastatic iCCA patients were enrolled in this study, including 1,120 who received ablation, arterially directed therapy (ADT), or external beam radiation therapy (EBRT) combined with ST. The meta-analysis showed that the LRT + ST group had a better OS (HR = 0.51; 95% CI = 0.41–0.64; p value < 0.001), PFS (HR = 0.40, 95% CI = 0.22–0.71, p value = 0.002) and ORR (RR = 1.68; 95% CI = 1.17–2.42; p value = 0.005). Subgroup analysis showed that both ST combined with ADT (HR = 0.42, 95% CI = 0.31–0.56, p value < 0.001) and EBRT (HR = 0.67, 95% CI = 0.63–0.72, p value < 0.001) could improve OS. Neutropenia, thrombocytopenia, anemia, anorexia, and vomiting did not show significant differences between the groups (p value > 0.05). Conclusions. Compared with only ST, LRT + ST improved survival outcomes for unresectable and metastatic iCCA patients without increasing severe AEs, which can further provide a basis for guidelines. Key words: unresectable iCCA; locoregional therapy; systemic therapy; meta-analysis Introduction Intrahepatic cholangiocarcinoma (iCCA), which develops from the bile duct within the hepatic pa- renchyma, has been increasing in incidence and mortality in recent years.1-3 Due to the absence of symptoms at the initial stage, only approximately 22% of iCCA patients are resectable at the primary diagnosis.4 The ABC-02 trial established gemcit- abine plus cisplatin (GemCis) as the first-line treat- Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma420 ment for locally advanced or metastatic biliary tract cancer, with a median overall survival (OS) was 11.7 months.5 The recent TOPAZ-1 trial dem- onstrated that using durvalumab plus GemCis improved patient survival compared with GemCis alone, bringing a median survival benefit of 1.3 months compared to GemCis.6 With the rapid development of equipment and technology, locoregional therapies, such as abla- tion7,8, arterially directed therapies (ADTs)9-13, and external beam radiation therapy (EBRT)14-17, have shown excellent effects on iCCA. However, there have been conflicting opinions about the effective- ness and safety of locoregional therapy combined with systemic therapy (LRT + ST) in unresect- able or metastatic iCCA because of the absence of randomized controlled trials or meta-analyses. Evaluating this combination therapy method’s ef- fectiveness, long-term survival, and safety is dif- ficult because most evidence is derived from ret- rospective cohort studies, case reports, or series. Thus, this study was performed to explore the feasibility and survival benefits of LRT + ST for unresectable and metastatic iCCA via a systematic review and meta-analysis. Material and methods This systematic review and meta-analysis were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines18 and registered in PROSPERO (registration: CRD42020162902). Literature search A literature search was conducted in PubMed, Web of Science, EMBASE, and the Cochrane Library on November 3, 2022, without language or geographic restrictions. The search terms included “unresectable intrahepatic cholangiocarcinoma”, “locoregional therapy”, “systemic therapy”, and their synonyms. The detailed search strategies FIGURE 1. Systematic Reviews and Meta-Analyses (PRISMA) flow chart. Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma 421 are provided in Supplementary Table 1. The refer- ence lists of the final included studies were also checked for possible additional records. Study selection and eligibility criteria EndNote X9.1 (Version 19.1.0) was used to iden- tify and remove duplicates. The remaining stud- ies from the databases were filtered by their titles, abstracts, and keywords independently by two au- thors. Then, a comprehensive review of the stud- ies’ full texts was conducted. If there was an over- lap in research, the most recent and most extensive studies were selected for this meta-analysis. Ten studies were included in the final analysis. The PRISMA study selection flowchart is shown in Figure 1. The inclusion criteria were as follows: (a) stud- ies involved patients with a confirmed diagnosis of unresectable or metastatic iCCA and compared LRT + ST with ST treatment; and (b) studies report- ed clinical outcomes including any of the follow- ing: OS, progression-free survival (PFS), objective response rate (ORR) and adverse events (AEs). A study was excluded if it met any of the following criteria: (a) nonhuman studies; (b) population-level studies; (c) inadequate description of materials and methods; (d) raw data unavailable (letters, editori- als, conference abstracts, posters, commentaries, and reviews); (e) clinical outcomes not reported for LRT + ST or ST; and (f) studies included patients with all types of cholangiocarcinoma and did not distinguish the clinical outcomes of patients with iCCA. Quality assessment The studies were evaluated according to the Newcastle‒Ottawa Scale standards for cohort studies.19 Two authors independently assessed the quality of the studies, and any disagreements were resolved by discussion and consensus with a third author. Three columns comprise the scale: selection, comparability, and outcome. The score is rated out of 9 stars: >6 stars indicate a low risk of bias, 4–6 stars indicate a moderate risk of bias, and <4 stars indicate a high risk of bias. Out of the ten studies included20-29 in the systematic review, three studies24-26 were determined to be of good quality, and seven studies20-23,27-29 were determined to be of acceptable quality. Details of the quality assess- ment are shown in Supplementary Table 2. TABLE 1. Main study characteristics of included studies Study Country Treatment Sample Age (years) Sex (F:M) Outcomes LRT + ST ST LRT + ST ST LRT + ST ST LRT + ST ST Yang, 202220 China ADT: DEB-TACE (Doxorubicin) ICIs (Camrelizumab or Sintilimab) Gemcitabine + Cisplatin 20 20 59 59 9:11 7:13 OS, PFS, ORR, AEs Yan, 202221 China Ablation: RFA / MWA Gemcitabine* Gemcitabine* 36 36 NR NR 14:22 15:21 OS Sun, 202122 China ADT: TACI (5-Fluoruracil + cisplatin) Gemcitabine + S-1 Gemcitabine + Cisplatin + S-1 33 33 NR NR NR NR OS, PFS, ORR, AEs Gairing, 202123 Germany ADT: TACE (Mitomycin C / Doxorubicin) Gemcitabine * Gemcitabine* 14 59 61.3 66.8 8:6 29:30 OS Hu, 202024 China ADT: DEB-TACE (Gemcitabine + Cisplatin) / cTACE (Gemcitabine + Cisplatin + lipiodol) Apatinib Apatinib 13 10 55.9 58.7 7:6 2:8 OS, PFS, ORR, AEs Verma, 201825,& America EBRT SYS SYS 666 2176 65 65 309:357 1095:1081 OS Chang, 201826,& China EBRT: CCRT / CTRT Fluoropyrimidine * / Gemcitabine* Fluoropyrimidine* / Gemcitabine* 211 211 60.11 60.80 81:130 84:127 OS Konstantinidis, 201627 America ADT: HAI (Floxuridine*) Gemcitabine* / Irinotecan* / 5-Fluoruracil* Gemcitabine* / 5-Fluoruracil* 78 26 62 26 47:31 13:13 OS, ORR Edeline, 201528,& France ADT: 90Y SIRT Gemcitabine* / 5-Fluoruracil* Gemcitabine + Cisplatin# 24 33 NR NR NR NR OS, PFS Kim, 201329 Korea EBRT: CCRT Capecitabine + Cisplatin Capecitabine + Cisplatin 25 67 56 58 6:19 14:53 OS, PFS, ORR, AEs ADT = arterially directed therapy; AEs = adverse events; CCRT = concurrent chemoradiation therapy; cTACE = conventional transcatheter arterial chemoembolization; CTRT = sequential chemotherapy and radiotherapy; DEB-TACE = TACE with drug-eluting beads; 90Y SIRT = Yttrium-90 selective internal radiotherapy; EBRT = external beam radiation therapy; F = female; HAI = hepatic arterial infusion; ICIs = immune checkpoint inhibitors; LRT + ST = locoregional therapy combined with systemic therapy; M = male; MWA = microwave ablation; NR = not reported; ORR = objective response rate; OS = overall survival; PFS = progression-free survival; RFA = radiofrequency ablation; ST = systemic therapy; SYS = systemic chemotherapy; TACI = transarterial chemoinfusion; TACE = transarterial chemoembolization; * = major drugs in the treatment regime; # = data from the ABC-02 study; & = multi-canter study. All studies included were retrospective cohort studies. Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma422 FIGURE 2. Forest plots for overall survival (OS) in unresectable and metastatic intrahepatic cholangiocarcinoma (iCCA) patients treated by locoregional therapy combined with systemic therapy versus only systemic therapy. 95% CI = 95% confidence intervals; DL = DerSimonian–Laird method; HR = hazard ratio; IV = inverse variance method Data acquisition The extracted primary information was as follows: (a) basic information, such as title, first author, journal, country or region, and publication date; (b) baseline characteristics of the study popula- tion, including sample size, age, gender, duration of follow-up, physical status, etc.; (c) interventions, including the type of medication, measurement, periodicity, frequency, etc.; (d) observed outcome data, including OS, PFS, ORR, AEs, etc.; (e) ele- ments related to the inclusion and exclusion crite- ria; and (f) elements of the risk of bias evaluation. If the required information, such as the hazard ratio (HR) and 95% confidence interval (95% CI), were provided in the article, they were extracted direct- ly; otherwise, they were calculated using the digi- tal tools in Tierney’s article.30 Engauge Digitizer (version 11.1) was used for digitizing the survival curves and then transforming the digital informa- tion to obtain the HR and 95% CI. Two authors in- dependently extracted data from the papers, and any discrepancies were resolved through a con- sensus meeting. Statistical analysis Meta-analysis was only performed when data from at least three studies were available. StataMP 17 (Version 521.17.0.104) was used to conduct a meta- analysis. OS and PFS are time-to-event data evalu- ated by the HR. The ORR and AEs are enumeration data evaluated by the relative risk (RR). The 95% CI was used for interval estimation. Heterogeneity between studies was analyzed by Cochran’s Q test with a significance level of α = 0.1, and the degree of heterogeneity was assessed using I2 statistics. If p ≥ 0.10 and I2 ≤ 50%, heterogeneity was consid- ered minor, and a fixed-effects model was used for analysis. Heterogeneity was considered major if p < 0.10 or I2 > 50%. The random-effects or fixed- effects model and sensitivity analysis were used to evaluate the stability of the outcome and identify articles with high heterogeneity. When necessary, subgroup analysis was performed. A P value < 0.05 was considered statistically significant. Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma 423 Results Baseline characteristics Ten cohort studies were deemed eligible and in- cluded in the descriptive analysis; of the 3,791 pa- tients in these studies, 1,120 (29.5%) were treated with LRT + ST. ADT was performed in six arti- cles20,22-24,27,28, EBRT in three25,26,29, and ablation in one21 (Table 1). Further details of the included lit- erature are provided in Supplementary Table 3. OS A total of 3,791 patients from all ten studies were included in the meta-analysis. The pooled HR in- dicated that compared with ST, LRT + ST highly significantly improved OS (HR = 0.51; 95% CI = 0.41–0.64; p value < 0.001), reducing the risk of death by 49%. High heterogeneity existed among the ten studies (I2=78.1%, p < 0.001). Changing to a fixed- effects model for HR pooling showed that the out- FIGURE 3. Subgroup analysis of overall survival (OS) in unresectable and metastatic intrahepatic cholangiocarcinoma (iCCA) patients according to types of locoregional therapy combined with systemic therapy (ablation, ADT, RT). 95% CI = 95% confidence intervals. ADT = arterially directed therapy; DL = DerSimonian–Laird method; HR = hazard ratio; IV = inverse variance method; EBRT= external beam radiation therapy Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma424 come remained stable (HR = 0.65; 95% CI = 0.61– 0.70; p value < 0.001) (Figure 2). Sensitivity analysis was then performed and showed that the pooled HR was still reliable after deleting any of the arti- cles (Supplementary Figure 1). Subgroup analysis according to locoregional therapies showed high heterogeneity in the ADT group (Supplementary Figure 2). Based on the above analysis, in addition to locoregional treatment, the heterogeneity main- ly came from Hu’s article24, which may be related to the intervention (apatinib) they adopted. When this article was removed, the heterogeneity was markedly reduced (I2 = 47.7%, p = 0.054). Subgroup analysis of the remaining nine articles showed that ST combined with ADT (HR = 0.42, 95% CI = 0.31–0.56, p value < 0.001) or EBRT (HR = 0.67, 95% CI = 0.63–0.72, p value < 0.001) improved patients’ OS and reduced the risk of death by 58% or 33% (Figure 3). PFS The data on PFS were obtained from five stud- ies20,22,24,28,29 with 278 patients. Among them, 115 (41.4%) were treated with LRT + ST. LRT + ST re- duced the risk of tumor recurrence and metas- tasis by 60% more than ST (HR = 0.40, 95% CI = 0.22–0.71, p value = 0.002). The five studies had high heterogeneity (I2=74.9%, p = 0.003). A fixed-effects model showed that the outcome remained sta- ble (HR = 0.49; 95% CI = 0.37–0.64; p value < 0.001) (Figure 4). Sensitivity analysis showed that the pooled HR was still reliable after deleting any of the articles (Supplementary Figure 3). Considering that the primary source of heterogeneity was still Hu’s article24, the heterogeneity almost completely disappeared after omitting it (I2=0.0%, p = 0.609) (Supplementary Figure 4). ORR Five studies20,22,24,27,29 with 327 patients were used to analyze the ORR. 181 (55.4%) were treated with LRT + ST. The ORR of the LRT + ST group was bet- ter than that of the ST group (RR = 1.68, 95% CI = 1.17–2.42, p value = 0.005) (Figure 5). AEs Four articles20,22,24,29, comprising a total of 221 pa- tients, reported data on the occurrence of post- treatment neutropenia, thrombocytopenia, and anemia. Among these patients, 91 individuals (70.0%) received LRT + ST. Additionally, three ar- ticles20,24,29, involving 155 patients, presented data on post-treatment anorexia and vomiting, with 58 patients (37.4%) receiving LRT + ST. Neutropenia (RR = 1.48, 95% CI = 0.46–4.69, p value = 0.509), thrombocytopenia (RR = 1.05, 95% CI = 0.76–1.45, p value = 0.763), anemia (RR = 1.32, 95% CI = 0.94– 1.86, p value = 0.112), anorexia (RR = 1.31, 95% CI = 0.89–1.93, p value = 0.167), and vomiting (RR = 1.40, FIGURE 4. Forest plots for progression-free survival (PFS) in unresectable and metastatic intrahepatic cholangiocarcinoma (iCCA) patients treated by locoregional therapy combined with systemic therapy versus only systemic therapy. 95% CI = 95% confidence intervals; DL = DerSimonian–Laird method; HR = hazard ratio; IV = inverse variance method Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma 425 95% CI =0.91–2.16, p value = 0.130) did not show sig- nificant differences between the LRT + ST group and ST group (Figure 6). Furthermore, among the four studies investigating treatment-related AEs, no instances of severe AEs, such as acute portal vein thrombosis, bleeding, biloma, abscess forma- tion, bone marrow suppression, or pancreatitis, were observed. There were no reported cases of AEs-related death. More detailed data on AEs are recorded in Supplementary Table 4. Discussion For unresectable or metastatic iCCA, the primary recommendation is GemCis or GemCis plus dur- valumab. Additionally, combination or mono- therapy regimens based on fluorouracil, capecit- abine, and gemcitabine are also recommended. Entrectinib, larotrectinib, pembrolizumab, and pralsetinib may be used as first-line drugs for patients with specific gene expressions or immu- nohistochemical phenotypes. Besides these ST op- tions, the current National Comprehensive Cancer Network® (NCCN®) guidelines also recommend locoregional therapies or LRT + ST to treat unre- sectable or metastatic iCCA.31 According to pre- vious studies, the median OS for advanced iCCA patients who were treated with ST was 5.2 to 15.4 months.32-39 The emergence of locoregional thera- pies may bring more survival benefits to patients with unresectable or metastatic iCCA. Locoregional therapies, including ablation, ADT, and EBRT, have emerged as promising treat- ments for iCCA. Ablation is widely used for single tumors smaller than 3 cm in nonsurgical candi- dates with iCCA.7,8 ADT can increase the local con- centration of chemotherapeutic drugs while mini- mizing systemic adverse effects.10-13 EBRT, espe- cially combined with standard or high-dose fluo- rouracil chemotherapy, has been recommended by the NCCN guidelines as one of the standard treat- ments for locally advanced iCCA.14,17,40 However, the current NCCN guidelines lack specific recom- mendations regarding the treatment approach for locoregional therapies in combination with sys- temic therapy, aside from chemoradiotherapy, due to insufficient evidence-based medicine. This systematic review and meta-analysis evaluated the clinical outcomes of LRT + ST and ST using data from 10 cohort studies with 3,791 patients. The OS and PFS in the LRT + ST group were much better than those in the ST group, and the ORR was improved. Subgroup analysis based on the type of locoregional therapy indicated that the combination of ST with ADT or EBRT might increase the OS of patients and lower their risk of death. The sole trial of ST combined with ablation21 showed that LRT + ST reduced the risk of death by 47%. Particularly worth mentioning is that Hu’s study24 was highly heterogeneous among the ten included articles, which is likely attributed to the use of apatinib as a targeted drug rather than the first-line chemotherapy primarily recommended in the guidelines. Apatinib is a novel, small mol- ecule, selective vascular endothelial growth fac- tor receptor-2 (VEGFR-2) tyrosine kinase inhibitor and has been confirmed to be effective in various FIGURE 5. Forest plots for objective response rate (ORR) in unresectable and metastatic intrahepatic cholangiocarcinoma (iCCA) patients treated by locoregional therapy combined with systemic therapy (LRT + ST) versus only systemic therapy (ST). 95% CI = 95% confidence intervals; MH = Mantel–Haenszel model; RR = relative risk. Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma426 FIGURE 6. Forest plots for the incidence of neutropenia (A), thrombocytopenia (B), anemia (C), anorexia (D), and vomiting (E) in unresectable and metastatic intrahepatic cholangiocarcinoma (iCCA) patients treated by locoregional therapy combined with systemic therapy (LRT + ST) versus only systemic therapy (ST). 95% CI = 95% confidence intervals; DL = DerSimonian–Laird method; MH = Mantel–Haenszel model; RR = relative risk. A B C D E Radiol Oncol 2023; 57(4): 419-429. Zhang M et al. / LRT + ST for unresectable and metastatic intrahepatic cholangiocarcinoma 427 advanced cancers including gastric cancer, hepato- cellular carcinoma, non-small cell lung cancer, and breast cancer.41-44 In biliary tract cancer, apatinib is also in the clinical exploration stage and has not yet been listed as standard treatment for iCCA in guidelines.45-47 Complete resection is the only potential curative treatment for iCCA, whereas systemic therapy and locoregional treatments are considered palliative measures for patients diagnosed with unresectable or metastatic disease. Notably, in Konstantinidis’ study, eight patients initially deemed unresectable iCCA underwent curative-intent surgical resec- tion. Among these cases, four patients received systemic chemotherapy, three underwent systemic chemotherapy combined with hepatic arterial in- fusion (HAI), and one received isolated HAI. Their postoperative median overall survival was 36.9 months (range: 10.4-92.3 months).27 In the Phase II single-arm MISPHEC trial, the combination of radioembolization with Y-90 microspheres with GemCis served as a first-line treatment approach for 41 unresectable iCCA patients, resulting in tumor downstaging and subsequent surgery for nine patients (22%).10 Currently, the NCCN guide- lines do not provide recommendations for neoad- juvant/conversion therapy strategies for unresect- able/metastatic iCCA. However, the integration of LRT + ST may represent a promising direction for achieving downstaging to resection of initially un- resectable tumors in the future. In addition to evaluating the effectiveness of LRT + ST, this study focused on the occurrence of AEs. In this study, four studies20,22,24,29 reported the incidence of complications. Because few studies provided the same AE outcomes, meta-analyses were only performed on neutropenia, throm- bocytopenia, anemia, anorexia, and vomiting. It was confirmed that LRT + ST did not increase the incidence of AEs compared with ST, consist- ent with the results of the four studies included. Particularly worth mentioning is a statistically significant difference in the incidence of neutrope- nia between the two groups in Yang’s and Kim’s studies. However, in Yang’s study, the incidence in the ST group was higher, while in Kim’s study, the incidence in the LRT + ST group was higher, which may be related to the different interven- tions they used. Both Yang’s and Kim’s ST groups were treated with GemCis, but Kim’s experimen- tal group was treated with concurrent chemora- diation therapy, while Yang’s experimental group was treated with transarterial chemoembolization with drug-eluting beads (DEB-TACE) + immune checkpoint inhibitors (ICIs). In addition, in Kim’s study, the incidence of hand-foot syndrome was higher in the LRT + ST group. In Yang’s study, the incidences of leukopenia, hypothyroidism, and reactive cutaneous capillary endothelial prolifera- tion (RCCEP) were significantly different between the two groups, but this may be due to the use of ICIs. In conclusion, based on the scant data avail- able, there is no solid evidence that LRT + ST will bring an additional AE burden to iCCA patients. This meta-analysis study also had several limi- tations. First, the LRT + ST used in the study was diverse and nonstandard treatment. Among the ten original studies incorporated, there were vari- ations in the selection of locoregional treatment modalities, the timing of incorporating locore- gional therapies, and the local pharmacological interventions for interventional therapy. Future guidelines should specify when and what locore- gional therapies should be combined. Second, the application of chemotherapy schemes included in the study was not completely consistent, which led to strong heterogeneity between different studies and the failure to obtain a specific survival time. Extensive randomized controlled trials are needed to confirm the findings of this study. In conclusion, LRT + ST resulted in more sur- vival benefits than ST without increasing the inci- dence of complications for unresectable and meta- static iCCA, which can be used as a supplement to the practice guidelines. Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References 1. Florio AA, Ferlay J, Znaor A, Ruggieri D, Alvarez CS, Laversanne M, et al. Global incidence and trends in intra- and extrahepatic cholangiocarcinoma from 1993 to 2012. Cancer 2020; 126: 2666-78. doi: 10.1002/cncr.32803 2. Kim D, Konyn P, Cholankeril G, Bonham CA, Ahmed A. 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A study of second-line treatment with apatinib in patients with advanced intrahepatic cholangiocarcinoma. 2023. [cited 2023 Sep 10]. Available at: https://clinicaltrials.gov/study/NCT03251443 Radiol Oncol 2023; 57(4): 430-435. doi: 10.2478/raon-2023-0045 430 review Retropharyngeal calcific tendinitis in the neurological emergency unit, report of three cases and review of the literature Tatjana Filipovic1, Jernej Avsenik2 1 Institute of Clinical Neurophysiology, Division of Neurology, University Medical Centre, Ljubljana, Slovenia 2 Institute of Radiology, University Medical Centre, Ljubljana, Slovenia Radiol Oncol 2023; 57(4): 430-435. Received 4 July 2023 Accepted 17 August 2023 Correspondence to: Tatjana Filipović, M.D., Ph.D., Institute of Clinical Neurophysiology, University Medical Centre, 1000 Ljubljana, Zaloška cesta 7, SI-1000 Ljubljana, Slovenia. E-mail: tatjana.filipovic@kclj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Retropharyngeal calcific tendinitis (RCT) is a relatively benign condition of calcination of the longus colli muscle tendon of unknown origin, which causes severe acute neck pain. However, it is often not recognised, which leads to delayed diagnosis and unnecessary treatment. Patients and methods. We have searched PubMed and Google Scholar for publications which reported at least one patient with RCT and were published in the last 20 years. The literature was then analysed according to the PRISMA-S protocol. We also report three patients with RCT presenting at the Neurological Emergency Unit, University Medical Centre, Ljubljana, Slovenia, from 1 January 2020 to 1 June 2022. We discuss their clinical presentation and differential diagnosis, explain our decision-making process, and briefly describe the clinical course. Case reports have been performed according to the CARE protocol. Results. We have analysed a total of 112 titles with 231 patients. The most frequent symptoms and signs were: neck pain, neck stiffness and odynophagia, as was the case in our reported cases. Conclusions. RCT is a dramatic yet self-limiting condition, often not recognised in time. An effort should be made to increase neurologists’ awareness about this condition. Key words: retropharyngeal calcific tendinitis Introduction Calcifications in the retropharyngeal space as a cause of severe acute head and neck pain have long been recognised, but have only recently been incor- porated in the latest (3rd) International Classification of Headache Disorders (ICHD-3). Retropharyngeal calcific tendinitis (RCT) is, according to ICHD-3 cri- teria, a “headache caused by inflammation or calci- fication in the retropharyngeal soft tissues”.1 It oc- curs as a result of poorly understood mechanisms in the upper fibres of the longus colli muscle. Data from a similar condition affecting the shoulder joint showed neovascularisation and new nerve growth as a result of the innate immune response.2 This often dramatic but self-limiting condition is often not recognised among physicians, which leads to unnecessary diagnostic procedures and delayed treatment.3,4 Besides neurologists, other specialists are also involved in treatment of this condition, es- pecially otorhinolaryngologists (ENT)3,4 and ortho- paedic surgeons.5 The annual incidence of RCT is estimated to be from 0.5 cases per 100 000, up to 1.1 case per 1000.4,6 The aim of the present study was to review the literature data and present our own experience with RCT in order to increase neurologists’ aware- ness about the condition. Radiol Oncol 2023; 57(4): 430-435. Filipovic T and Avsenik J / Retropharyngeal calcific tendinitis review 431 Patients and methods On 7 September 2022 we have searched the PubMed and Google Scholar with the keywords “retropharyngeal calcific tendinitis”, “longus colli tendinitis” and “acute neck pain”. The search was conducted for the titles published in the last 20 years (2002–2022). The analysis of the literature was performed according to the PRISMA-S proto- col.7 We analysed the frequency of reports of RCT regarding the facility where patients were first registered and treated. In addition, we report three cases of ret- ropharyngeal calcific tendinitis (RCT) that were referred to the Neurological Emergency Unit (NEU), Division of Neurology, University Medical Centre Ljubljana, Slovenia, from 1 January 2020 to 1 June 2022. The reports follow the CARE proto- col.8 Patients NEU is a tertiary medical facility for over 300 000 inhabitants where 25 016 patients were exam- ined in the time period described above, of which 2073 were discharged with the main diagnosis “Headache, unspecified” (G 44.8 or R 51) accord- ing to ICD-10 and only three were diagnosed with RCT (M 65.2). Patient 1 A previously healthy 40-year-old female with sus- pected meningitis was referred to our institution in November 2020. She was experiencing excru- ciating throbbing neck pain, which had devel- oped spontaneously within 12 hours without any trauma or heavy mechanical load. She complained that swallowing was painful and that the pain in- creased with any attempt to move the head. A neu- rological exam showed severe neck stiffness with immobility in all directions as well as dysesthesia over the vertex and occipital regions. Laboratory workup revealed only mildly increased C reactive protein (CRP) of 20 mg/l (normal value < 5 mg/l) and white blood cell count (WBC) of 10.2 (normal < 10x109/l). Although the patient was afebrile, the re- tropharyngeal abscess was still considered in the differential diagnosis. Magnetic resonance imag- ing (MRI) of the neck showed fluid collection and swelling in the cranial part of the longus capitis/ colli muscle on the left (Figure 1). A lumbar punc- ture was also performed, but the CSF was normal. She was treated in the NEU and discharged with ketoprofen 200 mg daily and a soft neck collar. The pain resolved in one month. Patient 2 A 51-year-old female with a history of arterial hy- pertension was referred to the NEU in November 2021 with clinical suspicion of meningitis. Five days prior to referral, she experienced a sudden mild pain in the right posterior neck, radiating to the occipital region. In the following three days, the pain became unbearable. It increased during swallowing and with eye movements. Due to the chronic neck pain, the patient had an MRI of the neck 9 months prior to the examination. Broad- based protrusions of the C5/6 and C6/7 interver- tebral discs without compromise of neural struc- tures were described, but no other abnormalities FIGURE 1. MRI in a 40-year-old female showed short tau inversion recovery sequence (STIR) hyperintensity in the upper part of her left Longus colli muscle, suggesting an oedema (A), with thin prevertebral effusion on sagittal images (B). After intravenous gadolinium contrast injection, a small area of enhancement was observed in the medial aspect of the muscle (C), but no peripherally enhancing collection to suggest an abscess was present. Diffusion-weighted imaging was normal, excluding the presence of pus (D). A B C D Radiol Oncol 2023; 57(4): 430-435. Filipovic T and Avsenik J / Retropharyngeal calcific tendinitis review432 were noted at the time. She was afebrile at pres- entation. A neurological exam showed limited mobility of the neck and reduced light touch sense over the right lower extremity. Laboratory workup was normal. Head CT and computed tomography angiography (CTA) of the aortocervical and intracranial vessels revealed no vascular abnormalities. MRI of the cervical spine revealed prevertebral oedema from the C1 to the C4 level (Figure 2), not seen on previous MRIs. In addition, calcifications in front of the C1 arc on CTA was noted. The patient had been treated three days in the hospital and discharged with ibupro- fen, 1800 mg daily. The pain gradually subsided over the next two months. The sensory distur- bance over the left leg remained unexplained. Patient 3 In May 2022, a previously healthy 43-year-old female experienced pain in the right posterior neck which evolved gradually over a period of 48 hours. She described an electric shock-like pain that was radiating to the occipital area at any at- tempt of head extension, and even if she tried to hold the head in the neutral position. Laboratory workup at the Medical Emergency Unit revealed only slightly elevated CRP. Pain medication (met- amizole 2.5 g IV) was minimally effective. Three days after the onset of pain the patient was ex- amined at the NEU where occipital neuralgia was suspected, and outpatient MRI of the head and neck was suggested. As the pain continued, she came to the NEU on day 5 and spondylodiscitis was added to differential diagnosis. A neck CT revealed calcifications in the region of alar liga- ments and repeated laboratory results showed el- evated CRP of 20 mg/l (normal < 5 mg/l), with no other abnormalities. The patient was discharged with ibuprofen 1200 mg daily. As the pain con- tinued without relief, she returned to the NEU on day 7. This time RCT was diagnosed and a short course of corticosteroids was prescribed (dexa- FIGURE 2. STIR (short tau inversion recovery) imaging in sagittal (A) and axial (B) plane demonstrated prevertebral soft tissue swelling and oedema in a 51-year- old female, suggesting retropharyngeal calcific tendinitis as the underlying cause. Calcifications in the medial aspect of the longus colli muscle in front of the C1 arc were noted on computed tomography angiography (CTA) (C), confirming the diagnosis. A B C TABLE 1. Results from literature analysis N % SPECIALITY REPORTS 112 100 Otorhinolaryngology (ENT) 32 28.6 Emergency medicine 26 23.2 Orthopaedic surgery 24 21.4 Other 19 17 Neurology 11 9.8 PATIENTS TOTAL 231 100 Sex: women:men 121:110 52.4:47.6 Age (years) Median 22–78 46.7 No comorbidities 224 96 Acute onset (24–72 hours) 208 91 LEADING SYMPTOMS Neck pain 231 100 Neck immobility 222 96 Odynophagia 210 91 Trismus 35 15 Torticollis 11 5 Stridor 1 0.4 Dysarthria 1 0.4 Vertigo 1 0.4 DIAGNOSTIC WORKUP Mild to moderate increase in CRP and/or total leucocyte count 216 93 CT 111 43 CT + MR 120 47 Aspiration biopsy 7 3 DIFFERENTIAL DIAGNOSIS Retropharyngeal abscess 134 58 Spondylodiscitis 28 12 Meningitis 25 11 Neck artery dissection 4 1,7 COURSE Marked improvement within 2 weeks 221 95 Radiol Oncol 2023; 57(4): 430-435. Filipovic T and Avsenik J / Retropharyngeal calcific tendinitis review 433 methasone, 4 mg per os daily). The pain disap- peared within one week. Results The literature search returned no randomised controlled trials, meta analyses, clinical trials or systematic reviews. We retrieved a total of 198 ti- tles (Online Resources- 1 and 2), all of which were case and series reports and reviews. Of those, 112 were eligible for the study. The details of article selection are given in Figure 3. The total number of reported cases was 231. The results of literature analysis are summarised in Table 1. Discussion According to ICHD-3 criteria, RCT represents a “headache caused by inflammation or calcifica- tion in the retropharyngeal soft tissues”. The pain is usually severe, continuous, throbbing or elec- trising in quality. Trigeminal afferent fibres from dura and cervical afferent fibres from the skin and muscular tissue in the cervical region converge to synapse onto the same second-order neurons in the trigeminocervical complex.9 This at least par- tially explains occipital (headache) and pharyn- geal (odynophagia) irradiation of the pain as well as neck stiffness (or decreased range of motion), as seen in our patients. Torticollis is sometimes reported, but may represent exaggerated neck stiffness. A detailed review of the (rare) causes of craniofacial and neck pain can be found in the lit- erature.10 Little is known about the causes of calcium dep- osition or inflammation in the longus colli mus- cle. One report on histological findings in the re- tropharyngeal tissue of RCT patients has revealed a foreign-body type of inflammation around hy- droxyapatite crystals.11 Immunological mecha- nisms involving the innate immune system in the form of new nerve growth and neovascularisation within the tendon in calcific tendinitis of the shoul- der joint have been reported.12 Similar processes in the retropharyngeal space may be a plausible ex- planation in patients with RCT. However, further studies are needed to confirm these speculations. TABLE 2. Differential diagnosis of the Retropharyngeal calcific tendinitis (RCT) Feature RCT Meningitis Abscess Discitis Dissection GON,CH Neck pain +++ ++ +++ +++ ++ ++ Fever - + + + - - Photophobia - + - - - - Nausea - + - - -/+ - Decreased ROM +++ + (flexion) ++ ++ - -/+ Odynophagia ++/+ - ++ -/+ - - Long tract signs - - - -/+ + - CH = cervicogenic headache; GON = greater occipital nerve neuralgia; ROM = range of movement TABLE 3. Radiological clues for differential diagnosis Differential diagnosis Modality RCT ABSCESS TUMOUR X-RAY May show calcifications Prevertebral swelling - Prevertebral swelling CT Calcifications LCM oedema +Peripheral enhancement +Lymphadenopathy +Soft tissue mass (Variable enhancement) +Lymphadenopathy MR May suggest calcifications LCM oedema +Diffusion restriction (pus) (Superior contrast resolution) LCM = longus colli muscle FIGURE 3. Flowchart of article selection. Radiol Oncol 2023; 57(4): 430-435. Filipovic T and Avsenik J / Retropharyngeal calcific tendinitis review434 Differential diagnosis includes other similar conditions that should be promptly recognised by neurologists (Table 2). Meningitis and meningoencephalitis are usu- ally accompanied by fever, photophobia, nausea and/or vomiting, or even an altered mental state1, not typically seen in RCT. They are also accompa- nied by neck stiffness, but only on flexion, not on extension or rotational movements.1 Odynophagia is often seen in RCT, but is a hall- mark of retropharyngeal abscess, where in most cases, the blood count shows elevated leucocytes above 12 x 10 9/l.3,13 Spondylodiscitis, another pos- sible cause of acute severe neck pain, is usually accompanied by clinical and laboratory signs of systemic inflammation, and diagnosis is made af- ter appropriate imaging.14,15 Spontaneous carotid or vertebral artery dissection could present as an isolated head and/or neck pain, sometimes accom- panied by nausea, but no neck stiffness has been reported.16 Lastly, greater occipital nerve (GON) neuralgia and cervicogenic headache (CH), which are relatively benign conditions, characterised by prominent neck pain, should also be kept in mind1.1,17 However, pain in GON neuralgia emerg- es in the form of short (seconds to minutes) attacks, leaving tenderness or allodynia in the region of GON.1 Therefore, when pain evolves over a period of several hours or even days, other causes should be considered. CH is a broad category of chronic conditions, with some diagnostic criteria overlap- ping with RCT. Radiologically, differential possibilities in pa- tients with suspected RCT include retropharyn- geal abscess, tumour or even trauma; therefore, fa- miliarity with typical imaging findings facilitates early diagnosis and may prevent inappropriate therapeutic procedures (Table 3). As plain film radiography may miss subtle calcifications within the tendon, CT is the pre- ferred imaging modality due to its supreme reso- lution and multiplanar capabilities.18 CT reliably confirms the location of calcifications within the superior fibres of the longus colli tendons and may show soft tissue swelling and/or small ret- ropharyngeal effusion as well.19 Nowadays, the first imaging modality that these patients undergo is usually MRI, which demonstrates oedema and retropharyngeal effusion clearly. However, as cal- cifications are much subtler on MRI in comparison to CT, a high level of clinical suspicion is needed for the correct diagnosis. On the other hand, the presence of peripheral postcontrast enhancement or the evidence of pus on diffusion-weighted im- aging should suggest infection as the cause.14 CT is immediately available and cheap but exposes the patient to ionizing radiation, which may be inap- propriate in women in the childbearing age. MRI is not as accessible as CT, is expensive, and lasts longer, which may pose a problem due to inabil- ity of the patient to lay still in the supine position. Finally, MRI may be contraindicated in the cases of metallic implants or claustrophobia. Conclusions RCT is a rare disorder that neurologists should be familiar with. It is also a relatively new subject for neurologists: it has been recognised as a neurolog- ic disorder only in 2013, when the ICHD-3 criteria were published. Typically, the patient is a previ- ously healthy middle-aged person, presenting with the triad of neck pain, neck immobility and painful swallowing. Mildly or moderately elevat- ed CRP and leucocytes are usually present. Neck MRI and/or CT imaging should be performed to confirm diagnosis. Treatment with oral steroids or non-steroidal anti-inflammatory drugs should lead to prompt symptom alleviation. References 1. Headache Classification Committee of the International Headache Society (IHS). The international classification of headache disorders, (beta version). Cephalalgia 2013; 33: 629-808. doi: 10.1177/0333102417738202 2. Oliva F, Via A.G, Maffulli N. Physiopathology of intratendinous calcific depo- sition. BMC medicine 2012; 10: 1-10. doi: 10.1186/1741-7015-10-95 3. Langner S, Ginzkey C, Mlynski R; Weiss L. Differentiation of retropharyngeal calcific tendinitis and retropharyngeal abscess: a case series and review of the literature. Eur Arch Otorhinolaryngol 2020; 277: 2631-6. doi: 10.1007/ s00405-020-06057-w 4. Horowitz G, Ben-Ari O, Brenner A, Fliss DM, Oshri Wasserzug O. Incidence of retropharyngeal calcific tendinitis (longus colli tendinitis) in the gen- eral population. Otolaryngol Head Neck Sug 2013, 148: 955-8. doi: 10.1177/0194599813482289 5. Suh B, Eoh J, Shin J. Clinical and imaging features of longus colli calcific tendinitis: an analysis of ten cases. Clin Orthop Surg 2018, 10: 204-9. doi: 10.4055/cios.2018.10.2.204 6. Boardman J, Kanal E, Aldred P, Boonsiri J, Chijindu Nworgu C, Zhang F. Frequency of acute longus colli tendinitis on CT examinations. Emerg Radiol 2017, 24: 645-51. doi: 10.1007/s10140-017-1537-z 7. Rethlefsen ML, Kirtley S, Waffenschmidt S, Ayala AP, Moher D, Page MJ, et al. PRISMA-S: an extension to the PRISMA statement for reporting literature searches in systematic reviews. Syst Rev 2021; 10: 39. doi: 10.1186/s13643- 020-01542-z 8. Gagnier JJ, Kienle G, Altman DG, Moher D, Sox H, Riley D, et al. The CARE guidelines: consensus-based clinical case reporting guideline development. Headache 2013; 53:1541-7. doi: 10.1111/head.12246. PMID: 24266334 9. Bartsch T, Goadsby PJ. Stimulation of the greater occipital nerve induces increased central excitability of dural afferent input. Brain 2002; 125: 1496- 509. doi: 10.1093/brain/awf166 Radiol Oncol 2023; 57(4): 430-435. Filipovic T and Avsenik J / Retropharyngeal calcific tendinitis review 435 10. Aydil U, Kizil Y, Köybaşioğlu A. Less known non-infectious and neuromus- culoskeletal system-originated anterolateral neck and craniofacial pain disorders. Eur Arch Otorhinolaryngol 2012; 269: 9-16. doi: 10.1007/s00405- 011-1746-0 11. Ring, D, Vaccaro A R, Scuderi G, Pathria MN, Garfin SR. Acute calcific retropharyngeal tendinitis. Clinical presentation and pathological charac- terization. J Bone Joint Surg 1994; 76: 1636-42. doi: 10.2106/00004623- 199411000-00006 12. Hackett L, Millar N L, Lam P, Murrel GAC. Are the symptoms of calcific tend- initis due to neoinnervation and/or neovascularization? J Bone Joint Surg 2016; 98: 186-92. doi: 10.2106/jbjs.o.00417 13. Jain H, Knorr TL, Sinha V. Retropharyngeal abscess. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing 2022. [cited 2022 Sep 10]. Available at: https://www.ncbi.nlm.nih.gov/books/NBK441873/, [Updated 2022 Apr 28] 14. Salaffi F, Ceccarelli L, Carotti M, Di Carlo M, Polonara G, Facchini G, et al. Differentiation between infectious spondylodiscitis versus inflammatory or degenerative spinal changes: how can magnetic resonance imaging help the clinician? Radiol Med 2021; 126: 843-59. doi: 10.1007/s11547-021-01347-7 15. Estimable K, Rizk C, Pujalte GG. A rare case of neck pain: acute longus colli calcific tendinitis in a possibly immunocompromised individual. J Am Board Fam Med 2015; 28: 146-50. doi: 10.3122/jabfm.2015.01.140124 16. Arnold M, Cumurciuc R, Stapf C, Favrole P, Berthet K, Bousser MG. Pain as the only symptom of cervical artery dissection. J Neurol Neurosurg Psychiatry 2006; 77: 1021-4. doi: 10.1136/jnnp.2006.094359 17. Barmherzig R, Kingston W. Occipital neuralgia and cervicogenic headache: diagnosis and management. Curr Neurol Neurosci Rep 2019, 19: 1-8. doi: 10.1007/s11910-019-0937-8 18. Offiah CE, Hall E. Acute calcific tendinitis of the longus colli muscle: spec- trum of CT appearances and anatomical correlation. Br J Radiol 2009; 82: e117-21. doi: 10.1259/bjr/19797697 19. Eastwood JD, Hudgins PA, Malone D. Retropharyngeal effusion in acute calcific prevertebral tendinitis: diagnosis with CT and MR imaging. AJNR Am J Neuroradiol 1998; 19:1789-92. PMID: 9802506 Radiol Oncol 2023; 57(4): 436-445. doi: 10.2478/raon-2023-0055 436 research article Quantitative SSTR-PET/CT for predicting response and survival outcomes in patients with pancreatic neuroendocrine tumors receiving CAPTEM Maria Ingenerf1, Homeira Karim1, Christoph Auernhammer3,4, Matthias Zacherl2, Vera Wenter2, Michael Winkelmann1, Jens Ricke1,3, Frank Berger1, Christine Schmid-Tannwald1,3 1 Department of Radiology, University Hospital, LMU Munich, Munich, Germany 2 Department of Nuclear Medicine, University Hospital, LMU Munich, Munich, Germany 3 ENETS Centre of Excellence, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System at the University Hospital of Munich (GEPNET-KUM), University Hospital of Munich, Munich, Germany 4 Department of Internal Medicine, University Hospital, LMU Munich, Munich, Germany Radiol Oncol 2023; 57(4): 436-445. Received 5 June 2023 Accepted 25 September 2023 Correspondence to: Dr. Christine Schmid-Tannwald, Department of Radiology, University Hospital, LMU Munich, Ziemssenstr. 5, 81377 Munich, Germany. E-mail: Christine.schmid-tannwald@med.uni-muenchen.de Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. This study aimed to evaluate the predictive and monitoring role of somatostatin receptor (SSTR) posi- tron emission tomography-computed tomography (PET/CT) and clinical parameters in patients with neuroendocrine liver metastases (NELM) from pancreatic neuroendocrine tumors (pNET) receiving capecitabine and temozolomide (CAPTEM). Patients and methods. This retrospective study included twenty-two patients with pNET and NELM receiving CAPTEM who underwent pre- and post-therapeutic 68Ga-DOTATATE/-TOC PET/CT. Imaging (including standardized uptake value [SUV] of target lesions [NELM and pNET], normal spleen and liver) and clinical (Chromogranin A [CgA], Ki-67) parameters were assessed. Treatment outcome was evaluated as response according to RECIST 1.1, progres- sion free survival (PFS) and overall survival (OS). Results. The median PFS (mPFS) was 7 months. Responders had a significantly longer mPFS compared to non- responders (10 vs. 4 months p = 0.022). Median OS (mOS) was 33 months (mOS: responders = 80 months, non-re- sponders = 24 months p = 0.182). Baseline imaging showed higher SUV in responders, including absolute SUV, tumor- to-spleen (T/S), and tumor-to-liver (T/L) ratios (p < 0.02). All SUV parameters changed only in the responders during follow-up. Univariable Cox regression analysis identified baseline Tmax/Smean ratio and percentage change in size of pNETs as significant factors associated with PFS. A baseline Tmax/Smean ratio < 1.5 was associated with a shorter mPFS (10 vs. 4 months, (p < 0.05)). Prognostic factors for OS included age, percentage change in CgA and in T/S ratios in univariable Cox regression. Conclusions. SSTR-PET/CT can be useful for predicting response and survival outcomes in pNET patients receiving CAPTEM: Higher baseline SUV values, particularly Tmax/Smean ratios of liver metastases were associated with better response and prolonged PFS. Key words: prognosis; positron emission tomography–computed tomography; neuroendocrine tumors; capecit- abine/temozolomide Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM 437 Introduction Capecitabine/temozolomide (CAPTEM) has shown to be effective and safe in advanced NETs, particularly in well-differentiated pNETs.1,2 CAPTEM is included in national and international guidelines for the treatment of gastroenteropan- creatic neuroendocrine neoplasms, such as those by the European Society for Medical Oncology (ESMO).3,4 In vitro studies have demonstrated an apoptotic synergism between capecitabine and temozolomide (CAPTEM), although the exact mechanism of action in NETs remains unclear.3,5 Capecitabine incorporates 5-fluorodeoxyuridine triphosphate into DNA, inhibiting thymidylate synthase and attenuating the repair activity of methylguanine DNA methyltransferase (MGMT).3 Temozolomide exerts a cytotoxic effect through DNA alkylation/methylation at the O6 and N7 po- sitions of guanine, leading to DNA mismatch and tumor cell death.6 To improve the selection of patients who would benefit from this cytotoxic regimen and avoid un- necessary toxicity due to treatment failure, predic- tive biomarkers need to be identified.7 Potential predictive biomarkers, such as MGMT expression, tumor grade and serum alanine aminotransferase (ALT) activation, have been investigated; however, the results have been controversial. A study by Cives et al. did not recommend biomarker-driven selection criteria for the use of the CAPTEM regi- men.6 Conversely, Wang et al. identified the Ki-67 index as the only independent prognostic factor for overall survival and PFS.7 In addition to the size based RECIST 1.1. criteria, other imaging parameters are increasingly being evaluated for predicting and monitoring onco- logic therapy concepts. The Choi criteria, which integrate changes in tumor density show a better correlation with OS than RECIST in the therapy evaluation of pNET under sunitinib.8 This may be attributed to antiproliferative or antiangiogenic ef- fects, particularly in slow-growing tumors such as NETs.9-11 Approximately 80-95% of good to moderately differentiated NETs overexpress somatostatin receptors (SSTRs) on cell surfaces. PET/CT with 68Ga-labeled somatostatin analogues (SSA) (68Ga- DOTA-TATE, -DOTA-NOC and -DOTA-TOC) al- lows visualization of SSTRs and correlates with the histopathological expression of SSTRs.12-14 SSTR-PET/CT enables detection of NET and its me- tastases with high sensitivity and specificity15 and it is recommended for initial staging and follow-up of gastroenteropancreatic neuroendocrine tumors (GEP-NET) by the European Society for Medical Oncology Guidelines Working Group.4 Although quantitative evaluation of SSTR imag- ing has not yet been standardized, several studies suggest that these tracers could serve as param- eters for therapy monitoring and response predic- tion in various therapeutic approaches in NET pa- tients16 including those undergoing peptide-recep- tor-radionulcide therapy (PRRT)17, or transarterial radioembolization (TARE).18,19 However, to date, no study has investigated the role of SSTR-PET/CT parameters in predicting and assessing tumor response in patients with pNET with liver metastases treated with CAPTEM. Therefore, the aim of this study was to evaluate morphologic and functional imaging factors for predicting and monitoring the therapy response in patients with metastatic pNET treated with CAPTEM. Patients and methods Patients This retrospective study included consecutive patients with histologically proven pNETs who received CAPTEM treatment and underwent pre- and post-therapeutic 68Ga-DOTA-TATE or -DOTA-TOC PET/CT imaging at our department with therapy start between 2012 and 2020. Patient selection for CAPTEM therapy was based on con- sensus decisions made in an interdisciplinary tu- mor conference certified for NETs at our ENETS Center of Excellence. The study was approved by the local research ethics committee (#20-1077), and written informed consent was waived due to the retrospective nature of the study. All procedures performed in studies involving human partici- pants were in accordance with the 1964 Helsinki declaration and its later amendments or compara- ble ethical standards. PET/CT Whole-body PET scans were conducted using ei- ther a GE Discovery 690 (GE Healthcare, Little Chalfont, United Kingdom) or a Biograph 64 TruePoint PET/CT scanner (Siemens Healthcare, Erlangen, Germany) in 3-D mode with a duration of 3 minutes per bed position. The emission se- quence began 60 minutes after intravenous admin- istration of approximately 200 MBq of 68Ga-DOTA- TATE or 68Ga-DOTA-TOC, along with a possible Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM438 administration of 20 mg of furosemide. Emission data were reconstructed with attenuation correc- tion based on a diagnostic CT scan. PET/CT scans encompassed the neck, thorax, abdomen, and pelvis, utilizing a diagnostic CT scan protocol (100−190 mAs, 120 kV, collimation 2 × 5 mm, pitch of 1.5). An iodine-based contrast agent (Ultravist 300TM; Bayer Healthcare, Berlin, Germany; 1.5 mL/kg body weight) was intrave- nously injected at a rate of 2.5 mL/s with a delay of 80−110 seconds to visualize the portal venous phase of the liver. PET images were reconstructed using specific parameters: a transaxial 256 × 256 matrix with VPFX (2 iterations, 36 subsets, 3D Gauss postfilter of 6.5 mm full-width half maxi- mum) for the GE scanner and a transaxial 168 × 168 matrix with TrueX (3 iterations, 21 subsets, 3D Gauss postfilter of 2.0 mm full-width half maxi- mum) for the Biograph scanner. Standardized up- take values (SUV) were calculated using the pa- tient’s body weight (SUVbw). Image analysis PET/CT scans were reviewed by two board-certi- fied radiologists in consensus, who were blinded to the clinical history of the patients, except for the diagnosis of pNET. Two target neuroendocrine liv- er metastases (NELM) larger than 1 cm in size per patient were defined as target lesions based on vis- ibility on CT scan and visual positive somatostatin receptor (SSTR) uptake compared to normal liver parenchyma, with no artifacts within the lesions. The image review process consisted of two sep- arate sessions conducted at a 3-week interval (1. analysis of pre-therapeutic PET/CT and 2. analy- sis of post-therapeutic PET/CT). In each session, the size and density (measured in HU) of the se- lected liver metastases and pNET were recorded. Additionally, the HU of the normal liver parenchy- ma and spleen were measured. The hepatic tumor burden was assessed visually. Semi-quantitative measurements were performed by placing circular volume of interest (VOI) in the respective target le- sions/organs to obtain maximum and mean stand- ardized uptake values (SUVs) of the liver metasta- ses, pNET, normal liver parenchyma, and healthy spleen parenchyma. SUV tumor-to-organ ratios, including tumor-to-spleen (T/S) ratio and tumor- to-liver (T/L) ratio, were calculated as SUVmax (liver metastasis) divided by SUVmean (normal liver or spleen respectively) and SUVmean (liver metastasis) divided by SUVmean (liver or spleen) for normalization purposes. Standard of reference and response to treatment Clinical, histopathological, surgical records of each patient were collected by a third radiologist. The histopathological diagnosis of pNET and the Ki-67 labeling index of the primary tumor were confirmed for all patients. Tumor grading was performed based on WHO Tumor Classification Guideline, which categorized tumors into G1 (Ki- 67 Index < 3%), G2 (Ki-67 Index 3−20%), and G3 NET/NEC (Ki-67 Index > 20%). The treatment response was assessed using RECIST 1.1 criteria. Patients were classified as re- sponders (R) if they achieved a complete response (CR) or partial response (PR) based on the first follow-up PET/CT scan. Non-responders were de- fined as patients with stable disease (SD) or pro- gressive disease (PD). Progression-free survival (PFS) was calculated in months from the initiation of CAPTEM treatment until progression, as de- termined by imaging and clinical parameters ac- cording to the local interdisciplinary tumor board. Overall survival (OS) was measured in months from the start of CAPTEM treatment until death from any cause. Patients who were still alive at the last follow-up in December 2022 were censored. Statistical analysis All data were presented as mean or median val- ues with standard deviation (SD) or interquartile range [IQR], respectively. The normal distribution of continuous variables was assessed by visually inspecting the frequency distribution using histo- grams. To compare imaging parameters such as SUV, SUV tumor-to-spleen (T/S) ratios, SUV tumor- to-liver (T/L) ratios, size, HU, and quantitative clinical parameters before and after therapy, a Wilcoxon signed rank test was employed. The Mann-Whitney test was used to compare these pa- rameters between different response groups. OS and PFS were analysed using the Kaplan- Meier method, and survival curves were com- pared using the Breslow-Wilcoxon test. Prognostic clinical and imaging parameters for PFS and OS were analysed using Cox proportional hazards regression. In the multivariable model, variables with a p-value ≤ 0.05 in the univariable analysis were included using a stepwise approach. A statis- tical significance level of p ≤ 0.05 was considered significant. Statistical analyses were conducted using commercially available software, including Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM 439 GraphPad Prism Version 6 (San Diego, CA) and SPSS version 25 (Chicago, IL). Results Patients characteristics 22 patients with a total of 44 target NELM and 16 pNETs were included for PET/CT analysis. For den- sity measurements, two patients were disregarded due to non-contrast-enhanced CT scans. Six pa- tients underwent surgery with resection of pNET and three patients underwent splenectomy. The baseline PET/CT scans were obtained 58 d prior to CAPTEM initiation (IQR 32 -103d), and follow-up PET/CT scans were performed 132 d after the start of therapy (IQR 88 – 192d). The majority of patients were male (77%), and most had G2 tumors (77%), with a minority having G3 tumors (18%). Detailed patient characteristics are presented in Table 1. OS, PFS and Treatment response according to RECIST 1.1 According to RECIST 1.1 criteria, 10 patients exhib- ited a partial response (PR), 11 patients had stable disease (SD), and 1 patient showed progressive disease (PD) on the first follow-up PET/CT scan. The objective response rate (ORR) was 46%, with PD and SD classified as non-responders. The overall median PFS was 7 months (95% CI: 1.5–12.5 months). Among the responders (n = 10), the median PFS was 10 months (95% CI: 6.9–13.1 months), while in the non-responder group (n = 12), it was 4 months (95% CI: 3.3–4.7 months). The difference in PFS between the two groups was statistically significant (p = 0.022). The overall median OS was 33 months (95% CI: 0–77 months). Among the responders (n = 10), the median OS was 80 months (95% CI: 11–149 months), whereas in the non-responder group (n = 12), it was 24 months (95% CI: 14–34 months). Although the difference in OS between the two groups was not statistically significant (p = 0.182). Response according to RECIST 1.1. Prognostic imaging and clinical parameters Significant differences were observed in the re- sponse groups based on RECIST 1.1 criteria. Responding NELM exhibited higher baseline TABLE 1. Patient characteristics Sex Male 17 (77%) Female 5 (23%) Median age, years (range) 66 (40-85) Grading G1 1 (5%) G2 17 (77%) G3 4 (18%) Median Ki-67 (ng/ml, range) 12 (2–40) Treatment with CAPTEM Duration of treatment (month, range) 7.5 (3−20) Prior treatment 16 (73%) pNET resected 6 (27%) Prior medical treatment 9 (41%) Prior PRRT 6 (27%) Prior liver targeted therapy 6 (27%) CAPTEM = capecitabine and temozolomide; pNET = pancreatic neuroendocrine tumors; PRRT = peptide-receptor-radionulcide therapy A B C D FIGURE 1. 72-year-old male with responding liver metastases of pancreatic neuroendocrine tumor. on the pretherapeutic PET/CT (A, B) there were high tumor-to- liver (t/l) ratios. after three months of treatment with CAPTEM, both liver metastases showed a shrinkage in size, but also a significantly reduced uptake of 68ga-dotatate compared to pretherapeutic PET/ CT (C, D). CAPTEM = capecitabine and temozolomide Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM440 SUVmax (47 vs. 24, p < 0.02) and higher baseline SUVmean (30 vs. 14, p < 0.004) compared to non-re- sponding lesions (Table 2). Furthermore, the SUV ratios of Tumor/Liver (T/L ratio) and Tumor/Spleen (T/S ratio) were significantly different between the response groups on pretreatment imaging, with responding NELM demonstrating higher ratios. For instance, the Tmean/Lmean ratio was 4.8 (R) compared to 2.1 (NR) (p = 0.006). However, there were no significant differences in SUVmax and SUVmean of the primary neuroendocrine tumor (pNET) between the response groups. Baseline clinical and laboratory parameters did not differ between the response groups (Table 2). Changes between baseline and follow-up imaging Significant changes were observed in SUVmax and SUVmean of the liver metastasis between baseline and follow-up imaging in responders, whereas no changes were observed in non-responders (Figure 1). Similarly, the T/L and T/S ratios showed a significant decrease in responders, while there was no relevant change in non-responders, except for the Tmean/Lmean ratio, which also demon- strated a decrease in non-responder. The HU of the NELM exhibited a slightly significant decrease in non-responder (p = 0.02), while no significant change was observed in responding lesions. In TABLE 2. Imaging and clinical parameters on baseline and follow-up imaging Responder Non-Responder Baseline Rvs. NR Baseline Follow-up p-valuebaseline vs. FU Baseline Follow-up p-value baseline vs. FU p-value Age (years) 66 (56−75) 69 (57−82) 0.44 Grading 13 (7−20) 10 (4−15) 0.33 G1 0 1 G2 6 11 G3 4 0 Ki-67 (%) 12.5 (7.3–20) 10 (4.3–15) 0.33 Male sex 7 (70%) 10 (83%) Chromogranin A 796 (512–2756) 270 (102–1136) < 0.04 178 (90–845) 198 (96–1071) < 0.02 0.06 Bilirubin (mg/dl) 0.6 (0.60.88) 0.85 (0.5–1.1) 0.64 0.6 (0.43–0.9) 0.55 (0.33–0.88) > 0.99 0.45 Hepatic tumor burden (%) 35 (5–40) 13 (5–33) 0.16 13 (5–38) 15 (5–38) 0.25 0.42 SUVmax LM 47 (24–62) 21 (13–46) < 0.04 24 (13–43) 21 (11–39) 0.15 < 0.02 SUVmean LM 30 (15–38) 15 (11–24) 0.04 14 (9–22) 11 (8–17) 0.40 < 0.0004 Tmax/Lmean 6.9 (3.2–11.3) 2.8 (1.6–8.3) 0.03 3.6 (2.0–5.8) 3.2 (1.5–5.5) 0.6 0.0192 Tmean/Lmean 4.8 (2.0–6.8) 1.9 (1.2–3.9) 0.02 2.1 (1.4–2.9) 1.6 (1.1–2.6) 0.05 0.0061 Tmax/Smean 2.0 (1.4–4.1) 0.9 (0.5–3.0) 0.01 1.3 (0.5–1.9) 1.3 (0.4–2.2) 0.12 0.0469 Tmean/Smean 1.4 (0.8–2.1) 0.7 (0.4–1.5) 0.007 0.7 (0.4–1.1) 0.6 (0.3–1.1) 0.19 0.0094 Size LM (mm) 32 (24–42) 20 (14–32) < 0.0001 27 (17–36) 30 (18–43) 0.12 0.10 HU LM 106 (88–116) 106 (95–126) 0.41 92 (85–108) 90 (68 –104) 0.02 0.09 SUVmax pNET 26 (14–47) 26 (16–41) 0.94 30 (12–59) 28 (16–45) 0.2 0.68 SUVmean pNET 17 (9–22) 18 (13–28) 0.81 15 (9–33) 15 (15–30) 0.57 0.98 Size pNET 35 (25–38) 20 (14–37) 0.03 34 (20–47) 34 (23–54) 0.81 0.92 HU pNET 105 (77–113) 94 (88–98) 0.79 107 (81–117) 92 (81–100) 0.15 0.89 FU = Follow-up; HU = Hounsfied unit, L = liver; LM = liver metastases; pNET = pancreatic neuroendocrine tumor; SUV = standardized uptake value; S = spleen; T = tumor Data are given as median (25th and 75th percentile) or number (percentage) Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM 441 terms of clinical parameters, CgA demonstrated a significant decrease of 72% in responders, whereas there was a slight increase of 41% non-responders (Table 2). Cox regression analysis for progression free survival (PFS) In the univariable analysis, both the baseline Tmax/ Smean ratio and the percentage change in the size of pNETs demonstrated a significant association with PFS (Table 3). However, in the multivariable model, none of the parameters remained statisti- cally significant. Nonetheless, it is worth noting that the baseline Tmax/Smean ratio showed a bor- derline association (p = 0.09), which may be attrib- uted to the limited sample size. Subsequently, a re- ceiver operating characteristic (ROC) analysis was performed, revealing a baseline Tmax/Smean ratio of < 1.5 as the optimal cutoff for identifying pa- TABLE 3. Cox regression analysis for progression free survival (PFS) Univariable Multivariable Exp (B) 95% CI p-value Exp (B) 95% CI p-value Baseline Age (years) 1.037 0.995−1.082 0.084 Sex 1.105 0.324−2.528 0.849 Grading G1 0.251 G2 0 0 0.985 G3 2.913 0.827−10.625 0.096 Ki-67 1.02 0.978−1.063 0.362 Chromogranin 1 1 0.989 Hepatic tumor burden 0.98 0.955−1.006 0.125 SUVmax LM 0.979 0.951−1.007 0.134 SUVmean LM 0.982 0.94−1.025 0.41 SUVmax Pancreas 0.983 0.956−1.01 0.21 SUVmean Pancreas 0.975 0.93−1.023 0.3 Tmax/Lmean 0.922 0.821−1.037 0.175 Tmean/Lmean 0.886 0.72−1.09 0.251 Tmax/Smean 0.588 0.373−0.927 0.022 0.626 0.365−1.076 0.09 Tmean/Smean 0.474 0.223−1.004 0.051 % change CgA 1.004 1−1.008 0.056 SUVmax LM 1.002 0.99−1.013 0.792 SUVmean LM 0.998 0.986.1.009 0.686 HU LM 0.999 0.975−1.024 0.948 SUVmax Pancreas 1.002 0.996−1.008 0.451 SUVmean Pancreas 1.002 0.995−1.009 0.512 Tmax/Lmean 1.003 0.992−1.013 0.623 Tmean/Lmean 0.99 0.989−1.009 0.836 Tmax/Smean 1.006 0.99−1.013 0.079 Tmean/Smean 1.006 0.998−1.013 0.149 Size LM (mm) 0.999 0.983−1.015 0.908 Size Pancreas 1.018 1.001−1.034 0.037 1.009 0.99−1.009 0.37 Size total (RECIST) 1.014 0.993−1.036 0.183 HU = Hounsfied unit; L = liver; LM = liver metastases; pNET = pancreatic neuroendocrine tumor; SUV = standardized uptake value; S = spleen; T = tumor Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM442 tients with a shorter median PFS (sensitivity: 60%, specificity: 89%), with a median PFS of 10 months compared to 4 months in the lower ratio group (p = 0.047). These results were comparable to the PFS when classified by RECIST with a median PFS of 10 months (95% CI: 6.9–13.1) in the responder (R) group (n = 10), whereas it was 4 months in the non-responder (NR) group (n = 12) (95% CI: 3.3–4.7, p = 0.022). Cox regression for overall survival (OS) In the univariable analysis, patient age, percent change of CgA, percent changes of Tmax/Smean ratio, and Tmean/Smean ratio were found to be sig- nificant factors associated with survival (Table 4). However, in the multivariable analysis, none of these parameters remained significant, although patient age showed borderline significance with a TABLE 4. Cox regression analysis for overall survival (OS) Univariable Multivariable Exp (B) 95% CI p-value Exp (B) 95% CI p-value Baseline Age (years) 1.059 1.003-1.118 0.039 1.054 0.996-1.115 0.071 Sex 2.326 0.486-11.4 0.291 Grading G1 0.64 G2 0 0 0.988 G3 2.137 0.442-10.325 0.345 Ki-67 1.033 0.979-1.09 0.24 Chromgranin 1 1.0-1.0 0.572 Hepatic tumor burden 1.005 0.974-1.036 0.776 SUVmax LM 0.982 0.949-1.016 0.294 SUVmean LM 0.951 0.894-1.012 0.114 SUVmax Pankreas 0.962 0.916-1.011 0.125 SUVmean Pankreas 0.912 0.813-1.023 0.115 Tmax/Lmean 0.988 0.856-1.142 0.874 Tmean/Lmean 0.879 0.653-1.184 0.396 Tmax/Smean 0.83 0.516-1.336 0.443 Tmean/Smean 0.615 0.239-1.581 0.313 % change CgA 1.006 1.0-1.012 0.046 1.003 0.996-1.01 0.448 SUVmax LM 1.004 0.99-1.018 0.563 SUVmean LM 1.006 0.99-1.022 0.493 HU LM 0.986 0.956-1.017 0.374 SUVmax Pankreas 1.006 0.998-1.013 0.118 SUVmean Pankreas 1.007 0.998-1.015 0.132 Tmax/Lmean 1 0.989-1.011 0.986 Tmean/Lmean 1.001 0.988-1.013 0.933 Tmax/Smean 1.014 1.002-1.026 0.02 1.008 0.971-1.046 0.68 Tmean/Smean 1.017 1.003-1.032 0.02 1.011 0.973-1.05 0.58 Size LM (mm) 1.011 0.991-1.031 0.289 Size Pancreas 1.004 0.986-1.023 0.672 Size total (RECIST) 1.009 0.988-1.031 0.409 HU = Hounsfied unit; L = liver; LM = liver metastases; pNET = pancreatic neuroendocrine tumor; SUV = standardized uptake value; S = spleen; T = tumor Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM 443 p-value of 0.07. In a subsequent receiver operating characteristic (ROC) analysis, a percent change of Tmean/Smean ratio ≥ -35 was identified as the op- timal cutoff for stratifying patients with a shorter median OS after treatment. This cutoff had a sensi- tivity of 80% and specificity of 67%. Mean OS was 71 months (95% CI: 10–53 months) compared to 44 months (95% CI: 14–17 months) in the lower per- centage decrease group (p = 0.034). No significant difference in OS was observed between responder (R) and non-responder (NR) groups according to RECIST 1.1. Mean OS was 60 months (95% CI: 9–42 months) in the R group compared to 56 months (95% CI: 15–28 months) in the NR group (p = 0.182). Discussion In this study we investigated the use of clinical, morphological, and functional imaging param- eters for response assessment and prediction of pNETs treated by CAPTEM. Our findings high- light the potential of quantitative SSTR-PET/CT as a valuable tool for predicting and monitoring treatment response and survival in pNET patients receiving CAPTEM therapy. The overall median PFS in our cohort was 7 months, which appears relatively low compared to the range of 6 to 34 months reported in the lit- erature for advanced neuroendocrine neoplasms regardless tumor site of origin.3 However, report- ed PFS times vary considerably and are likely in- fluenced by factors such as tumor grading, prior treatments, and variations in the administration of the CAPTEM regimen across studies. Our cohort consisted of rather heavily pretreated patients, which may contribute to these differences, as al- so observed in a retrospective work by D’Alpino Peixoto et al.20 The overall median OS in our popu- lation was 33 months, consistent with recent stud- ies reporting median OS times ranging from 29 to 75 months.3,21 Generally, pNETs are known to have a better response to the CAPTEM regimen com- pared to non-pancreatic NETs.20.21 The ORR according to RECIST was rather high in our cohort, reaching 45%, while other studies fo- cusing on pNETs reported ORRs between 21% and 54%.3,21 In our study, treatment response based on RECIST 1.1 showed a slightly improved PFS of 10 months compared to 4 months; however, this did not result in a significant increase in OS. It should be noted that the assessment of OS can be chal- lenging in slow-growing tumor types like NET, where patients often have long survival times and receive a variety of different post-progression therapy regimens. Interestingly, in our study, baseline imaging re- vealed significantly higher SUV in liver metasta- ses that responded to treatment compared to non- responding lesions. Moreover, all calculated SUV ratios including tumor-to-liver (T/L) ratios and tumor-to-spleen (T/S) ratios, were significantly higher in responding lesions. We also found that a higher baseline Tmax/Smean of NELM was as- sociated with longer PFS in our univariable Cox regression analysis (HR 0.59; 95% CI 0.37−0.93, p = 0.02). Using a cutoff of > 1.5 for baseline Tmax/ Smean yielded similar median PFS times as re- sponse classification according to RECIST 1.1, with 10 months compared to 4 months. Several studies investigating peptide-receptor- radionulcide therapy (PRRT) have also reported higher baseline SUV and pretherapeutic T/L ra- tios as prognostic factors for a better treatment response, suggesting cutoffs of SUVmax between 13 and 18 to distinguish responders from non-re- sponders.23-25 This observation can be explained by the fact that PRRT is a receptor-directed treatment approach, where SUV values roughly correspond to the dose delivered by PRRT.23 However, the un- derlying mechanism in the context of cytoreduc- tive therapy, such as CAPTEM, remains unclear. One possible explanation is that a target lesion with a higher SSTR-expression might be more dif- ferentiated, although this contrasts with the com- mon observation that particularly high-grade NET profit from CAPTEM treatment. On follow-up imaging, we observed signifi- cant changes in SUV parameters in NELM that responded to treatment, whereas non-responding NELM showed no changes between baseline and follow-up imaging. However, despite this finding, we did not find an association between percent- age changes in SUV parameters and PFS in our regression analysis. Interestingly, we did find that percentage changes in Tmax/Smean and Tmean/ Smean were significantly associated with OS in our univariable analysis (e.g., Tmean/Smean HR 1.017, 95% CI 1.003– 1.032, p = 0.02), although these association did not remain significant in our mul- tivariable analysis. A percentual decrease in the Tmean/Smean ratio ≤ -35% was associated with a slightly longer mean OS of 71 months compared to 44 months (p = 0.03). In relation to FDG PET/CT, several studies have demonstrated that SUV reduction after treatment can predict survival. For example, this has been observed in patients with liver metastasis of pan- Radiol Oncol 2023; 57(4): 436-445. Ingenerf M et al. / SSTR-PET/CT for predicting response and outcomes in patients with pNET receiving CAPTEM444 creatic cancer treated with TARE and in breast cancer patients receiving targeted therapies.26,27 Regarding the primary tumor we did not de- tect any statistically significant changes in SUV during treatment and between response groups. Only the size of the pNETs decreased signifi- cantly in responders while there was no change in non-responders. These observations might be related to the small number of total pNET (n = 16). Another possibility is that functional / morpho- logical changes might be different between liver metastasis and the primary tumor as discussed by Ingenerf et al.28 The Choi et al. response criteria suggested that changes in tumor attenuation could better repre- sent treatment response to imatinib in gastrointes- tinal stromal tumors.29 While Choi et al. found a decrease in tumor density of more than 15% on CT had a sensitivity of 97% and a specificity of 100% in identifying PET responders versus 52% and 100% by RECIST, we observed a slight, but statis- tically significant decrease in HU values in non- responding lesions (p = 0.02), while no significant change was observed in responding lesions. Regarding clinical parameters, changes in CgA were significantly different between response groups according to RECIST (responders: -72% vs. non-responders: + 41%). Also, percentage changes of CgA were identified as predictive factor for OS, although statistical significance was not reached in the multivariable model. It should be noted that CgA levels can be influenced by various condi- tions such as gastritis and liver cirrhosis, limiting its use as a tumor marker despite its correlation with tumor progression in several studies.30,32 Another potential biomarker, Ki-67, has con- troversial applicability in patients with neuroen- docrine tumors (NETs) receiving CAPTEM thera- py.3,33,34 Therefore, at present, no biomarker-driven selection criteria for use of the CAPTEM regimen can be recommended.6 In our patient cohort, we found no differences in Ki-67 between response groups, and no correlation with OS or PFS. It is im- portant to note that the small number of patients with G1 (n = 1) and G3 (n = 4) tumors in our cohort might have limited the ability to detect significant associations. The main limitations of this study were its small patient cohort and its retrospective nature, accom- panied by heterogeneous time intervals between PET/CTs and CAPTEM initiation, as well as het- erogenous prior therapies. Additionally, while the quantification of SUVs is well-established for FDG PET/CT using the PERCIST criteria35, its applica- tion to SSTR-PET/CT is less established. Therefore, caution must be exercised when interpreting SUV changes, as they can be attributed to tumor re- gression or dedifferentiation.23 Some authors have suggested that normalized SUV measures, such as tumor-to-spleen, liver, or blood pool ratios, may be more reliable than absolute SUV measurements.36,37 Another limitation is that patients underwent scans using different scanners and different so- matostatin analogs, which further supports the preference for tumor-to-organ ratios. Furthermore, in some cases, the pre- and post-treatment scans from the same patient was performed on different scanners. Our study highlights the potential value of quantitative SSTR-PET/CT in predicting response and survival outcomes in patients with pNETs receiving CAPTEM. Responders exhibited higher SUV values on baseline imaging, and the baseline Tmax/Smean ratio showed a significant associa- tion with PFS. Moreover, the observed significant decrease in SUV values in responding NELM dur- ing follow-up imaging supports the utility of these parameters for treatment monitoring. These find- ings provide valuable insights into non-invasive tools for guiding treatment strategies, monitor- ing response, and predicting patient outcomes. Further research is needed to validate and expand upon these findings. Incorporating these param- eters into routine clinical practice could enhance patient care and enable more personalized treat- ment approaches. References 1. Dogan I, Tastekin D, Karabulut S, Sakar B. 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Strosberg JR, Cives M, Brelsford M, Black M, Meeker A, Ghayouri M. Identification of response predictors to capecitabine/temozolomide in metastatic pancreatic neuroendocrine tumors. J Clin Oncolog 2015; 33: 4099. doi: 10.1200/jco.2015.33.15_suppl.4099 34. Spada F, Antonuzzo L, Marconcini R, Gelsomino F, Fumagalli C, Messerini L, et al. Chemotherapy with capecitabine plus temozolomide (CAP-TEM) in patients with advanced neuroendocrine neoplasms (NENs): an Italian multi- center retrospective analysis. J Clin Oncol 2015; 33: e15174-e. doi: 10.1200/ jco. 2015. 33.15_suppl.e15174 35. O JH, Lodge MA, Wahl RL. Practical PERCIST: a simplified guide to PET Response Criteria in Solid Tumors 1.0. Radiology 2016; 280: 576-84. doi: 10.1148/radiol.2016142043 36. Opalińska M, Morawiec-Sławek K, Kania-Kuc A, Al Maraih I, Sowa-Staszczak A, Hubalewska-Dydejczyk A. 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J Nuclear Med 2020; 61: 217-21. doi: 10.2967/jnumed.119.228072 Radiol Oncol 2023; 57(4): 446-454. doi: 10.2478/raon-2023-0051 446 research article Breast-lesion assessment using amide proton transfer-weighted imaging and dynamic contrast-enhanced MR imaging Lulu Zhuang1,2, Chun Lian1,2, Zehao Wang2, Ximin Zhang1,2, Zhigang Wu3, Rong Huang1 1 Department of Radiology, Peking University Shenzhen Hospital, Shenzhen, China 2 Shantou University, Shantou University Medical College, Shantou, China 3 Clinical & Technical Support, Philips Healthcare (Shenzhen) Ltd., China Radiol Oncol 2023; 57(4): 446-454. Received 23 May 2023 Accepted 1 September 2023 Correspondence to: Rong Huang, Department of Radiology, Peking University Shenzhen Hospital, China. E-mail: huangrong0260@outlook.com Disclosure: No potential conflicts of interest were disclosed. Lulu Zhuang and Chun Lian are co-first authors on the paper. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Previous studies have indicated that amide proton transfer-weighted imaging (APTWI) could be utilized for differentiating benign and malignant tumors. The APTWI technology has increasingly being applied to breast tumor research in recent years. However, according to the latest literature retrieval, no relevant previous studies compared the value of APTWI and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) in distinguishing benign lesions from malignant lesions. In the present study, the application of APTWI and DCE for differentiating the benign and malignant breast lesions was investigated. Patients and methods. APTWI was performed on 40 patients (42 lesions) who were enrolled in this prospective study. The lesions were split into two groups, one with malignant breast lesions (n = 28) and the other with benign breast lesions (n = 14), based on the results of the histology. The measured image characteristics (APT value, apparent diffusion coefficient [ADC] value, and time-of-intensity-curve [TIC] type) were compared between the two groups, and the ROC curve was used to quantify the diagnostic performance on the basis of these factors. The correlation between the APT values and the estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 expression levels and histological grades was examined using Spearman’s correlation coefficient. Results. The measured APT and ADC values showed a strong inter-observer agreement according to the intraclass correlation coefficients (0.954 and 0.825). Compared to benign lesions, malignant lesions had significantly higher APT values (3.18 ± 1.07 and 2.01 ± 0.51, p < 0.001). Based on APTWI, DCE, diffusion-weighted imaging (DWI), and ADC + APTWI, ADC + DCE, and DCE + APTWI, the area-under-the-curve values were 0.915, 0.815, 0.878, 0.921, 0.916, and 0.936, respectively. Conclusions. APTWI is a potentially promising method in differentiating benign and malignant breast lesions, and may it become a great substitute for DCE examination in the future. Key words: amide proton transfer; breast lesions; dynamic contrast-enhanced; magnetic resonance imaging Introduction Breast cancer is the most prevalent malignant neo- plasm all over the world.1 Magnetic resonance im- aging (MRI), a non-invasive technique with excep- tional soft tissue resolution, plays a significant role in diagnosis, treatment, and prognosis assessment of breast diseases.2-4 In an effort to standardize the imaging strategy for breast lesions, the American College of Radiology created the Breast Imaging Radiol Oncol 2023; 57(4): 446-454. Zhuang L et al. / Amide proton transfer-weighted imaging 447 Reporting Data System (BI-RADS) in 1992.5 However, some imaging features of benign and malignant lesions still overlap. False-positive re- sults from conventional breast MRI could result in unnecessary invasive biopsies being performed.6 Dynamic contrast-enhanced MRI (DCE-MRI) is used to obtain focal information by injecting a contrast agent, and it has been widely used for de- termining prognosis, monitoring therapy, and di- agnosing many diseases.7 The study of Alkhunizi SM et al. provided insights into the consequences of gadolinium-based contrast agent (GBCA) deliv- ery by showing considerable retention of gadolini- um in the spinal cord and peripheral nerves 1 day after dose.8 Meanwhile, after multiple GBCA injec- tion, the dentate nucleus and pallidum showed ab- normally high signals under T1-weighted imaging sequence.9 Therefore, the search for a safe tool for evaluating breast lesions is of great importance. In 2000, the first MR contrast images of several small molecules were acquired by Wolff et al., who called this novel molecular imaging technology chemical exchange saturation transfer (CEST).10 Amide proton transfer-weighted imaging (APTWI) technique is a new molecular MRI sequence that is based on CEST, focusing on the exchange between amide protons and bulk water and thus generat- ing image contrast at 3.5 parts per million (ppm) away from water frequency.11 Various preclinical and clinical research investigations have been conducted on this imaging technology12, making it a potential molecular imaging tool that is now used in clinics.13 Meanwhile, in recent years, the differentiation of tumor subtypes and grades and the assessment of therapy efficacy are two applica- tions of APTWI in breast cancer that have drawn increasing attention.14-17 However, no research has compared the value of APTWI in diagnosing be- nign and malignant breast diseases with it in DCE. The purpose of this study was to investigate the potential of APTWI in breast-lesion diagnosis and compare the performance of DCE and APTWI in identifying benign from malignant breast lesions. Patients and methods Patients The local Ethics Committee of Peking University Shenzhen Hospital granted approval for this pro- spective trial (reference number 2022-073) and each subject provided their informed consent. Patients with mammary lesions were enrolled from June 2022 to December 2022 in compliance with the following standards: 1) no contraindication to MRI examinations; 2) suspicious breast lesions discovered by mammogram and/or ultrasound; 3) no previous surgical procedure, chemotherapy, or radiotherapy before MRI examination; 4) the histo- pathology of each mammary lesion was validated by a biopsy or surgical specimen. The flowchart of patient enrollment is represented in Figure 1. MR examination The entire examinations were conducted on a 3.0T MRI scanner (Ingenia CX, Philips Healthcare) equipped with a 16-channel breast coil. Both breasts were draped naturally over the center of the coil while the patients were lying in the prone position with the feet entering first. The tradi- tional sequences, widely used in clinical practice, were performed firstly, including T1-weighted imaging, T2-weighted imaging, and diffusion- weighted imaging (DWI). Following completion of the traditional sequences, all slices containing lesion tissues were subjected to three-dimensional (3D) APTWI examinations using the images from the traditional sequence as the reference. Finally, DCE was performed with the injection of GBCA (gadodiamide; 0.2 mL/kg body weight; Bayer AG). Table 1 lists the imaging parameters in detail. Imaging analysis All data were analyzed using the IntelliSpace Portal (Philips Healthcare, Cleveland, OH, USA) workstation. First, the APTWI pseudo-color images were merged with the DCE images. Next, using the TABLE 1. Imaging protocol parameters APTw DWI DCE TR [ms] 5500 6356 4 TE [ms] 9 84 2 Field of views [mm2] 120 × 120 340 × 309 260 × 340 Voxel size 1.9 × 2.0 × 7 2.7 × 2.3 × 4 0.9 × 0.9 × 3.5 Flip angle 90 90 11 Matrix (mm2) 64 × 60 128 × 133 280× 368 Reconstructed voxel size 0.94 × 0.94 × 7 0.97 × 0.97 × 4 0.61 × 0.61 × 3.5 b-value [s/mm2] NA 0,50,800 NA Bandwidth [pixel/Hz] 406.9 23.1 783.2 Scan duration 5 min 03 s 2 min 14 s 7 min 44 s APTWI = amide proton transfer-weighted imaging; DCE = dynamic contrast enhancement; DWI = diffusion-weighted imaging; NA = not applicable Radiol Oncol 2023; 57(4): 446-454. Zhuang L et al. / Amide proton transfer-weighted imaging448 plain scan and DWI used as references, regions of interest (ROIs) were manually delineated by high- lighting the solid portion of the lesion tissue on the axial DCE images. On the DCE images, on the slice that showed the largest lesion area, the cap- sule’s necrotic and hemorrhagic areas were kept as far away from as possible.18 Two radiologists sepa- rately assessed the ROI (L.Z. and R.H., with 3 and 25 years of breast imaging diagnostic experiences, respectively). For the purpose of measuring appar- ent diffusion coefficient (ADC) value, the identical ROI was converted to an ADC image. The formula below was used to calculate the APT value: APT value = MTRasym (3.5 ppm) (%) = [Ssat (−3.5 ppm) − Ssat (+3.5 ppm)]/S0 Note: Ssat = the signal intensity after applying the saturation pulse; S0 = the signal intensity with- out the saturation pulse; MTRasym (3.5 ppm) = the magnetization transfer ratio asymmetry at 3.5 ppm Time–intensity curves (TICs) were generated from the DCE images, and they were separated in- to three categories: I = Persistent, II = Plateau, and III = Washout. Disagreements regarding the type of TIC were resolved by discussion. On the DCE images, the maximum diameter of each lesion was measured. Pathological grade and stage All diagnoses of breast lesions were confirmed by biopsy or surgical histopathology. According to pathological standards, grade I breast cancer was defined as well-differentiated tumors. Grades II and III refer to moderately and poorly differenti- ated tumors, respectively. The following were the explanation criteria for the estrogen receptor (ER) and progesterone receptor (PR) status: ≥ 10 percent of the tumor cells exhibited positive results, while < 10 percent showed negative results.19 The human epidermal growth factor receptor-2 (HER-2) ex- pression status was considered as positive when samples scored +++ or when HER2 gene amplifica- tion was proven in case of a ++ score.20 A critical point of 14% separates high-expression and low- expression values for Ki-67.21 Statistical analysis SPSS 19.0 (IBM) and MedCalc 20.0 were used to analyze the data. In order to evaluate inter-ob- server reliability, the intra-class correlation coef- ficient (ICC) was used. Kolmogorov–Smirnov test was utilized to ascertain if the quantitative data followed a normal distribution. The variations in FIGURE 1. Flowchart of patient selection. Radiol Oncol 2023; 57(4): 446-454. Zhuang L et al. / Amide proton transfer-weighted imaging 449 each parameter across several groups were com- pared using t-test or Mann–Whitney U test. Each parameter’s diagnostic efficacy was assessed us- ing ROC curves. Meanwhile, Delong tests were used to evaluate whether one parameter’s area under the ROC curve (AUC) differed from the oth- ers. By using Spearman’s correlation coefficient, the correlations between APT values and various clinicopathological variables were calculated. The composite diagnosis of multiple indices was de- termined using a logistic regression. P < 0.05 was considered statistically significant. Results Clinicopathological data According to the inclusion and exclusion criteria, a total of 40 patients with 42 lesions were enrolled. All lesions were classified into two groups: benign group (n = 14; including seven fibroadenomas, one juvenile fibroadenoma, two adenoses, one intra- ductal papilloma, one benign phyllodes tumor, and two fibroadenomas mixing adenoses) and ma- lignant group (n = 28; including 24 invasive breast carcinomas and four ductal carcinoma in situ). The summary of patient characteristics and the clini- cal and pathological data of breast cancer are dis- played in Tables 2 and 3, respectively. Consistency test The two observers had solid agreements. The ICCs were 0.954 for the APT values and 0.825 for the ADC values. As a result, the final evaluation indi- ces were calculated using the averages of the two radiologists’ measurements of the parameters. Comparison of MRI parameters Malignant group had significantly higher APT values than benign group (3.18 ± 1.07 and 2.01 ± 0.51, p < 0.001). It was found that the ADC values for the malignant group were lower than those FIGURE 2. Magnetic resonance imaging (MRI) from a 42-year-old woman with invasive breast carcinoma. (A) DWI = diffusion- weighted imaging (B) apparent diffusion coefficient (ADC) (C) dynamic contrast-enhanced (DCE) (D) The amide proton transfer-weighted imaging (APTWI) pseudo-color image was merged with the DCE images and the APT value was 3.9%. (E) The type of time-intensity curves (TIC) was plateau. A B C D E Radiol Oncol 2023; 57(4): 446-454. Zhuang L et al. / Amide proton transfer-weighted imaging450 for the benign group (1.13 ± 0.36 and 1.64 ± 0.41, p < 0.001). The type of TIC was considered statisti- cally significant (p < 0.001, Table 4). Representative images for the malignant and benign groups are shown in Figures 2 and 3. ROC curve analysis The APT value, TIC, and ADC value imaging shown AUC values of 0.915, 0.815, and 0.878, re- spectively, in distinction between the malignant lesions and the benign lesions. The AUC values of ADC + APTWI, ADC + DCE, and DCE + APTWI were 0.921, 0.916, and 0.936, respectively. However, only the differences between the AUC of TIC and DCE + APT and between the AUC of TIC and DCE + DWI were significant (Z = 1.987, p = 0.0470; Z = 2.049, p = 0.0405). The variations in AUC among various parameters are displayed in Table 5 and Figure 4. Correlation analysis ER, PR, HER-2, and Ki-67 expression, as well as histological grade, did not significantly correlate with APT value (r = 0.254, 0.278, −0.222, −0.219, 0.029, respectively; p = 0.202, 0.161, 0.265, 0.273, 0.895, respectively). Discussion In this prospective study, a pilot research of the vi- ability of using APT value in conjunction with DWI and DCE sequences for the distinction between benign and malignant mammary lesions was suc- cessfully conducted. The correlation between the APT value and the pathological factors of breast cancer was explored. The feasibility of 3D APTWI MRI for the distinguishing between benign and malignant mammary lesions was demonstrated. With the clinical feasibility of APTWI in breast a concern among researchers, this study showed an excellent agreement in APT value measurement and the high diagnostic efficiency of APT values, similar to the efficiency of TIC, indicating that valuable diagnostic information could be obtained without using GBCA. Thus, APT value could be regarded as a non-invasive biomarker for differen- tiating mammary lesions. In this study, the malignant lesions typically had higher APT values than the benign lesions, consistent with what is generally known about malignant tumors in other diseases.22-25 APTWI is a molecular MRI technique that is based on chemi- cal exchange saturation transfer that could detect endogenous mobile proteins and peptides at low molecular concentrations. The high intensity in APTWI was made possible by the fact that malig- nant lesions were highly cellular and that several proteins were overexpressed in comparison to be- nign lesions. Due to the high levels of hemoglobin and albumin in blood, angiogenesis is another component that may contribute to enhanced pro- TABLE 2. Summary of patient characteristics Benign lesions (n = 14) Malignant lesions (n = 28) Age (years) 35 ± 14 (13–68) 49 ± 12 (30–77) Largest diameter (cm) 2.19 ± 2.40 3.31 ± 1.25 Histology Fibroadenoma (n = 7) Invasive breast carcinoma (n = 24) Juvenile fibroadenoma (n = 1) Ductal carcinoma in situ (n = 4) Adenosis (n = 2) Fibroadenoma + adenosis (n = 2) Intraductal papilloma (n = 1) Benign phyllodes tumor (n = 1) TABLE 3. Clinical and pathological data of breast cancer Variable Malignant breast tumors (n = 28) Tumor diameter < 2 cm 3 (10.71) ≥ 2 cm 25 (89.29) Metastatic status of axillary lymph node 15 (53.57) Calcification 16 (57.14) Grade of IBC (N, %) Grade I 1 (4.17) Grade II 12 (50) Grade III 11 (45.83) Receptor status (N, %) ER+ 20 (71.43) PR+ 20 (71.43) HER-2+ 9 (32.14) Ki67+ 25 (89.29) One patient without the result of immunohistochemical ER = estrogen receptor; HER-2 = human epidermal growth factor receptor-2; PR = progesterone receptor Radiol Oncol 2023; 57(4): 446-454. Zhuang L et al. / Amide proton transfer-weighted imaging 451 tein signaling in malignancies.13 However, this dis- covery differs from the results of earlier research by Meng et al.14,16 They suggested that one reason could be that some benign lesions’ secretory abili- ties were intact, whereas the secretory abilities of some malignant lesions were damaged, leading to lower protein and polypeptide concentration. This difference could result from variations in study participants. Loi L et al. demonstrated that breast cancer displayed a substantially higher APT value than typical fibro-glandular tissue26, which fully confirms the results of the present study. The findings showed that APT value has a strong diagnostic performance similar to TIC, and it could be employed for the differential diagnosis of mammary lesions. The combination of DWI and APT may increase the specificity of the diagno- sis. DCE examination was conducted by injecting GBCA. As is well known, the injection of GBCA has many side effects, such as allergic as well as aller- goid reactions, including anaphylactic reactions27, contrast-induced nephropathy28, nephrogenic sys- temic fibrosis29, and gadolinium retention/deposi- tion.30 On the contrary, APTWI does not require the use of GBCA, thus saving costs and avoiding side effects. For patients with a history of allergies or those refusing to undergo DCE, APTWI could be a great substitute for DCE examination. In this study, the APT value showed no corre- lations with ER, Ki-67 expression and histological grades, inconsistent with the results of previous studies. Liu Z et al. found a weakly positive con- nection between APT value and Ki-67 expression.31 A notable detail that they used a 20% threshold to report comparable APT values between groups with varying Ki-67 proliferation levels. This con- flict could result from the differing Ki-67 prolifera- tion index levels employed for group classification. Zhang N et al. reported that the APT value and ER expression had a negative connection, may be be- FIGURE 3. Magnetic resonance imaging (MRI) from a 26-year-old woman with intraductal papilloma. (A) Diffusion-weighted imaging (DWI); (B) apparent diffusion coefficient (ADC); (C) dynamic contrast-enhanced (DCE); (D) the amide proton transfer- weighted imaging (APTWI) pseudo-color image was merged with the DCE images and the APT value was 1.6%; (E) the type of time-intensity curves (TIC) was persistent. A B C D E Radiol Oncol 2023; 57(4): 446-454. Zhuang L et al. / Amide proton transfer-weighted imaging452 cause ER inhibits lesion angiogenesis by regulating the production of the vascular endothelial growth factor.32 On the contrary, the results of syudy by Meng N et al. showed that there was no correla- tion between the APT value and ER expression.14 Consequently, a larger sample size is required for further research. Meng N et al. showed that only a weakly positive correlation existed between the pathogenic grade and APT value; they hypoth- esized that the cause is that high-level tumor cells have a high density, evident nuclear atypia, as well as increased tissue necrosis, all of which alters the proteins and peptides in the nearby microenviron- ment, in addition to the rate and distribution of in- ternal water molecule diffusion and movement.16 Nevertheless, our grouping of histological grades was I, II, and III while their grouping of histologi- cal grades was low and high, which could explain the differences in the results. In accordance with previous researches, the APT value showed no correlations with PR and Her-2.32,33 This study has several limitations. First, breast lesions and some subtypes of breast cancer had relatively small sample sizes, which could cause the AUC of APTWI to be overestimated. Future research may also be warranted on the genomic subtyping capabilities of APTWI for breast can- cers. Therefore, more research involving a bigger sample size is required. Second, the diagnostic efficacy of DCE assessed using only TIC curves may be underestimated. Liang X et al. recently re- vealed that the quantitative and semi-quantitative parameters of DCE provided great diagnostic per- formance. Third, breast tissue has a substantial amount of fat, which could affect the accuracy of APT values.34 Obviously, in the present study, pa- tients with fatty breast tended to have poorer im- age quality. All CEST signals become essentially dependent on the amount of fat per voxel due to the ubiquitous fat signal’s erroneous normaliza- tion of the Z-spectrum. Finally, bias may have ex- isted in the selection of patients. Breast MRI is usu- ally conducted in high-risk population.35 Patients receiving MRI in clinical practice had a consider- ably higher chance of malignant tumor than those receiving mammography or ultrasound, which could induce background bias (more malignant cases than benign cases). Conclusions The capability of identifying benign from malig- nant breast lesions could be achieved using APTWI and DCE, and APTWI may be a great supplement or even replacement for DCE sequence. The find- ings still need to be confirmed by future investiga- tions on patient cohorts with larger breast lesions. TABLE 4. Comparison of different parameters between benign lesions and malignant lesions Measurement index Benign lesions (n = 14) Malignant lesions (n = 28) P APT value 2.01 ± 0.51 3.18 ± 1.07 < 0.001 Type of TIC < 0.001 I 11(78.6%) 5(17.9%) II 3(21.4%) 20(71.4%) III 0 3(10.7%) ADC value 1.64 ± 0.41 1.13 ± 0.36 < 0.001 ADC = apparent diffusion coefficient; APT = amide proton transfer; Type of time-intensity curves (TIC): (I = Persistent; II = Plateau; III = Washout) TABLE 5. ROC analysis of the performance in separating breast cancer from benign lesions using various criteria and techniques alone or in combination Multi parameters Cutoff Sensitivity Specificity AUC 95%CI Parameters APT value > 2.35 85.71% 92.86% 0.915 0.786–0.978 TIC > 1 82.14% 78.57% 0.815 0.665–0.918 ADC value ≤ 1.26 89.29% 92.86% 0.878 0.739–0.958 Methods ADC+APTWI / 85.71% 96.43% 0.921 0.795–0.982 ADC+DCE / 71.43% 89.29% 0.916 0.788–0.979 DCE+APTWI / 78.57% 92.86% 0.936 0.816–0.988 ADC = apparent diffusion coefficient; APTW = amide proton transfer-weighted imaging; AUC = area under the ROC curve; dynamic contrast-enhanced (DCE); TIC = time-intensity curves FIGURE 4. The graph displays ROC curves to evaluate the usefulness of various parameters for differentiating between malignant and benign lesions. Radiol Oncol 2023; 57(4): 446-454. 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Received 1 June 2023 Accepted 22 July 2023 Correspondence to: Dr. Chunhong Hu, Department of Radiology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, Jiangsu, China. Phone: +(0)512 6778 1214; E-mail: sudahuchunhong@163.com and Dr. Yu Zhang, Department of Radiology, Dushu Lake Hospital Affiliated to Soochow University, Suzhou, 215006, Jiangsu, China. Phone: +(0)512 6595 5080; E-mail: zhangyusdfyy@163.com The two authors Xing Xiong and Yuzhu Ma contributed equally to this work. Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY li-cense (https://creativecommons.org/licenses/by/4.0/). Background. The purpose of our study was to explore and compare the tumor burden of different bone marrow infiltration patterns and evaluate the feasibility of apparent diffusion coefficient (ADC) value to identify patterns in multiple myeloma (MM). Patients and methods. Ninety-three patients with newly diagnosed multiple myeloma and 23 controls had under- gone routine magnetic resonance imaging (MRI) and diffusion-weighted MRI (DWI) from January 2019 to November 2020. Five bone marrow (BM) infiltration patterns were allocated according to routine MRI. The laboratory data and ADC values of patterns were analyzed and compared. ROC analysis was used to establish the best diagnostic ADC threshold value for identifying these patterns and distinguishing normal pattern from controls. Besides, the correlation between the ADC values of diffuse pattern and the plasma cells ratio was assessed. Results. The values of hemoglobin, beta-2 microglobulin (β2-MG), plasma cell, M protein, the percentages of stage, high-risk fluorescence in situ hybridization, and ADC values showed significant difference among patterns. ADCmean at a specific value (368.5×10-6 mm2/s) yielded a maximum specificity (95.5%) and sensitivity (92.0%) in diagnosing MM. A specific value (335.5×10-6mm 2/s) yielded a maximum specificity (84.7%) and sensitivity (88.0%) in discriminating visually normal pattern in MM from controls. There was a moderate positive correlation between the plasma cells ratio and ADCs of diffuse infiltration patterns (r = 0.648, P < 0.001). Conclusions. The bone marrow infiltration patterns in MM patients can indicate the tumor burden and ADC value has the ability to discriminate these patterns objectively. Key words: multiple myeloma; bone marrow; infiltration pattern; tumor burden; apparent diffusion coefficient Introduction Multiple myeloma (MM) is a malignant hemato- logic disease, and incidence is increasing gradu- ally in recent years.1 It originates from B cells (plas- ma cells) and mainly invades bone marrow (BM).2 Tumor burden is important for treatment regimen and prognosis in MM patients. In clinical prac- tice, tumor burden is assessed mainly by serum markers and BM biopsy.3-5 With the exposure of laboratory examination defects and the rapid de- velopment of imaging techniques, visual evalua- Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM456 tion of BM infiltration degree and pattern becomes possible using imaging methods directly. As one of the most sensitive imaging methods, magnetic resonance imaging (MRI) can provide relevant information of BM infiltration.6 Whole body (WB) MRI has been listed as the golden standard for the detection of MM lesions by International Myeloma Working Group (IMWG), and many authorities around the world also supports its application in clinical practice for MM assessment and manage- ment.7-9 The latest IMWG guidelines divide the BM in- filtration into five patterns based on MRI findings: focal involvement (F), pure diffuse infiltration (D), “salt-and- pepper” pattern (SP), combined diffuse/ focal infiltration (M) and visually normal pattern (N).10 The pattern of BM infiltration at initial di- agnosis could indicate the patient’s tumor burden, which has important prognostic significance for MM patients.11 However, BM infiltration pattern recognition is a subjective assessment based on visual criteria. For example, D pattern is recog- nized by comparing with the signal intensity (SI) of the intervertebral disc or paravertebral muscle, and N pattern is not visually distinguishable from healthy BM.12,13 The lack of objective criteria may affect the interpretation of results and lead to false positive/negative diagnosis. With the appearance of diffusion-weighted MRI (DWI), it shows further sensitivity to detect MM lesions, and quantitative analysis of apparent diffusion coefficient (ADC) value becomes the hot spot for current research.14,15 However, the quantitative standards of ADC val- ues of five patterns is lacking, especially for N pat- tern. Furthermore, the IMWG criteria only consid- ers the occurrence of F lesions as the beginning of clinical treatment, while the tumor burden and therapeutic value of other patterns, which in- cludes D, SP, M, N, are still unclear. Our previous study found that the proportion of non-F patterns was significantly higher than that of F pattern.16 Therefore, the purpose of our study was to compre- hensively explore and compare the tumor burden of different BM infiltration patterns. Furthermore, to evaluate the feasibility of ADC value in identify- ing these patterns in MM quantitatively. Subjects and methods Subjects This study was approved by the Ethics Committee of the local institution (registration number: 000/2021). The newly diagnosed MM patients admitted to our institute from January 2019 to November 2020 were collected retrospectively. Inclusion criteria: (1) patients with MM confirmed by BM biopsy, immunofixation electrophoresis, se- rum protein electrophoresis and other laboratory examinations; (2) the patient underwent diffusion- weighted whole body MRI (WB-DWI) within one week before treatment; (3) Complete histological and laboratory data. Exclusion criteria: (1) patients with a history of additional malignant tumors; (2) patients who have received radiation therapy; (3) patients who have received granulocyte colony- stimulating factors or bisphosphonates; (4) patients with compressed fractures of vertebral body. At the same time, healthy controls were included. Inclusion criteria: (1) no history of malignant tu- mors; (2) no evidence of anemia in clinical and lab- oratory examinations; (3) age between 50 and 80. Histological and laboratory data Marrow plasma cell ratio (%), M protein (g/L), β2- microglobulin (mg/L), hemoglobin (g/L), lactate dehydrogenase (LDH) (U/L), creatinine (µmol/L) levels, International Staging System (ISS) and Revised International Staging System (R-ISS) stage were collected. Fluorescence in situ hybridization (FISH) analysis after CDl38 separation, includ- ing Del(17p), t(4; 14), t(14; 16), t(14;20) and gain(1p) was also reported. MM patients were divided into high-risk cytogenetics (HRC) and standard-risk cytogenetics (SRC) groups on the basis of the FISH results. MR scanning protocol All examinations were performed on a 3.0T MRI scanner (Magnetic Verio, Siemens Healthcare, Erlangen, Germany). Patients were in supine posi- tion with head first and arms placed on both sides of the body. The scanning parameters were as fol- lows: coronal T2 TIRM sequence, repetition time: 7110 ms; echo time: 84 ms; slice thickness: 5 mm; slice spacing: 1.5 mm; FOV: 480 mm. The scan cov- ered the skull, whole spine, pelvis and upper fe- mur. DWI sequence, b values are 50 s/mm2 and 700 s/mm2, respectively. The scan range was the same as above. Sagittal T1 FSE sequence, repetition time: 1700 ms; echo time: 8.6 ms; slice thickness: 4 mm; slice spacing: 0.8 mm. Sagittal T2 FS sequence, rep- etition time: 3000 ms; echo time: 91 ms; slice thick- ness: 4 mm; slice spacing: 0.8 mm. The scan range was T11-S1. All DWI data was transferred to Syngo Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM 457 FIGURE 1. Five bone marrow infiltration patterns of multiple myeloma patients and healthy controls: pure diffuse pattern (A, B), combined diffuse/ focal pattern (C, D), focal pattern (E, F), salt and pepper pattern (G, H), normal pattern (I, J), healthy control (K, L). T1 weighted(WI) image (A, C, E, G, I, K), T2WI FS image (B, D, F, H, J, L). A B C D E G H I J K L F Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM458 MultiModality station, and the Funtool software was used to process and generate ADC images. Image analysis Images were analyzed by two radiologists with more than 10 years of experience. They were blind- ed to the laboratory data. Assessment differences between two radiologists were resolved by con- sensus. According to MRI findings, BM infiltration was divided into five patterns, including focal pattern (F), pure diffuse pattern (D), combined diffuse/fo- cal pattern (M), salt and pepper pattern (SP) and normal pattern (N) (Figure 1). The detailed defini- tion of patterns are as follows: F pattern was de- fined as signal intensity (SI) of nodular lesion less than or equal to the disc or surrounding muscles SI on T1WI and higher than that on T2 turbo in- version recovery magnitude (TIRM) images with diameter > 5mm. DWI (b = 700 s/mm2) images showed higher SI compared to peripheral BM. SP pattern was defined as sparse small foci with T1WI hypointense and T2 TIRM hyperintense with the background of normal vertebral body SI. D pattern was defined as the vertebral SI on the TIWI im- age less than or equal to the undegenerated disc or surrounding muscles SI without normal fat signal visible; on the T2 TIRM image, it shows diffusely higher SI. The M pattern was defined as the ap- pearance of D pattern on the T1WI image and on the T2 TIRM image as one or more nodular higher SI within the vertebral body, where focal and dif- fuse changes were superimposed. The N pattern was defined as no visible SI change on vertebral T1WI and T2 TIRM images, which could not be distinguished from healthy BM by conventional MRI. Since lumbar vertebra are one of the main sites for monoclonal plasma cell infiltration and charac- terized by lower amount of red marrow and more adipogenesis due to obvious mechanical stress and local ischemia, so we have chosen lumbar ver- tebra as representative background BM region. For MM patients with D, SP and M patterns, an oval ROI with 100 mm2 was placed in each vertebral body at the central slice in the ADC image with the aid of DWI (b values, 700) in conjunction with the anatomic (T1WI and T2WI FS) images by us- ing image linking and scrolling workstation facili- ties and co-registration tools. Only when lesions detectable in these images, we will conduct ADC measurements. For MM patients with N pattern and healthy controls, we only need to place ROI with 100 mm2 in each vertebral body at the central slice in the ADC image since there is no abnormal SI detectable in these images. For F pattern, the le- sions with clear boundaries and diameter of at least 5mm in the ADC image which also confirmed by other images were selected. ROI was placed on the largest slice of the lesion and contoured around the lesions as far as possible. Vertebral edge and BM edema, Schmorl’s nodules and hemangioma were strictly avoided. ADC value of each ROI was meas- ured three times at the same slice, and the average value of the three measurements was calculated and recorded. Statistical analysis Statistical analysis was performed using IBM SPSS 25.0 software (Chicago, USA) and GraphPad Prism 5.0 software (GraphPad Software, California, TABLE 1. The laboratory data of patients Characteristics Number / mean ± standard deviation MM subtypea lgG-K 28 lgG-λ 21 IgA-K 8 lgA-λ 11 IgM-K 1 K 5 λ 19 ISS stagea I/ II/ III 18/ 48/ 27 R-ISS stagea I/ II/ III 14/ 63/ 16 FISHa HRC/ SRC 20 / 73 Plasma cells in BM (%)b 34.4 ± 22.3 Serum M protein b 36.6 ± 23.7 Hemoglobin b 93.3 ± 23.1 Serum β2-MGb 6.6 ± 8.5 Serum creatinineb 127.3 ± 143.3 LDHb 197.9 ± 115.1 a categorical variables shown with number; b continuous variables shown with mean ± standard deviation β2-MG = beta-2 microglobulin; BM = bone marrow; FISH = fluorescence in situ hybridization; HRC = high-risk cytogenetics; ISS = international staging system; LDH = lactate dehydrogenase; MM = multiple myeloma; R-ISS = revised international staging system; SRC = standard- risk cytogenetics Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM 459 USA). The normality of the distribution was as- sessed using the Kolmogorov-smirnov test. One- way analysis of variance and Kruskal-Wallis H test were used to evaluate the differences between laboratory data of patients with different infiltra- tion patterns. Chi-square test and Fisher’s exact test were used to evaluate the difference of ISS stage, R-ISS stage and HRC status. Kruskal-Wallis H test was used to evaluate the difference of the mean ADCs between the control group and the MM group, and between different infiltration patterns. Pairwise comparison between multiple groups was performed by Bonferroni correction. Spearman correlation analysis was used to explore the correlation between ADC values of N, D pat- terns and the plasma cells ratio in BM. A P-value less than 0.05 was considered significant. ROC curve was plotted to determine the cut-off values of ADC between healthy BM and MM BM, healthy BM and N pattern BM, N pattern BM and D pattern BM based on different research main concerns. Result Patients Ninety-three MM patients were enrolled in this study and the laboratory data of these patients were collected (Table 1). The average age was 56.2 years and ranged from 31 to 85 years. Among them, 45 were males with an average age of 56.6 years, ranging from 34 to 85 years. There were 48 females with a mean age of 55.8 years, ranging from 31 to 79 years. At the same time, 23 healthy controls were included in the study with an aver- age age of 59.6 years, ranging from 50 to 73 years. Among them, 11 were males with an average age of 59.1 years, ranging from 50 to 69 years. There were 12 female patients with a mean age of 60.0 years, ranging from 51 to 73 years. Among all pa- tients, 23 were D pattern, 19 were M pattern, 17 were F pattern, 14 were SP pattern and 20 were N pattern. Clinical variables difference of patients with different infiltration patterns based on MRI The relevant clinical variables of patients with different infiltration patterns were as follows: hemoglobin level was 93.3 ± 23.1 g/L, and the com- parison of hemoglobin level among different in- filtration patterns showed significant difference TABLE 2. Hemoglobin characteristics of multiple myeloma (MM) patients with different magnetic resonance imaging (MRI) infiltration patterns MRI pattern Haemoglobina (g/L) P D M F SP N D 77.0 ± 19.5 > 0.05 0.001 0.014 < 0.001 M 88.9 ± 21.1 > 0.05 > 0.05 0.009 F 100.8 ± 17.8 > 0.05 > 0.05 SP 97.6 ± 20.0 > 0.05 N 106.7 ± 23.8 a continuous variables shown with mean ± standard deviation D = pure diffuse pattern; F = focal pattern; M = combined diffuse/focal pattern; N = normal pattern; SP = salt and pepper pattern TABLE 3. Beta-2 microglobulin (β2-MG) characteristics of multiple myeloma (MM) patients with different magnetic resonance imaging (MRI) infiltration patterns MRI pattern β2-MGa (mg/L) P D M F SP N D 9.3 ± 8.8 > 0.05 < 0.05 < 0.05 < 0.05 M 10.7 ± 13.9 < 0.05 < 0.05 < 0.05 F 4.4 ± 5.2 > 0.05 > 0.05 SP 2.9 ± 1.5 > 0.05 N 3.6 ± 2.4 D = pure diffuse pattern; F = focal pattern; M = combined diffuse/focal pattern; N = normal pattern; SP = salt and pepper pattern FIGURE 2. ROC curve analysis of discriminating multiple myeloma (MM) infiltration patterns of D from N by quantitative parameters of apparent diffusion coefficient (ADC). D = pure diffuse pattern; N = normal pattern Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM460 (Table 2). A total of 57 patients showed anemia clinically and the hemoglobin level decreased successively with the order of N, F, SP, M and D patterns. The creatinine level was 127.3 ± 143.3 g/L. There was no significant difference in serum creatinine level among different infiltration pat- terns, but the creatinine level still increased suc- cessively in SP, N, F, D and M patterns. The beta-2 microglobulin (β2-MG) level was 6.6 ± 8.5 mg/L. The comparison results of β2-MG level among pa- tients with different infiltration patterns showed significant difference (Table 3). The β2-MG level increased successively according to the N, SP, F, D and M patterns. The plasma cells ratio in BM was 34.4 ± 22.3%. The comparison results among patients showed significant difference (Table 4). The plasma cells ratio in BM increased according to N, SP, F, D and M patterns. The M protein level was 36.6 ± 23.7 g/L. The comparison results among patients showed significant difference (Table 5). Serum M protein level increased according to N, F, SP, D and M patterns. The LDH level was 197.9 ± 115.1 U/L. There was no significant difference in LDH level among different infiltration patterns, but it still increased successively in N, SP, F, D and M patterns. ISS, R-ISS stage and HRC characteristics of MM patients with different infiltration patterns based on MRI There were significant differences in ISS stage be- tween groups (supplement Table 1). D, M and F patterns were mainly located on stage II and stage III, accounting for 91.3%, 94.7% and 82.4%, respec- tively. While N and SP patterns were mainly lo- cated on stage I and stage II, accounting for 85.0% and 92.9%, respectively. There were significant differences in R-ISS stage between groups (Supplement Table 1). D and M patterns were mainly located on stage II and III, accounting for 100% and 94.7%, respectively. While F, N and SP patterns were mainly located on stage I and II, accounting for 94.1%, 90.0% and 100%, respectively. A total of 20 patients with HRC status were detected with positive rate of 21.5%. The posi- tive rate of HRC was 36.8% in M group, 30.4% in D group, 23.5% in F group and 10.0% in N group, respectively and no HRC cases were found in SP group. There were significant differences between groups and the detailed information of each pa- tient with HRC status was collected (Supplement Table 2). TABLE 4. Plasma cells characteristics of multiple myeloma (MM) patients with different magnetic resonance imaging (MRI) infiltration patterns MRI pattern plasma cellsa (%) P D M F SP N D 46.9 ± 20.1 > 0.05 > 0.05 < 0.05 < 0.001 M 50.3 ± 23.1 < 0.05 < 0.05 < 0.001 F 34.9 ± 17.8 > 0.05 < 0.05 SP 18.8 ± 8.3 < 0.05 N 15.7 ± 11.2 a continuous variables shown with mean ± standard deviation D = pure diffuse pattern; F = focal pattern; M = combined diffuse/focal pattern; N = normal pattern; SP = salt and pepper pattern TABLE 5. Serum M protein characteristics of multiple myeloma (MM) patients with different magnetic resonance imaging (MRI) infiltration patterns MRI pattern M protein a (g/L) P D M F SP N D 46.9 ± 27.6 > 0.05 > 0.05 > 0.05 < 0.05 M 49.8 ± 18.9 > 0.05 > 0.05 > 0.05 F 33.4 ± 20.8 > 0.05 > 0.05 SP 40.3 ± 18.8 < 0.05 N 19.9 ± 16.9 a continuous variables shown with mean ± standard deviation D = pure diffuse pattern; F = focal pattern; M = combined diffuse/focal pattern; N = normal pattern; SP = salt and pepper pattern FIGURE 3. ROC curve analysis of discriminating multiple myeloma (MM) infiltration patterns of N from healthy controls by quantitative parameters of apparent diffusion coefficient (ADC). N = normal Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM 461 ADC characteristics of MM patients with different infiltration patterns and controls based on MRI A total of 440 ROIs were collected, of which 113 ROIs were collected from 23 patients with D pat- tern, 76 ROIs from 17 patients with F pattern, 100 ROIs from 20 patients with N pattern, 83 ROIs from 19 patients with M pattern and 68 ROIs from 14 patients with SP pattern. 111 ROIs were collect- ed from 23 healthy controls. The mean ADC value of the ROIs in MM pa- tients was (631.4 ± 240.1)×10-6 mm2/s. Among them, the mean ADC values of D pattern were (607.8 ± 73.1) ×10-6 mm2/s, M pattern were (783.9 ± 196.4) ×10- 6 mm2/s, F pattern were (967.8 ± 185.3) ×10-6 mm2/s, SP pattern were (463.9 ± 59.1) ×10-6 mm2/s, N pat- tern were (389.9 ± 63.8) ×10-6 mm2/s. The mean ADC of control group was (268.5 ± 63.6) ×10-6 mm2/s. Pairwise comparison of ADC values for different infiltration patterns and controls showed signifi- cant differences between groups. Diagnostic efficacy of quantitative ADC on distinguishing healthy BM and different BM infiltration patterns of MM patients An ADC value of 480.5 × 10-6 mm2/s was the op- timal cut-off value for distinguishing D pattern from N pattern. The corresponding sensitivity was 97.3%, specificity 94.0%, and the area under the curve (AUC) was 0.994 (Figure 2). When the ADC value ≥ 522.5×10-6 mm2/s, the specificity of diagnosing D pattern was 100%. An ADC value of 335.5×10-6 mm2/s was the optimal cut-off value for distinguishing healthy BM from N pattern. The sensitivity was 88.0%, specificity 84.7%, and the AUC was 0.912 (Figure 3). When the ADC value ≥ 421.0×10-6 mm2/s, the specificity of diagnosing N pattern was 100%. An ADC value of 368.5×10- 6 mm2/s was the optimal cut-off value for distin- guishing healthy BM from MM BM, with diag- nostic sensitivity of 92.0%, specificity of 95.5%, and AUC of 0.979 (Figure 4). When the ADC value ≥ 420.0 × 10-6 mm2/s, the specificity of diagnosing MM BM was 100%. Correlation between ADC value of D, N patterns and plasma cell ratio in BM ADC value of D, N patterns and plasma cell ratio in MM patients showed a moderate correlation with r = 0.648 (significance level P < 0.001) which indicates a positive correlation between plasma cell ratio and ADC value (Figure 5). Discussion In this study, we aimed to explore the tumor bur- den of different patterns in MM patients and the ability of quantitative ADC values for discriminat- FIGURE 4. ROC curve analysis of discriminating healthy controls from multiple myeloma (MM) infiltration bone marrow by quantitative parameters of apparent diffusion coefficient (ADC). FIGURE 5. Correlation analysis of bone marrow plasma cell ratio and mean apparent diffusion coefficient (ADCmean) values of diffuse infiltration patterns (N and D) in multiple myeloma (MM) patients. D = pure diffuse pattern; N = normal pattern Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM462 ing different infiltration patterns and healthy BM. It showed that five different patterns in MM pa- tients had different degrees of tumor burden, and it could be identified by quantitative analysis of ADC values. Some studies have pointed out that the occur- rence of MM is closely related to the BM micro- environment, and the serum tumor markers such as hemoglobin, β2-MG, creatinine could contribute to early clinical diagnosis and response evalua- tion.17 Anemia is one of the most common clinical presentations in patients and hemoglobin is used to evaluate anemia clinically.18 Renal function im- pairment is also one of the common clinical com- plications of MM. β2-MG and creatinine level have important implications in renal function impair- ment, and can also reflect the tumor burden of MM patients.19-21 Our results showed that D and M pat- terns had the highest β2-MG and creatinine levels and the lowest hemoglobin, which suggested that MM bone marrows of D or M patterns had more severe tumor burden than N and SP. The latest R-ISS stage incorporates cytogenetic abnormalities and elevated serum LDH based on the ISS stage.22 LDH can reflect the proliferative activity of tu- mors, which has been widely used in hematologi- cal malignancies such as MM, non-Hodgkin lym- phoma and leukemia.23,24 Colovi and Perez-Andres found that LDH level is an independent prognostic indicator of MM through multivariate analysis.25,26 Our studies showed that the ISS and R-ISS stages of D and M patterns were significantly higher than the other three, While the other three types have no significant difference. It means that the prog- nosis of D and M patterns is worse than that of N, SP and F patterns. Moulopoulos et al. found that the survival period of D pattern was significantly shorter than those with F and N patterns.5 Tian et al. also demonstrated that D pattern had a worse prognosis than those with F pattern.27 These find- ings were consistent with our analysis, which may be related to the increased BM neoangiogenesis and advanced stage in MM patients with D pat- tern. Despite the important prognostic significance of infiltration pattern for MM patients, it is still a subjective assessment based on the visual stand- ard of disc or paravertebral muscle SI as a refer- ence. The lack of objective criteria may affect the interpretation of the diagnostic results, especially for N pattern, which could not be distinguished from healthy BM by conventional MRI. With the development of DWI techniques, quantitative analysis of infiltrated BM is possible by meas- urement of ADC values. The results of this study showed that the healthy BM had the lowest ADC value. N pattern, unable to be visually recognized, had significantly higher ADC than that of healthy BM. The ADC value of MM BM increased sequen- tially based on N, SP, D, M, and F patterns. It is well known that normal fatty BM has low ADC value due to low water content and impeded water movement. In MM patients, increased ADC val- ues are found which are characterized by reduced number of adipocytes, increased water content, and increased proportion of plasma cells.15 Thus, the more tumor cells infiltrate the vertebral body, the higher the ADC value and the tumor burden are. D pattern was divided into different grades ac- cording to its severity by some scholars, including low grade, medium grade, high grade.28 The me- dium to high grade is defined as the lower SI of vertebral bone compared to the intervertebral disc on TlWI image. However, low-level infiltration has similar SI to healthy BM and is difficult to distin- guish visually. Histological studies found that for diffuse infiltrating BM, when the plasma cells ratio was >50%, SI of vertebral body in TlWI was lower than the intervertebral disc. When plasma cells ra- tio is between 20% and 50%, the SI in TlWI image was slightly reduced and MM BM is unrecogniz- able visually from healthy BM.29 In our study, the plasma cells ratio of D pattern was 46.9 ± 20.1%, the vertebral SI was equal to or lower than that of the intervertebral disc in TlWI. The ratio of N pattern was 15.7 ± 11.23%, and the SI was higher than disc in T1WI. Since D and N patterns showed diffuse and homogenous performance in the conventional MRI images, we assumed that the examination of plasma cell ratio in BM could be replaced by use of ADC values for D and N patterns noninvasively. The results showed that the moderate correlation between ADC values of D, N patterns, and plasma cell ratio. Therefore, we believe that the N pattern defined in MRI corresponds to the lower level D pattern in histology, representing the initial stage of diffuse MM bone marrow infiltration. At this stage, BM plasma cell infiltration is mild, adipo- cytes is slightly reduced and the proportion of fat/ water has little change, so the MM BM is indistin- guishable visually. However, through the quanti- tative analysis of ADC value, it can be identified from the healthy BM, and its therapeutic value needs further exploration and research. This study had several limitations. First, the scanning sequence does not include dynamic en- hancement and in/out phase proposed in the lit- Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM 463 erature because some MM patients with bone pain cannot bear long time scan and patients with renal insufficiency should not be injected intravenous contrast. Second, pathology cannot be performed on each lesion. Third, ADC cutoff values partly de- pend on the choice of b values of DWI images used for calculations. In our study, 2 b values (50 and 700) were used which may not reach an agreement in other institutions. Fourth, we have focused on the clinical variables which could reflect progno- sis indirectly. Last, the observation range was lim- ited to the lumbar spine since some studies found that it is the greatest important site of MM infiltra- tion. In the future, we will expand the scope of the study and conduct a comprehensive and in-depth analysis in the prospective study with the aid of AI. In summary, MM patients presenting with dif- ferent BM infiltration patterns have different tu- mor burden and ADC values are able to identify infiltrated BM and further distinguish these pat- terns. Acknowledgement The study has received funding by Gusu health tal- ent project of Suzhou (GSWS2020003) and Suzhou Science and Technology Project (SKJY2021025). Reference 1. Terpos E, Christoulas D, Gavriatopoulou M. Biology and treatment of my- eloma related bone disease. Metabolism 2018; 80: 80-90. doi: 10.1016/j. metabol.2017.11.012 2. Hansford BG, Silbermann R. Advanced imaging of multiple myeloma bone disease. Front Endocrinol 2018; 9: 436. doi: 10.3389/fendo.2018.00436 3. Duvauferrier R, Valence M, Patrat-Delon S, Brillet E, Niederberger E, Marchand A, et al. Current role of CT and whole body MRI in multi- ple myeloma. Diagn Interv Imaging 2013; 94: 169-83. doi: 10.1016/j. diii.2012.12.001 4. Dimopoulos M, Terpos E, Comenzo RL, Tosi P, Beksac M, Sezer O, et al. International myeloma working group consensus statement and guidelines regarding the current role of imaging techniques in the diagnosis and moni- toring of multiple Myeloma. Leukemia 2009; 23: 1545-56. doi: 10.1038/ leu.2009.89 5. Moulopoulos LA, Gika D, Anagnostopoulos A, Delasalle K, Weber D, Alexanian R, et al. Prognostic significance of magnetic resonance imaging of bone marrow in previously untreated patients with multiple myeloma. Ann Oncol 2005; 16:1824-8. doi: 10.1093/annonc/mdi362 6. Messiou C, Hillengass J, Delorme S, Lecouvet FE, Moulopoulos LA, Collins DJ, et al. Guidelines for Acquisition, Interpretation, and Reporting of Whole-Body MRI in Myeloma: myeloma response assessment and di- agnosis system (MY-RADS). Radiology 2019; 291: 5-13. doi: 10.1148/ radiol.2019181949 7. Dimopoulos MA, Hillengass J, Usmani S, Zamagni E, Lentzsch S, Davies FE, et al. Role of magnetic resonance imaging in the management of patients with multiple myeloma: a consensus statement. J Clin Oncol 2015; 33: 657-64. doi: 10.1200/JCO.2014.57.9961 8. Messiou C, Kaiser M. Whole body diffusion weighted MRI – a new view of myeloma. Br J Haematol 2015; 171: 29-37. doi: 10.1111/bjh.13509 9. Kumar SK, Callander NS, Alsina M, Atanackovic D, Biermann JS, Castillo J, et al. NCCN Guidelines Insights: multiple myeloma, Version 3.2018. J Natl Compr Canc Netw 2018; 16: 11-20. doi: 10.6004/jnccn.2018.0002 10. Dutoit JC, Verstraete KL. MRI in multiple myeloma: a pictorial review of diagnostic and post-treatment findings. Insights Imaging 2016; 7: 553-569. doi: 10.1007/s13244-016-0492-7 11. Lecouvet FE, Vande Berg BC, Michaux L, Malghem J, Maldague BE, Jamart J, et al. Stage III multiple myeloma: clinical and prognostic value of spinal bone marrow MR imaging. Radiology 1998; 209: 653-60. doi: 10.1148/ radiology.209.3.9844655 12. Baur-Melnyk A, Buhmann S, Dürr HR, Reiser M. Role of MRI for the diag- nosis and prognosis of multiple myeloma. Eur J Radiol 2005; 55: 56-63. doi: 10.1016/j.ejrad.2005.01.017 13. Moulopoulos LA, Varma DG, Dimopoulos MA, Leeds NE, Kim EE, Johnston DA, et al. Multiple myeloma: spinal MR imaging in patients with untreated newly diagnosed disease. Radiology 1992; 185: 833-40. doi: 10.1148/radiol- ogy.185.3.1438772 14. Dutoit JC, Vanderkerken MA, Anthonissen J, Dochy F, Verstraete KL. The diagnostic value of SE MRI and DWI of the spine in patients with mono- clonal gammopathy of undetermined significance, smouldering myeloma and multiple myeloma. Eur Radiol 2014; 24: 2754-65. doi: 10.1007/s00330- 014-3324-5 15. Padhani AR, van Ree K, Collins DJ, D’Sa S, Makris A. Assessing the relation between bone marrow signal intensity and apparent diffusion coefficient in diffusion-weighted MRI. AJR Am J Roentgenol 2013; 200: 163-70. doi: 10.2214/AJR.11.8185 16. Zhang Y, Xiong X, Fu Z, Dai H, Yao F, Liu D, et al. Whole-body diffusion- weighted MRI for evaluation of response in multiple myeloma patients following bortezomib-based therapy: a large single-center cohort study. Eur J Radiol 2019; 120: 108695. doi: 10.1016/j.ejrad.2019.108695 17. Shaughnessy JD, Jr., Zhan F, Burington BE, Huang Y, Colla S, Hanamura I, et al. A validated gene expression model of high-risk multiple myeloma is de- fined by deregulated expression of genes mapping to chromosome 1. Blood 2007; 109: 2276-84. doi: 10.1182/blood-2006-07-038430 18. Nakaya A, Fujita S, Satake A, Nakanishi T, Azuma Y, Tsubokura Y, et al. Impact of CRAB symptoms in survival of patients with symptomatic myeloma in novel agent era. Hematol Rep 2017; 9: 6887. doi: 10.4081/hr.2017.6887 19. Kumar SK, Dispenzieri A, Lacy MQ, Gertz MA, Buadi FK, Pandey S, et al. Continued improvement in survival in multiple myeloma: changes in early mortality and outcomes in older patients. Leukemia 2014; 28: 1122-8. doi: 10.1038/leu.2013.313 20. Younes M, Hachfi H, Hammouda F, Younes K, Ben Hammouda S, Jguirim M, et al. [Survival prognosis factors in multiple myeloma]. [French]. Tunis Med 2014; 92: 399-405. PMID: 25741841 21. Yamasaki S, Kohno K, Kadowaki M, Takase K, Okamura S. Dose-adjusted le- nalidomide combined with low-dose dexamethasone rescues older patients with bortezomib-resistant multiple myeloma. Intern Med 2015; 54: 1711-5. doi: 10.2169/internalmedicine.54.4075 22. Greipp PR, San Miguel J, Durie BG, Crowley JJ, Barlogie B, Bladé J, et al. International staging system for multiple myeloma. J Clin Oncol 2005; 23: 3412-20. doi: 10.1200/JCO.2005.04.242 23. Palumbo A, Avet-Loiseau H, Oliva S, Lokhorst HM, Goldschmidt H, Rosinol L, et al. Revised international staging system for multiple myeloma: A report from International Myeloma Working Group. J Clin Oncol 2015; 33: 2863-9. doi: 10.1200/JCO.2015.61.2267 24. Barlogie B, Bolejack V, Schell M, Crowley J. Prognostic factor analyses of my- eloma survival with intergroup trial S9321 (INT 0141): examining whether different variables govern different time segments of survival. Ann Hematol 2011; 90: 423-8. doi: 10.1007/s00277-010-1130-y 25. Colović M, Janković G, Suvajdzić N, Milić N, Dordević V, Janković S. Thirty patients with primary plasma cell leukemia: a single center experience. Med Oncol 2008; 25: 154-60. doi: 10.1007/s12032-007-9011-5 Radiol Oncol 2023; 57(4): 455-464. Xiong X et al. / MRI-based pattern and ADC assesses tumor burden for MM464 26. Perez-Andres M, Almeida J, Martin-Ayuso M, De Las Heras N, Moro MJ, Martin-Nuñez G, et al. Soluble and membrane levels of molecules involved in the interaction between clonal plasma cells and the immunological microenvironment in multiple myeloma and their association with the characteristics of the disease. Int J Cancer 2009; 124: 367-75. doi: 10.1002/ ijc.23941 27. Tian C, Wang L, Wu L, Zhu L, Xu W, Ye Z, et al. Clinical characteristics and prognosis of multiple myeloma with bone-related extramedullary disease at diagnosis. Biosci Rep 2018; 38: BSR20171697. doi: 10.1042/BSR20171697 28. Baur A, Stäbler A, Nagel D, Lamerz R, Bartl R, Hiller E, et al. Magnetic reso- nance imaging as a supplement for the clinical staging system of Durie and Salmon? Cancer 2002; 95: 1334-45. doi: 10.1002/cncr.10818 29. Baur A, Stäbler A, Bartl R, Lamerz R, Reiser M. [Infiltration patterns of plas- macytomas in magnetic resonance tomography]. Rofo 1996; 164: 457-63. doi: 10.1055/s-2007-1015689 Radiol Oncol 2023; 57(4): 465-472. doi: 10.2478/raon-2023-0054 465 research article Assessment of short-term effect of platelet- rich plasma treatment of tendinosis using texture analysis of ultrasound images Karlo Pintaric1,2, Vladka Salapura1,2, Ziga Snoj1,2, Andrej Vovk3, Mojca Bozic Mijovski4,5, Jernej Vidmar1,6 1 Institute of Radiology, University Medical Center Ljubljana, Ljubljana, Slovenia 2 Department of Radiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Center of Clinical Physiology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia 4 Laboratory for Haemostasis and Atherothrombosis, University Medical Center, Ljubljana, Slovenia 5 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 6 Institute of Physiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2023; 57(4): 465-472. Received 14 June 2023 Accepted 06 August 2023 Correspondence to: Karlo Pintarić, M.D., Institute of Radiology, University Medical Center Ljubljana, Zaloška cesta 7, SI-1000 Ljubljana, Slovenia. E-mail: karlo.pintaric@gmail.com 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. Computer-aided diagnosis (i.e., texture analyses) tools are becoming increasingly beneficial methods to monitor subtle tissue changes. The aim of this pilot study was to investigate short-term effect of platelet rich plasma (PRP) treatment in supraspinatus and common extensor of the forearm tendinosis by using texture analysis of ultra- sound (US) images as well as by clinical questionnaires. Patients and methods. Thirteen patients (7 male and 6 female, age 36–60 years, mean age 51.2 ± 5.2) were fol- lowed after US guided PRP treatment for tendinosis of two tendons (9 patients with lateral epicondylitis and 4 with supraspinatus tendinosis). Clinical and US assessment was performed prior to as well as 3 months after PRP treatment with validated clinical questionnaires. Tissue response in tendons was assessed by using gray level run length matrix method (GLRLM) of US images. Results. All patients improved of tendinosis symptoms after PRP treatment according to clinical questionnaires. Almost all GLRLM features were statistically improved 3 months after PRP treatment. GLRLM-long run high gray level emphasis (LRLGLE) revealed the best moderate positive and statistically significant correlation after PRP (r = 0.4373, p = 0.0255), followed by GLRLM-low gray level run emphasis (LGLRE) (r = 0.3877, p = 0.05). Conclusions. Texture analysis of tendinosis US images was a useful quantitative method for the assessment of tendon remodeling after minimally invasive PRP treatment. GLRLM features have the potential to become useful imaging biomarkers to monitor spatial and time limited tissue response after PRP, however larger studies with similar protocols are needed. Key words: texture analysis; tendinosis; platelet-rich plasma; ultrasonography Introduction Tendinosis is a broad term encompassing pain and disability at the site of tendons associated with the histopathological findings of failed tendon healing response and no classical signs of inflammation. The most common sites of tendinosis involve the rotator cuff tendons (i.e., supraspinatus tendon), medial and lateral elbow epicondyles (i.e., common extensor tendon), patellar tendon, gluteal tendons Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma466 and the Achilles tendon).1 Several treatment op- tions exist in the process of tendinosis treatment. Conservative options with physiotherapy are widely accepted as the first line therapy, however most provide poor or only empirical evidence.2 Surgical management remains the last option due to the morbidity and inconsistent outcomes. Several minimally invasive treatments with ap- plication of different medication have been intro- duced, providing good patient outcome.3,4 Platelet rich plasma (PRP) injection therapy is effective in improving symptoms in supraspinatus tendinopa- thy and lateral epicondylitis.5,6 It is a good treat- ment option for patients with chronic changes of supraspinatus tendon and lateral epicondylitis who do not meet criteria for surgical management and are not content with the results of conservative treatments.7 Platelet-rich plasma is currently one of the most used and investigated therapeutic op- tions in clinical practice to target symptomatic ten- dinosis, however its effect was mostly monitored only clinically.8,9 PRP can be delivered directly into the lesion site and, once activated, the platelet con- centrate becomes a gel allowing the secretion of the bioactive molecules in situ and stimulates ten- don fibrillar remodulation. PRP is an autologous biotechnology that relies on the local delivery of a wide range of growth factors and cytokines with the aim of enhancing tissue healing.10 Gray-scale ultrasound (US) is the most common- ly used diagnostic tool to assess tendon pathology, particularly in superficial tendons. It has high level of evidence in shoulder and elbow examination.11 It is easily accessible, noninvasive and it has excel- lent spatial and contrast tissue resolution. It also enables dynamic assessment of tissues as well as can be used repeatedly without potentially harm- ful effects to the observed tissues. Additionally, it can also be used to guide and assess different treatments applied to tissues.12 However, US is a qualitative method and is entirely dependent on the performer with high interobserver variability ranging from poor to good.13,14 In order to over- come these limitations and to quantitatively assess tendon structure after specific treatment (i.e., PRP infiltration), several quantitative analyses of ten- don texture have already been introduced favor- ing gray level run length matrix method (GLRLM) due to its highest sensitivity, specificity and accu- racy.15,16 The aim of this study was to quantify short-term effect of PRP therapy in symptomatic supraspina- tus and elbow common extensors tendinosis using texture analysis of ultrasound images. Patients and methods Study population and study design Thirteen patients (7 male and 6 female), age 36–60 years (mean age 51.2 ± 5.2) were initially treat- ed with PRP injection therapy for tendinosis at Institute of Radiology, University Clinical Center Ljubljana from March 2019 till March 2020. Four supraspinatus tendons and nine common extensor tendons of the forearm were treated in this study. The inclusion criteria for patients with supraspina- tus tendinopathy was confirmed on MRI and per- sisting pain with disability for at least 3 months in the shoulder after at least one cycle of physio- therapy. The inclusion criteria for patients with lat- eral epicondylitis was confirmed by common ex- tensor tendinopathy with US and persisting pain with disability for at least 3 months after at least one cycle of physiotherapy. The exclusion criteria were rheumatoid arthritis, known malignancy, other joint injuries found on imaging, bleeding disorders, pregnancy and use of nonsteroidal anti- inflammatory drugs 7 days before PRP treatment as well as previous invasive treatments of the ten- dons. Patients were evaluated on the day of the PRP injection therapy and at 3 months follow up. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia (ap- proval number 0120 - 115/2018/4). Written informed consent was obtained from the patients or their authorized representatives in accordance with the Declaration of Helsinki. Patient evaluation Standardized questionnaires were used for the as- sessment of patient clinical outcome. Patients with lateral epicondylitis completed a Patient-rated tennis elbow evaluation (PRTEE) questionnaire, while patients with supraspinatus tendinopathy completed Shoulder pain and disability index questionnaire (SPADI).17,18 Both questionnaires were translated into Slovenian language and the scale was adjusted so that 0 points was assessed as the best score (i.e., no pain and no disability) and 100 points as the worst score with severe patient’s problems. Ultrasound imaging Real-time US examinations of the affected ten- dons were performed by one of three radiologists subspecialized in musculoskeletal imaging with at least 5 years of experience. US examinations Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma 467 were performed using a 13-15 MHz electronic linear-array transducer on a ProSound F75 scan- ner (Hitachi Aloka Medical, Ltd. Tokyo, Japan). The imaging resolution was approximately 1 mm in the longitudinal plane, scan width 38 mm and depth of field approximately 50 mm. Six consecu- tive US images of the tendons were acquired com- prising the most affected regions of tendinosis. Probe was placed longitudinally along the course of the tendon fibers with minimal probe move- ment to acquire the images. At the follow up, the second set of US images were acquired in the same manner. Patient’s bony and soft tissue landmarks were used to obtain comparable imaging location, angle and plane. PRP injection therapy Platelet-rich plasma preparation was obtained from patient autologous blood. Approximately 30 mL of blood was drawn from patient ś non-treated arm and mixed with 1 ml of anticoagulant solution (citrat dextrose). Centrifugation was performed using Harvest SmartPrep 2 (Terumo BCT, USA), which yielded approximately 3 mL of PRP. PRP in- jection therapy was performed under US guidance into the affected tendon. Patients were in supine position, surgical disinfection was performed and sterile probe sleeve was used. 3 mL of local anes- thetic (XylocaineTM) was infiltrated in subacromial- subdeltoid bursae in patients treating supraspina- tus tendinosis and in subcutaneous tissue around common extensor tendons in patients treating lat- eral epicondylitis.19,20 Approximately 2 ml of PRP was injected in the most heterogeneous area of the tendon on B mode. Small amount of PRP, which was not applied into the treated tendons, was analyzed in the Laboratory for Haemostasis and Atherothrombosis (University Medical Centre, Ljubljana, Slovenia) for PRP compounds (i.e., plate- let and leukocyte concentration). Image and statistical analysis Open source program for image processing, ImageJ (NIH programs, USA) was used. Region of interest (ROI) was selected in each 8-bit DICOM gray scale image from the set of 6 consecutive slices of each affected tendon as shown in Figure 1. Extracted ROI were then analyzed within sta- tistical program R v4.1.2 (R Core Team, Austria). Texture features extraction based on gray level run length matrix (GLRLM) were done with radiomics Image Processing Toolbox inside the R (Radiomics v 0.1.3) Statistical analysis involved mixed effect statistical modeling with the help of R package NLME i.e. (non-linear model effect, v 3.1–155). The assumptions about collinearity and homoscedas- FIGURE 1. US image of the representative supraspinatus tendinosis in longitudinal plane (A). Region of interest was selected from the footprint of the tendon to the myotendinous junction of supraspinatus muscle (encircled with red dots) and cropped for the purpose of texture analysis (B). A B Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma468 ticity were checked with residual and Q-Q plots. To achieve the normal distribution of data and to avoid outliers we selected maximum run length of 5 inside the GLRLM feature algorithm calculation of the study. Eight GLRLM features were extracted: grey level non-uniformity (GLN), long run empha- sis (LRE), high gray level run emphasis (HGLRE), long run high gray level emphasis (LRHGLE), long run low gray level emphasis (LRLGLE), low gray level run emphasis (LGLRE), run length non- uniformity (RLN) and run percentage (RP). Each of the features was tested for ability of prediction FIGURE 2. Box plots of the amplitude using gray level run length matrix method (GLRLM) features before and after platelet rich plasma (PRP) treatment (supraspinatus tendons encoded with blue and common extensor tendons encoded with pink). Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma 469 of tendon remodeling and clinical outcomes as as- sessed by questionnaires and p-value below 0.05 was considered as statistically significant. Results All patients stated at least some improvement of symptoms after PRP injection therapy. Laboratory analysis of PRP showed, that applied PRP consist- ed of average platelet concentration of 1954 x 103/ μL (range 1524 x 103/μL – 2163 x 103/μL) and aver- age leukocyte concentration of 24 x 103/μL (range 15,6 x 103/μL – 31,5 x 103/μL). Median score at PRTEE questionnaire for patients with epicondylitis before treatment was 65 (range 36 – 79), after treatment 26 (range 3 – 57) with a median improvement in score of 28. Median score at SPADI questionnaire for patients with shoulder problems before treatment was 47 (range 16 – 75), after treatment 12 (range 0–70) with a median improvement in score of 18. Figure 2 shows detailed statistical analysis of eight GLRLM features in treated tendons (i.e., com- mon extensor tendons and supraspinatus tendon) three months after PRP treatment. As can be seen from the Figure 2 values of almost all used GLRLM features were statistically significant after PRP treatment except for GLRLM-RP. Additionally, FIGURE 3. Clinical evaluation dependency of average texture feature per subject divided by tissue region and time. Tissues are coded with blue and pink lines, for supraspinatus and common extensor, while the time of measurement is indicated by a red circle for the first measurement before treatment and a green triangle for the measurement after treatment. Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma470 analysis of GLRLM features also showed that changes of their values occurred in similar trends in both studied tendon groups, i.e., supraspinatus (colored in blue) and common extensor tendon group (colored in green). Correlations according to the mixed effect model between clinical evaluation and changes in GLRLM features are shown in Figure 3. In the analysis it is seen that between among all used fea- tures GLRLM-LRLGLE has moderate positive and statistically significant correlation with clinical evaluation after PRP (r = 0.4373, p = 0.0255). Weak positive correlation is also seen for GLRLM-LGLRE (r = 0.3877, p = 0.0500) as well as weak negative cor- relation is seen for GLRLM-HGLRE, however, the later without statistical significance (r = -0.3210, p = 0.1098). Other used features showed weak corre- lations (i.e. positive as well as negative) but, also, without any statistical significance. Discussion This study aimed to perform texture analysis of symptomatic tendinosis US images of supraspi- natus and elbow common extensors tendons after PRP treatment. Several GLRLM features were ana- lyzed and found that almost all used features were significantly improved after PRP treatment. Computer-aided diagnosis tools (i.e., texture analyses) are becoming increasingly beneficial methods to monitor subtle tissue changes, i.e., small tendon tears or tissue repair. We used tools for segmentation, texture analysis and area com- putation, which allowed us to increase the accu- racy and more quantitative analysis of the changes in the tendons, otherwise hardly recognized with our eyes. Texture analysis enabled to quantitative- ly monitor the PRP effect in tendons. Almost all the applied features enabled to quantify intuitive qualities due to tissue changes in the observed ten- dons already shortly after PRP as a function of the spatial variation in pixel intensities of US images. Eight GLRLM features were used since it was al- ready shown that this set of GLRLM features pro- vides optimal sensitivity, specificity, and accuracy to characterize tissue properties.16 In our study most of the values of GLRLM fea- tures were significantly altered already in short- time interval (i.e., 3 months post PRP), as well as almost all treated patient reported good sympto- matic improvements of tendinosis. Although the process of tissue repair can take up to 12 months, we have already quantitatively observed signifi- cant tissue changes shortly after PRP. However, the most significant correlation was obtained only in features, which are linked to longitudinal ori- ented echotexture of the treated tendons. This seem reasonably since longitudinal reorientation is most comparable to architecture of normal ten- don. Specifically, GLRLM-LGLRE showed moder- ate positive correlation and GLRLM-HGLRE fea- ture showed weak positive correlation with clini- cal questionnaires. Both of these features describe changes of low gray level emphasis, which in our case turned out to be the most important param- eter of texture analysis to monitor progress of tis- sue response. In comparison, GLRLM-LRLGLE was favored since it depicted tissue changes (i.e., reduction of hypoechoic areas) in a longitudinal direction. Our results seem to be in accordance with previous studies, which reported similar reduction of hypoechoic areas in tendinosis after PRP application, however these observations were semiquantitative and not linked to texture orien- tation.19,20 In a normal tendon the fibers are usu- ally arranged longitudinally in a fibrillar pattern, forming a relatively homogeneous tissue texture. In the tendinosis, the fibers lose their longitudinal fibrillar pattern and tissue texture turns out to be more heterogeneous and deranged. In the normal healing process of the tendons the fibers remodel their orientation again more longitudinally.21 In our study similar alterations of the fibrillar pattern were observed after PRP treatment. So far, several studies have been performed to follow-up the effect of various treatment options of tendinopathies. However, mostly these were semi- quantitative evaluations based on standard US cri- teria for tendinopathy, such as focal or diffuse loss of uniform tendon echotexture, thickening of the tendon, loss of fibrillar structure and neovascular- ity.22 In our study GLRLM avoids semi-quantita- tive assessment of tendinopathy based on “classic” US features and enabled to quantify properties of the tendon by their texture content. This approach enabled us to analyze tissue imaging and clinical outcome after PRP more objectively. Despite still uncertain steps of PRP effects, in the short-term the factors released by the platelets most likely lower tissue inflammation and modu- late the pain receptors sensibility.23 Afterwards, PRP favors the cell proliferation with collagen and matrix deposition, as well as tissue remodeling. The final outcome is tissue scar formation, which can help to restore tendon function. In the study, two groups of tendons were treat- ed (i.e., symptomatic supraspinatus and common Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma 471 extensor tendinosis) without any major differences between the groups comparing the initial or post- treatment texture analyses. This could be attribut- ed to a small number of subjects and consequently making difficult to observe any major considera- tion about the differences between tendon groups. On the other hand, we hypothesized that similar, spatial and time related tissue changes occurred in the both tendon groups before and after PRP, which are not significantly discernable by texture analysis. Tendons with potential to assess its re- modeling after PRP treatment with GLRLM are preferably superficially lying tendons like rotator cuff tendons, common extensor and flexor tendons of the forearm, patellar ligament, where high-res- olution US images with optimal signal-to-noise ra- tio (SNR) could be obtained in a repetitive manner. Deeply lying tendons with complex anatomical structures e.g., proximal hamstrings tendons, are probably less suitable for texture analysis due to the lack of optimal SNR which is a basic prerequi- site for texture analysis. All treated patients improved of tendinosis symptoms in rather short time prior to complete tendon remodeling. This effect could be associ- ated also with high leukocyte concentration of the obtained PRP. We assumed that the leukocyte- rich content of PRP influenced the concentration of various cytokines and modulate local immune and inflammatory response similar as reported in previous studies.24 The study had several limitations. Firstly, the studied sample in this pilot study is rather small since some of the treated patients did not return to the follow up. Secondly, the observation period was relatively short. Although we observed clini- cal improvements after three months, remodula- tion of the treated tendons was probably not yet completed in this short period of time by means of texture analysis. Therefore, the next step would be to perform a similar study in a larger group with intermediate as well as longer observation time period using a comparable concept of texture analysis and clinical questionnaires. In the study the intraobserver variability was also addressed. It was lowered as much as possible by taking mul- tiple consecutive slices of each tendon and placing the US probe in the similar positions regarding bony and soft tissue landmarks at the initial and posttreatment imaging. Finally, we studied only the effect of leukocyte-rich PRP compound and did not make any comparison between different PRP compound, i.e., leukocyte-poor PRP versus leukocytes-rich PRP. In conclusion, computer aided texture analysis of US tendinosis enabled to quantify tendon re- modeling in short time period after PRP treatment, with the most pronounced effect in features, sensi- tive to changes in longitudinal tissue orientation. Features of GLRLM analysis have the potential to become useful imaging biomarkers to monitor spatial and time limited tissue response, however larger studies with similar protocols are needed. References 1. Millar NL, Silbernagel KG, Thorborg K, Kirwan PD, Galatz LM, Abrams GD, et al. Tendinopathy. Nat Rev Dis Primers 2021; 7: 1. doi: 10.1038/s41572-020- 00234-1 2. Kuhn JE. Exercise in the treatment of rotator cuff impingement: a systematic review and a synthesized evidence-based rehabilitation protocol. J Shoulder Elb Surg 2009; 18: 138-60. doi: 10.1016/j.jse.2008.06.004 3. Sconfienza LM, Adriaensen M, Albano D, Allen G, Aparisi Gomez MP, Bazzocchi A, et al. Clinical indications for image-guided interventional proce- dures in the musculoskeletal system: a Delphi-based consensus paper from the European Society of Musculoskeletal Radiology (ESSR)-part I, shoulder. Eur Radiol 2020; 30: 903-13. doi: 10.1007/s00330-019-06419-x 4. Sconfienza LM, Adriaensen M, Albano D, Aparisi Gomez MP, Bazzocchi A, Beggs I, et al. Clinical indications for image-guided interventional proce- dures in the musculoskeletal system: a Delphi-based consensus paper from the European Society of Musculoskeletal Radiology (ESSR)-Part II, elbow and wrist. Eur Radiol 2020; 30: 2220-30. doi: 10.1007/s00330-019-06545-6 5. Chianca V, Albano D, Messina C, Midiri F, Mauri G, Aliprandi A, et al. Rotator cuff calcific tendinopathy: from diagnosis to treatment. Acta Biomed 2018; 89: 186-96. doi: 10.23750/abm.v89i1-S.7022 6. Ma KL, Wang HQ. Management of lateral epicondylitis: a narrative literature review. Pain Res Manag 2020; 2020: 6965381. doi: 10.1155/2020/6965381 7. Giovannetti de Sanctis E, Franceschetti E, De Dona F, Palumbo A, Paciotti M, Franceschi F. The efficacy of injections for partial rotator cuff tears: a systematic review. J Clin Med 2020; 10: 51. doi: 10.3390/jcm10010051 8. Kearney RS, Ji C, Warwick J, Parsons N, Brown J, Harrison P, et al. Effect of platelet-rich plasma injection vs sham injection on tendon dysfunction in patients with chronic midportion achilles tendinopathy: A randomized clini- cal trial. JAMA 2021; 326: 137-44. doi: 10.1001/jama.2021.6986 9. Rha DW, Park GY, Kim YK, Kim MT, Lee SC. Comparison of the therapeutic effects of ultrasound-guided platelet-rich plasma injection and dry needling in rotator cuff disease: a randomized controlled trial. Clin Rehabil 2013; 27: 113-22. doi: 10.1177/0269215512448388 10. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-rich plasma: new performance understandings and therapeutic considerations in 2020. Int J Mol Sci 2020; 21: 7794. doi: 10.3390/ijms21207794 11. Sconfienza LM, Albano D, Allen G, Bazzocchi A, Bignotti B, Chianca V, et al. Clinical indications for musculoskeletal ultrasound updated in 2017 by European Society of Musculoskeletal Radiology (ESSR) consensus. Eur Radiol 2018; 28: 5338-51. doi: 10.1007/s00330-018-5474-3 12. Tumpaj T, Potocnik Tumpaj V, Albano D, Snoj Z. Ultrasound-guided carpal tunnel injections. Radiol Oncol 2022; 56: 14-22. doi: 10.2478/ raon-2022-0004 13. Cavaggion C, Navarro-Ledesma S, Luque-Suarez A, Juul-Kristensen B, Voogt L, Struyf F. Subacromial space measured by ultrasound imaging in asymptomatic subjects and patients with subacromial shoulder pain: an inter-rater reliability study. Physiother Theory Pract 2023; 39: 2196-207. doi: 10.1080/09593985.2022.2072251 14. Ingwersen KG, Hjarbaek J, Eshoej H, Larsen CM, Vobbe J, Juul-Kristensen B. Ultrasound assessment for grading structural tendon changes in supraspi- natus tendinopathy: an inter-rater reliability study. BMJ Open 2016; 6: e011746. doi: 10.1136/bmjopen-2016-011746 Radiol Oncol 2023; 57(4): 465-472. Pintaric K et al. / Texture analysis of tendinosis after platelet rich plasma472 15. Paris MT, Mourtzakis M. Muscle composition analysis of ultrasound images: a narrative review of texture analysis. Ultrasound Med Biol 2021; 47: 880- 95. doi: 10.1016/j.ultrasmedbio.2020.12.012 16. Park BE, Jang WS, Yoo SK. Texture analysis of supraspinatus ultrasound image for computer aided diagnostic system. Healthc Inform Res 2016; 22: 299-304. doi: 10.4258/hir.2016.22.4.299 17. Breckenridge JD, McAuley JH. Shoulder pain and disability index (SPADI). J Physiother 2011; 57: 197. doi: 10.1016/S1836-9553(11)70045-5 18. Rompe JD, Overend TJ, MacDermid JC. Validation of the patient-rated tennis elbow evaluation questionnaire. J Hand Ther 2007; 20: 3-10; quiz 11. doi: 10.1197/j.jht.2006.10.003 19. Filardo G, Kon E, Di Matteo B, Pelotti P, Di Martino A, Marcacci M. Platelet- rich plasma for the treatment of patellar tendinopathy: clinical and imaging findings at medium-term follow-up. Int Orthop 2013; 37: 1583-9. doi: 10.1007/s00264-013-1972-8 20. Finnoff JT, Fowler SP, Lai JK, Santrach PJ, Willis EA, Sayeed YA, et al. Treatment of chronic tendinopathy with ultrasound-guided needle tenoto- my and platelet-rich plasma injection. PM R 2011; 3: 900-11. doi: 10.1016/j. pmrj.2011.05.015 21. Cook JL, Purdam CR. Is tendon pathology a continuum? A pathology model to explain the clinical presentation of load-induced tendinopathy. Br J Sports Med 2009; 43: 409-16. doi: 10.1136/bjsm.2008.051193 22. Docking SI, Ooi CC, Connell D. Tendinopathy: is imaging telling us the entire story? J Orthop Sports Phys Ther 2015; 45: 842-52. doi: 10.2519/ jospt.2015.5880 23. Abate M, Verna S, Di Gregorio P, Salini V, Schiavone C. Sonographic find- ings during and after platelet rich plasma injections in tendons. Muscles Ligaments Tendons J 2014; 4: 29-34. PMID: 24932444 24. Kobayashi Y, Saita Y, Nishio H, Ikeda H, Takazawa Y, Nagao M, et al. Leukocyte concentration and composition in platelet-rich plasma (PRP) influences the growth factor and protease concentrations. J Orthop Sci 2016; 21: 683-9. doi: 10.1016/j.jos.2016.07.009 Radiol Oncol 2023; 57(4): 473-486. doi: 10.2478/raon-2023-0061 473 research article The association of genetic factors with serum calretinin levels in asbestos-related diseases Cita Zupanc1,2, Alenka Franko2,3, Danijela Strbac2,4, Viljem Kovac2,4, Vita Dolzan5, Katja Goricar5 1 Military Medical Unit-Slovenian Army, Ljubljana, Slovenia 2 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 3 University Medical Centre Ljubljana, Clinical Institute of Occupational Medicine, Ljubljana, Slovenia 4 Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 University of Ljubljana, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Pharmacogenetics Laboratory, Ljubljana, Slovenia Radiol Oncol 2023; 57(4): 473-486. Received 19 October 2023 Accepted 31 October 2023 Correspondence to: Assist. Prof. Katja Goričar, Ph.D., University of Ljubljana, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Pharmacogenetics Laboratory, Vrazov trg 2, SI-1000 Ljubljana, Slovenia. E-mail: katja.goricar@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. Asbestos exposure is associated with different asbestos-related diseases, including malignant meso- thelioma (MM). MM diagnosis is confirmed with immunohistochemical analysis of several markers, including calretinin. Increased circulating calretinin was also observed in MM. The aim of the study was to determine if CALB2 polymor- phisms or polymorphisms in genes that can regulate calretinin expression are associated with serum calretinin levels or MM susceptibility. Subjects and methods. The study included 288 MM patients and 616 occupationally asbestos-exposed subjects without MM (153 with asbestosis, 380 with pleural plaques and 83 without asbestos-related disease). Subjects were genotyped for seven polymorphisms in CALB2, E2F2, MIR335, NRF1 and SEPTIN7 genes using competitive allele-specific polymerase chain reaction (PCR). Serum calretinin was determined with ELISA in 545 subjects. Nonparametric tests, logistic regression and receiver operating characteristic (ROC) curve analysis were used for statistical analysis. Results. Carriers of at least one polymorphic CALB2 rs889704 allele had lower calretinin levels (P = 0.036). Carriers of two polymorphic MIR335 rs3807348 alleles had higher calretinin (P = 0.027), while carriers of at least one polymorphic NRF1 rs13241028 allele had lower calretinin levels (P = 0.034) in subjects without MM. Carriers of two polymorphic E2F2 rs2075995 alleles were less likely to develop MM (odds ratio [OR] = 0.64, 95% confidence interval [CI] = 0.43-0.96, P = 0.032), but the association was no longer significant after adjustment for age (P = 0.093). Optimal serum calretinin cut-off values differentiating MM patients from other subjects differed according to CALB2, NRF1, E2F2, and MIR335 genotypes. Conclusions. The results of presented study suggest that genetic variability could influence serum calretinin levels. These findings could contribute to a better understanding of calretinin regulation and potentially to earlier MM diag- nosis. Key words: malignant mesothelioma; calretinin; CALB2; asbestos-related disease; polymorphism Introduction Prolonged asbestos exposure can lead to occur- rence of different asbestos-related diseases, in- cluding pleural plaques and asbestosis, as well as several cancers. Use and production of asbes- tos was largely banned after it was classified as a carcinogen, but it is still legally used in mostly developing countries and it can still be found in the environment.1,2 Asbestos-related diseases often Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases474 occur long after initial asbestos exposure and their incidence continues to rise.1 The most problematic asbestos-related disease is malignant mesothelioma (MM), a rare but very aggressive cancer. However, only a minority of asbestos-exposed people develops MM. Other fac- tors, such as genetic variability may contribute to carcinogenesis and development of MM.3 Among asbestos-exposed workers, several familial cases of MM were described, emphasizing that genetic factors could contribute to MM development.4 In recent years, germline BRCA1-associated protein 1 (BAP1) mutations were shown to predispose to the development of MM and other cancers. Additionally, studies suggest that numerous chro- mosomal deletions can accumulate in most MM cases, usually associated with the loss or inactiva- tion of tumor suppressor genes.5,6 Despite advanc- es in treatment, prognosis and survival of MM patients remain poor.7,8 Therefore, MM diagnosis and treatment have become increasingly focused on molecular mechanisms.9 To confirm MM diagnosis, several tumor mark- ers are routinely analysed using immunohisto- chemical staining.10 One of the established immu- nohistochemical markers is calretinin10, a calcium binding protein and calcium sensor crucial for neuron function that is also expressed on meso- thelial cells.11 It has been shown to affect mesothe- lial cell proliferation and migration and epithelial- to-mesenchymal transition. It was also associated with focal adhesion kinase signaling pathway and signaling pathways associated with response to asbestos.12 Calretinin is encoded by the CALB2 gene.13 As MM diagnosis is usually made when the dis- ease is already advanced, blood-based biomarkers such as mesothelin and fibulin-3 that would enable an earlier diagnosis and better prognosis of MM are extensively studied.14,15 Recently, calretinin was also proposed as a soluble biomarker in MM, as increased plasma or serum levels were observed in MM patients compared to subjects with other asbestos-related diseases or healthy controls.8,16-18 However, interindividual variability limits the sensitivity and specificity of calretinin as a diag- nostic biomarker and several clinical characteris- tics were previously associated with soluble cal- retinin levels.19 Low tumor calretinin expression was associated with lower protein concentration in the bloodstream, but there was no clear correla- tion with tumor size.20 Higher calretinin concen- trations were observed in patients with epithelioid or biphasic MM compared to patients with sarco- matoid MM.8,20,21 Calretinin levels were also higher in women compared to men and in subjects with renal dysfunction.22 Molecular mechanisms regulating calretinin expression in various tissues or in cancer could also contribute to interindividual variability of se- rum calretinin concentration, but the knowledge of these processes is limited.23 Calretinin expres- sion may be affected by several factors, including transcription factors or miRNAs. Among tran- scription factors, calretinin expression was found to be influenced by septin 7, E2F transcription fac- tor 2 (E2F2) and nuclear respiratory factor 1 (NRF- 1) in previous studies.23,24 Additionally, miR-335-5p was proposed as a regulator of CALB2 expression25 and miR-30e-5p was negatively correlated with the calretinin expression in pleural MM patient samples.26 Gene expression can also be modified by genetic variability in the promoter 5’ untrans- lated region (UTR) of the gene affecting binding of transcription factors, or genetic variability in the 3’ UTR affecting miRNA binding. Polymorphisms in genes coding for miRNAs or transcription factors involved in calretinin regulation could also influ- ence calretinin expression. In previous studies, ge- netic factors affecting expression and circulating levels of other important MM biomarkers such as mesothelin have already been identified.27-29 On the other hand, very little is known about the role of single nucleotide polymorphisms (SNPs) in the CALB2 gene. An intronic polymorphism in CALB2 gene was previously proposed as a risk factor for colon cancer.30 To date, no studies have been per- formed to evaluate if genetic factors influence cal- retinin expression or if they could modify suscep- tibility to develop asbestos-related diseases. Our aim was to determine whether genetic pol- ymorphisms in the CALB2 gene and in the genes coding for miRNA and transcription factors regu- lating calretinin expression are associated with MM susceptibility or serum calretinin levels in patients with asbestos-related diseases. Subjects and methods Study population Our retrospective study included patients with MM, subjects with asbestosis, subjects with pleu- ral plaques, and subjects that were occupationally exposed to asbestos but, did not develop any as- bestos-related disease. Patients with MM were treated at the Institute of Oncology Ljubljana between November 2001 and Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases 475 March 2019. The diagnosis of pleural or peritoneal MM was performed by thoracoscopy or laparos- copy, respectively, and confirmed histologically by an experienced pathologist, mostly in others ter- tiary institutions in Slovenia. Stage of MM was de- termined using the TNM staging system for pleu- ral MM. Performance status of MM patients was determined using Eastern Cooperative Oncology Group (ECOG) scores. Subjects with asbestosis, subjects with pleural plaques and asbestos-exposed subjects who did not develop any asbestos-related disease were selected from a cohort of occupationally exposed workers who were evaluated by the State Board for the Recognition of Occupational Asbestos Diseases at the Clinical Institute of Occupational, Traffic and Sports Medicine in Ljubljana between September 1998 and April 2007. The diagnosis of as- bestos-related diseases was based on the Helsinki Criteria for Diagnosis and Attribution of Asbestos Diseases31 and the American Thoracic Society rec- ommendations.32 Follow-up was performed for all subjects in 2018 to confirm they did not develop any other asbestos-related disease. For all subjects, data on demographic (sex, age, smoking) and clinical characteristics were obtained from the medical records or during an interview. All participants provided written in- formed consent. The study has been approved by the National Medical Ethics Committee of the Republic of Slovenia (31/07/04, 39/04/06 and 41/02/09) and was carried out according to the Declaration of Helsinki. Bioinformatic analysis Using bioinformatic analysis, we identified com- mon SNPs that could affect calretinin expression: SNPs in the 5’ UTR and 3’ UTR of the calretinin gene (CALB2) and SNPs in the genes coding for miRNAs and transcription factors involved in the regulation of calretinin expression. Experimentally confirmed miRNAs and transcription factors were selected using miRTarBase33 and literature screen- ing. Using LD Tag SNP Selection tool34 and dbSNP database35, we identified all SNPs in 5’ UTR, 3’ UTR and near gene regions (± 1000 base pairs) of CALB2 gene and all SNPs in 5’ UTR, 3’ UTR and coding regions of transcription factor coding genes with minor allele frequency (MAF) in European populations above 5%. Additionally, available lit- erature was screened for SNPs in miRNA coding genes.36 In silico predicted function of SNPs was assessed using SNP Function Prediction tool34 as well as HaploReg v4.137 and GTEx38 for SNPs in regulatory regions. Linkage disequilibrium (LD) between SNPs in one gene was evaluated using LD link tool.39 For genotyping, we selected only SNPs with in silico predicted functional role (non- synonymous SNPs, SNPs that influence transcrip- tion factor or miRNA binding or SNPs that influ- ence splicing). If more SNPs within one gene were in high LD (R2 > 0.8), only one SNP was selected for genotyping analyses. DNA extraction and genotyping Genomic DNA was extracted from peripheral ve- nous blood samples using Qiagen FlexiGene Kit (Qiagen, Hilden, Germany) according to the manu- facturer’s instructions. For a subset of subjects that did not develop any asbestos-related disease, DNA was extracted from capillary blood samples collect- ed on Whatman FTA cards using MagMaxTM DNA Multi-Sample Kit (Applied Biosystems, Foster City, California, USA). The genotyping of all selected SNPs was carried out using a fluorescence-based competitive allele-specific polymerase chain reac- tion (KASP) assay, according to the manufacturer’s instructions (LGC Genomics, UK). For all SNPs, 15% of samples were genotyped in duplicates. Genotyping quality control criteria were 100% du- plicate call rate and 95% SNP-wise call rate. Measurement of serum calretinin Serum samples were collected at diagnosis for MM patients and at inclusion in the study for all other subjects. Samples were prepared within 6 hours of blood collection and stored at -20°C. Serum calretinin levels were determined using a commercially available enzyme-linked immuno- sorbent Calretinin ELISA assay (DLD Diagnostika GmbH, Germany) according to the manufacturer’s instructions as previously described.8,16,21 Statistical Analysis Continuous and categorical variables were de- scribed using median with interquartile range and frequencies, respectively. Nonparametric Mann- Whitney test or Kruskal-Wallis test with post hoc Bonferroni corrections for pairwise comparisons were used to compare the distribution of continu- ous variables. Chi square test was used to compare the distribution of categorical variables among different groups and to evaluate deviation from Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases476 Hardy-Weinberg equilibrium. For all investigated SNPs, both additive and dominant models were used in the analysis. Univariate and multivariate logistic regression was used to compare genotype frequencies between groups and to determine odds ratios (ORs) and 95% confidence intervals (CIs). Demographic and clinical parameters, sig- nificantly associated with asbestos-related disease susceptibility in univariate analysis, were used for adjustment in multivariate models. Receiver oper- ating characteristic (ROC) curve analysis was used to determine area under the curve (AUC), sensitiv- ity and specificity. Cut-off values were determined as the values with the highest sum of sensitivity and specificity. All statistical tests were two-sided and the level of significance was set at 0.05. The statistical analyses were carried out by using IBM SPSS Statistics version 27.0 (IBM Corporation, Armonk, NY, USA). To assess the combined ef- fect of all CALB2 SNPs, we reconstructed haplo- types using Thesias software.40 Haplotypes with predicted frequency above 0.04 were included in the analysis and the most common haplotype was used as a reference. Results Subjects’ characteristics Among 904 subjects included in our study, 288 (31.9%) had MM. Among 616 non-MM subjects that were occupationally exposed to asbestos, 153 subjects had asbestosis, 380 subjects had pleural plaques and 83 did not develop any asbestos-relat- ed disease. Characteristics of each subject group are presented in Table 1. Patients with MM were older than all other groups (P < 0.001), but there were no significant differences regarding sex (P = 0.180) and smoking (P = 0.205). Among patients with MM, 217 (75.3%) patients had epithelioid histological type, 26 (9.0%) patients had biphasic type, and 26 (9.0%) patients had sar- comatoid type, while histological type could not be determined in 19 (6.6%) patients. According to cancer stage, 19 (6.6%) patients had stage 1 MM, 63 (22.0%) patients had stage 2 MM, 85 (29.6%) pa- tients had stage 3 MM, and 87 (30.3%) patients had stage 4 MM, while no data were available for one patient. Additionally, 33 (11.5%) patients had peri- toneal MM. Regarding ECOG performance status, 18 patients (6.3%) had score 0, 142 (49.5%) score 1, 110 (38.3%) score 2 and 17 (5.9%) score 3, while no data was available for one patient. Bioinformatic analysis Based on available literature and publicly available databases, we identified genes and SNPs that could influence calretinin expression and serum levels: SNPs in 5’ and 3’ UTR of CALB2 gene and SNPs in genes coding for transcription factors and miR- NAs associated with calretinin expression. Three miRNAs were experimentally associated with regulation of CALB2 expression: hsa-miR-9, hsa- miR-30e and hsa-miR-335-5p26 but common SNPs were only described in MIR335 gene. Additionally, three transcription factors were experimentally as- sociated with regulation of CALB2 expression: E2F transcription factor 2 (E2F2), nuclear respiratory factor 1 (NRF1), and septin 7 (SEPTIN7).23,24 In total, seven SNPs fulfilling all inclusion cri- teria were included in the study: CALB2 rs1862818, CALB2 rs889704, CALB2 rs8063760, E2F2 rs2075995, MIR335 rs3807348, NRF1 rs13241028, and SEPTIN7 rs3801339. Their role, predicted function and geno- type frequencies in the whole study group as well as minor allele frequency and agreement with Hardy-Weinberg equilibrium (HWE) in controls TABLE 1. Clinical characteristics of the subjects included in the study Characteristic Category/unit No disease(N = 83) Pleural plaques (N = 380) Asbestosis (N = 153) MM (N = 288) P Sex Male, N (%) 61 (73.5) 262 (68.9) 119 (77.8) 213 (74.0) 0.1801 Female, N (%) 22 (26.5) 118 (31.1) 34 (22.2) 75 (26.0) Age Median(25%−75%) 53.4 (48.5−59.2) 54.8 (48.8−62.7) 59.4 (51.3−66.1) 66.0 (59−73) < 0.001 2 Smoking No, N (%) 46 (55.4) 187 (49.2) 74 (48.4) 158 (56.4) [8] 0.2051 Yes, N (%) 37 (44.6) 193 (50.8) 79 (51.6) 122 (43.6) 1 calculated using chi-square test; 2 calculated using Kruskal-Wallis test. Number of missing data is presented in [] brackets. MM = malignant mesothelioma Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases 477 are presented in Table 2. All SNPs were in agree- ment with HWE in controls without asbestos relat- ed diseases and variant allele frequencies ranged between 14 and 63%. Association of selected SNPs with MM susceptibility In the whole study group, we evaluated if selected polymorphisms were associated with MM suscep- tibility. Genotype frequencies in MM patients and subjects without MM and are presented in Table 3. Carriers of two polymorphic E2F2 rs2075995 al- leles were less likely to develop MM (OR = 0.64, 95% CI = 0.43−0.96, P = 0.032), but the association was no longer significant after adjustment for age (OR = 0.68, 95% CI = 0.44−1.07, P = 0.093). No other SNP was significantly associated with MM susceptibility (Table 3). Additionally, we also com- pared MM patients to other subject groups sepa- rately. Genotype frequencies of SNPs among sub- jects with asbestosis, subjects with pleural plaques and subjects without asbestos-related diseases, are presented in Supplementary Table 1. When com- paring MM patients with subjects without any asbestos-related disease, carriers of two polymor- TABLE 2. Genotype frequencies of investigated single nucleotide polymorphisms (SNPs) in the whole study group, their variant allele frequency (VAF) and agreement with Hardy-Weinberg equilibrium (HWE) in subjects without any asbestos-related disease (controls) Gene SNP Nucleotide or amino acid change Predicted function Genotype N (%) VAF (controls) pHWE (controls) CALB2 rs1862818 c.-828C>T May influence transcription factor binding, may alter chromatin states and regulatory motifs CC 479 (53.0) 0.27 0.617 CT 346 (38.3) TT 79 (8.7) CALB2 rs889704 c.-634C>A May influence transcription factor binding, may alter chromatin states and regulatory motifs CC 708 (78.4) [1] 0.14 0.814 CA 182 (20.2) AA 13 (1.4) CALB2 rs8063760 c.*138T>C May influence miRNA binding, may alter regulatory motifs CC 527 (58.4) [2] 0.23 0.322 CT 319 (35.4) TT 56 (6.2) E2F2 rs2075995 c.678C>A, p.Gln226His Nonsynonymous, may influence splicing CC 187 (20.7) 0.61 0.209 CA 468 (51.8) AA 249 (27.5) MIR335 rs3807348 g.130496266G>A Downstream transcript variant, may influence transcription factor binding GG 228 (25.3) [3] 0.49 0.376 GA 446 (49.5) AA 227 (25.2) NRF1 rs13241028 c.*1321T>C May influence miRNA binding TT 547 (60.5) 0.22 0.061 TC 313 (34.6) CC 44 (4.9) SEPTIN7 rs3801339 c.1168-4451T>C Genic downstream transcript variant1 TT 164 (18.1) 0.63 0.187 TC 401 (44.4) CC 339 (37.5) 1 previously classified as a nonsynonymous variant. Number of missing data is presented in [] brackets. A = adenine; C = cytosine; G = guanine; SNP = single nucleotide polymorphisms; T = thymine Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases478 TABLE 3. Association of investigated single nucleotide polymorphisms (SNPs) with malignant mesothelioma (MM) susceptibility SNP Genotype Subjects without MM (N = 616) N (%) MM patients (N = 288) N (%) OR (95% CI) P OR (95% CI)adj Padj CALB2 rs1862818 CC 340(55.2) 139 (48.3) Reference Reference CT 226(36.7) 120 (41.7) 1.30 (0.97−1.75) 0.084 1.35 (0.97−1.87) 0.073 TT 50(8.1) 29 (10.1) 1.42 (0.86−2.34) 0.169 1.34 (0.77−2.32) 0.299 CT+TT 276(44.8) 149 (51.7) 1.32 (1.00−1.75) 0.052 1.35 (0.99−1.83) 0.059 CALB2 rs889704 CC 485(78.9) [1] 223 (77.4) Reference Reference CA 121(19.7) 61 (21.2) 1.10 (0.78−1.55) 0.602 1.03 (0.70−1.51) 0.899 AA 9(1.5) 4 (1.4) 0.97 (0.29−3.17) 0.955 0.55 (0.15−1.94) 0.349 CA+AA 130(21.1) 65 (22.6) 1.09 (0.78−1.52) 0.626 0.98 (0.67−1.42) 0.912 CALB2 rs8063760 CC 352(57.3) [2] 175 (60.8) Reference Reference CT 222(36.2) 97 (33.7) 0.88 (0.65−1.19) 0.398 0.91 (0.66−1.26) 0.576 TT 40(6.5) 16 (5.6) 0.80 (0.44−1.48) 0.483 0.82 (0.42−1.60) 0.554 CT+TT 262(42.7) 113 (39.2) 0.87 (0.65−1.15) 0.329 0.90 (0.65−1.23) 0.493 E2F2 rs2075995 CC 117(19.0) 70 (24.3) Reference Reference CA 319(51.8) 149 (51.7) 0.78 (0.55−1.11) 0.171 0.83 (0.56−1.23) 0.349 AA 180(29.2) 69 (24.0) 0.64 (0.43−0.96) 0.032 0.68 (0.44−1.07) 0.093 CA+AA 499(81.0) 218 (75.7) 0.73 (0.52−1.02) 0.067 0.78 (0.53−1.13) 0.182 MIR335 rs3807348 GG 158 (25.8) [3] 70 (24.3) Reference Reference GA 307(50.1) 139 (48.3) 1.02 (0.72−1.44) 0.902 1.00 (0.68−1.46) 0.980 AA 148(24.1) 79 (27.4) 1.20 (0.81−1.78) 0.352 1.22 (0.79−1.87) 0.376 GA+AA 455(74.2) 218 (75.7) 1.08 (0.78−1.50) 0.636 1.07 (0.75−1.52) 0.724 NRF1 rs13241028 TT 374(60.7) 173 (60.1) Reference Reference TC 210(34.1) 103 (35.8) 1.06 (0.79−1.43) 0.699 1.08 (0.78−1.50) 0.636 CC 32(5.2) 12 (4.2) 0.81 (0.41−1.61) 0.550 0.92 (0.44−1.93) 0.823 TC+CC 242(39.3) 115 (39.9) 1.03 (0.77−1.37) 0.853 1.06 (0.78−1.45) 0.711 SEPTIN7 rs3801339 TT 109 (17.7) 55 (19.1) Reference Reference TC 266(43.2) 135 (46.9) 1.01 (0.68−1.48) 0.976 1.05 (0.69−1.61) 0.815 CC 241(39.1) 98 (34.0) 0.81 (0.54−1.20) 0.291 0.76 (0.49−1.18) 0.218 TC+CC 507(82.3) 233 (80.9) 0.91 (0.64−1.30) 0.610 0.91 (0.61−1.35) 0.627 Number of missing data is presented in [] brackets. A = adenine; Adj = adjusted for age; C = cytosine; CI = confidence interval; G = guanine; OR = odds ratio; T= thymine Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases 479 TABLE 4. Association of selected SNPs with serum calretinin concentration SNP Genotype All subjects Subjects without MM MM patients Calretinin (ng/ml) Median (25−75%) Padd Pdom Calretinin (ng/ml) Median (25−75%) Padd Pdom Calretinin (ng/ml) Median (25−75%) Padd Pdom CALB2 rs1862818 CC 0.18 (0.11−0.34) 0.622 0.422 0.15 (0.09−0.22) 0.751 0.865 0.64 (0.22−1.45) 0.952 0.802 CT 0.19 (0.11−0.41) 0.16 (0.09−0.24) 0.51 (0.23−1.41) TT 0.18 (0.10−0.37) 0.13 (0.08−0.20) 0.38 (0.21−3.57) CT+TT 0.19 (0.11−0.40) 0.15 (0.09−0.24) 0.48 (0.23−1.43) CALB2 rs889704 CC 0.19 (0.11−0.37) 0.099 0.036 0.15 (0.10−0.23) 0.130 0.069 0.52 (0.25−1.43) 0.508 0.441 CA 0.17 (0.08−0.27) 0.16 (0.08−0.21) 0.44 (0.14−1.35) AA 0.21 (0.05−0.77) 0.10 (0.02−0.21) 1.07 (0.28−1.84) CA+AA 0.17 (0.08−0.28) 0.14 (0.07−0.21) 0.50 (0.15−1.51) CALB2 rs8063760 CC 0.18 (0.11−0.38) 0.955 0.770 0.14 (0.09−0.22) 0.382 0.647 0.53 (0.24−1.44) 0.326 0.768 CT 0.18 (0.12−0.32) 0.16 (0.1−0.24) 0.44 (0.19−1.30) TT 0.21 (0.06−0.51) 0.12 (0.05−0.22) 0.86 (0.50−2.30) CT+TT 0.19 (0.11−0.34) 0.16 (0.09−0.24) 0.51 (0.21−1.43) E2F2 rs2075995 CC 0.19 (0.10−0.46) 0.512 0.481 0.14 (0.08−0.2) 0.161 0.059 0.72 (0.33−1.45) 0.189 0.117 CA 0.18 (0.12−0.34) 0.16 (0.1−0.23) 0.53 (0.20−1.48) AA 0.18 (0.10−0.33) 0.14 (0.09−0.24) 0.40 (0.18−0.90) CA+AA 0.18 (0.11−0.34) 0.15 (0.1−0.23) 0.48 (0.20−1.44) MIR335 rs3807348 GG 0.18 (0.09−0.34) 0.057 0.151 0.14 (0.08−0.2) 0.027 0.081 0.44 (0.26−1.43) 0.400 0.978 GA 0.18 (0.11−0.34) 0.14 (0.09−0.22) AA vs. GG P = 0.029 0.50 (0.18−1.16) AA 0.21 (0.13−0.39) 0.18 (0.11−0.26) 0.65 (0.27−1.80) GA+AA 0.19 (0.11−0.37) 0.15 (0.1−0.23) 0.52 (0.22−1.44) NRF1 rs13241028 TT 0.19 (0.12−0.36) 0.272 0.144 0.16 (0.1−0.23) 0.096 0.034 0.52 (0.21−1.15) 0.381 0.672 TC 0.18 (0.10−0.33) 0.14 (0.08−0.21) 0.64 (0.25−1.67) CC 0.17 (0.07−0.36) 0.15 (0.07−0.3) 0.24 (0.07−1.18) TC+CC 0.18 (0.09−0.34) 0.14 (0.08−0.21) 0.46 (0.24−1.53) SEPTIN7 rs3801339 TT 0.18 (0.11−0.34) 0.403 0.419 0.14 (0.09−0.2) 0.424 0.288 0.35 (0.17−1.05) 0.079 0.080 TC 0.18 (0.11−0.33) 0.15 (0.09−0.22) 0.51 (0.21−1.23) CC 0.20 (0.11−0.45) 0.16 (0.09−0.25) 0.72 (0.38−1.48) TC+CC 0.19 (0.11−0.37) 0.15 (0.09−0.23) 0.64 (0.26−1.45) A = adenine; Add = additive model, calculated using Kruskal-Wallis test; C = cytosine; Dom = dominant model, calculated using Mann-Whitney test; G = guanine; MM = malignant mesothelioma, SNP = single nucleotide polymorphism, T = thymine Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases480 phic E2F2 rs2075995 alleles were less likely to de- velop MM (OR = 0.35, 95% CI = 0.16−0.78, P = 0.010), even after adjustment for age (OR = 0.35, 95% CI = 0.14−0.84, P = 0.019). The association with MM susceptibility was significant also in the domi- nant model, both in univariate (OR = 0.43, 95% CI = 0.21−0.87, P = 0.019) and multivariate (OR = 0.43, 95% CI = 0.19−0.94, P = 0.033) analysis. Compared to subjects with asbestosis, carriers of two polymor- phic MIR335 rs3807348 alleles were more likely to develop MM (OR = 1.82, 95% CI = 1.05−3.16, P = 0.033), even after adjustment for age (OR = 0.35, 95% CI = 1.10−3.50, P = 0.022). After adjustment for age, the association with MM susceptibility was significant also in the dominant model (OR = 1.62, 95% CI = 1.03−2.55, P = 0.037). None of the other SNPs was significantly associated with MM sus- ceptibility (Supplementary Table 2). Association of selected SNPs with serum calretinin levels Serum calretinin concentration was determined in 545 subjects. Calretinin concentration significantly differed among subject groups (P < 0.001): MM pa- tients (N = 163) had median calretinin concentra- tion 0.52 (0.23−1.43) ng/ml, subjects with asbestosis (N = 117) 0.13 (0.08−0.20) ng/ml, subjects with pleu- ral plaques (N = 195) 0.18 (0.12−0.25) ng/ml and sub- jects without disease (N = 70) 0.12 (0.07−0.19) ng/ml. TABLE 5. Receiver operating characteristic (ROC) curve analysis according to individual genotypes for selected single nucleotide polymorphisms: comparison of malignant mesothelioma (MM) patients with all other subjects SNP Genotype AUC (95% CI) P Calretinincut-off (ng/ml)1 Sensitivity Specificity Overall analysis in the whole group / 0.825 (0.781−0.868) < 0.001 0.32 0.681 0.887 CALB2 rs889704 CC 0.831 (0.782−0.880) < 0.001 0.32 0.695 0.876 CA 0.779 (0.667−0.891) < 0.001 0.31 0.607 0.935 AA2 0.958 (0.837−1.000) 0.019 0.21 1.000 0.833 CA+AA 0.801 (0.702−0.901) < 0.001 0.31 0.625 0.940 E2F2 rs2075995 CC 0.906 (0.845−0.968) < 0.001 0.26 0.810 0.903 CA 0.803 (0.736−0.869) < 0.001 0.32 0.671 0.888 AA 0.781 (0.686−0.876) < 0.001 0.33 0.615 0.877 CA+AA 0.797 (0.742−0.851) < 0.001 0.32 0.653 0.881 MIR335 rs3807348 GG 0.853 (0.766−0.940) < 0.001 0.29 0.757 0.872 GA 0.803 (0.739−0.867) < 0.001 0.32 0.643 0.892 AA 0.845 (0.765−0.925) < 0.001 0.35 0.738 0.881 GA+AA 0.815 (0.764−0.866) < 0.001 0.32 0.675 0.881 NRF1 rs13241028 TT 0.812 (0.754−0.871) < 0.001 0.32 0.693 0.884 TC 0.868 (0.804−0.931) < 0.001 0.23 0.818 0.798 CC3 0.664 (0.406−0.922) 0.203 0.18 0.714 0.700 TC+CC 0.842 (0.777−0.907) < 0.001 0.23 0.790 0.785 1 Cut-off with the highest sum of sensitivity and specificity; 2 based on 10 subjects, 3 based on 27 subjects. A = adenine; AUC = area under the curve; C = cytosine; G = guanine; SNP = single nucleotide polymorphism; T = thymine Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases 481 The association of selected SNPs with serum calretinin concentration is presented in Table 4 and Figure 1. In all subjects, carriers of at least one polymorphic CALB2 rs889704 A allele had lower calretinin than carriers of two wild-type alleles in the dominant model (P = 0.036), but no signifi- cant differences were observed if subjects without MM and MM patients were evaluated separately (P = 0.069 and 0.441, respectively). In the group of subjects without MM, carriers of two polymorphic MIR335 rs3807348 alleles had higher calretinin than carriers of two wild-type alleles (P = 0.027). In this group also carriers of at least one polymorphic NRF1 rs13241028 C allele had lower calretinin than TABLE 6. Association of CALB2 haplotypes with malignant mesothelioma (MM) susceptibility and serum calretinin concentration Haplotype Subjects without MM Predicted frequency MM patients Predicted frequency OR (95% CI) P OR (95% CI)adj Padj Serum calretinin concentration P CCC 0.457 0.431 Reference Reference TCC 0.245 0.294 1.26(0.0−991.60) 0.061 1.26 (0.97−1.64) 0.084 0.272 CCT 0.176 0.147 0.88 (0.65−1.20) 0.415 0.94 (0.66−1.34) 0.731 0.125 CAT 0.058 0.066 1.21 (0.77−1.89) 0.408 1.08 (0.64−1.81) 0.782 0.731 CAC 0.045 0.047 1.11 (0.64−1.91) 0.713 0.99 (0.55−1.79) 0.974 0.852 The SNPs are ordered from the 5′- to 3′-end as follows: rs1862818, rs889704, rs8063760. A = adenine; Adj = adjusted for age, C = cytosine; CI = confidence interval; MM = malignant mesothelioma; OR = odds ratio; SNP = single nucleotide polymorphism; T = thymine FIGURE 1. Association of selected single nucleotide polymorphisms (SNPs) with serum calretinin concentration: CALB2 rs889704 (A), E2F2 rs2075995 (B), MIR335 rs3807348 (C), NRF1 rs13241028 (D). A B C D Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases482 carriers of two wild-type alleles in the dominant model (P = 0.034), but no significant differences were observed in group of MM patients. Association of selected SNPs with serum cal- retinin concentration in subjects with asbestosis, subjects with pleural plaques and subjects with- out disease is shown in Supplementary Table 3. In subjects without asbestos-related disease, car- riers of at least one polymorphic CALB2 rs889704 A allele had lower calretinin than carriers of two wild-type alleles in the additive model (P = 0.014) and dominant model (P = 0.004), but no significant differences were observed in subjects with pleu- ral plaques (Padd = 0.060, Pdom = 0.300) and subjects with asbestosis (Padd = 0.290, Pdom = 0.279). In sub- jects with pleural plaques, carriers of at least one polymorphic NRF1 rs13241028 C allele had lower calretinin than carriers of two wild-type alleles in the dominant model (P = 0.025). In subjects with asbestosis, carriers of at least one polymor- phic E2F2 rs2075995 A allele had higher calretinin than carriers of two wild-type alleles in the ad- ditive model (P = 0.049) and dominant model (P = 0.017). With ROC curve analysis, we compared serum calretinin levels in MM patients with all other subjects according to individual genotypes for SNPs, which affected calretinin levels in at least one group. In almost all groups, calretinin concentration could significantly discriminate be- tween MM patients and other subjects with good sensitivity and specificity (Table 5). Optimal cal- retinin cut off values differed according to geno- type, even though the differences were small. For CALB2 rs889704, lower cut off was observed in carriers of two polymorphic alleles (0.21 vs. 0.32 ng/ml). For E2F2 rs2075995, higher cut off was ob- served in carriers of two polymorphic alleles (0.33 vs. 0.26 ng/ml). For MIR335 rs3807348, higher cut off was observed in carriers of two polymorphic alleles (0.35 vs. 0.29 ng/ml). For NRF1 rs13241028, lower cut off was observed in carriers of at least one polymorphic alleles (0.23 vs. 0.32 ng/ml) (Table 5). Haplotype analysis Analysis of CALB2 haplotypes identified eight SNP combinations. The most common haplotype was CCC with predicted frequency 0.449, fol- lowed by TCC (0.261), CCT (0.167), CAT (0.060), CAC (0.045), TCT (0.009), TAC (0.007) and TAT (0.003). Haplotype TCC was more common in MM patients, but the association was not statistically significant (P = 0.061, Table 6). CALB2 haplotypes were not associated with serum calretinin concen- trations (Table 6). Discussion In the present study, we evaluated the role of ge- netic variability in CALB2 and its regulatory miR- NA and transcription factors genes with serum calretinin levels and MM susceptibility. Genetic variability of CALB2 was associated with cal- retinin concentration, but not with MM suscep- tibility. For SNPs in genes regulating calretinin expression, differences in genotype frequencies among MM and other subjects were also observed. Additionally, genetic factors influenced optimal serum calretinin cut off values differentiating MM patients from other asbestos-exposed subjects. Using bioinformatic analysis, we identified sev- en common putatively functional SNPs that could affect calretinin expression: three SNPs in CALB2 gene, one SNP in transcription factor E2F2, one SNP in transcription factor NRF1, one SNP in tran- scription factor SEPTIN7 and one SNP in miRNA MIR335. In previous studies, demographic and clinical factors such as sex and renal function af- fecting plasma or serum calretinin concentration in asbestos-related diseases were already identi- fied21,22,41, but the role of genetic variability is large- ly unexplored. Among CALB2 SNPs investigated in our study, CALB2 rs1862818 and CALB2 rs889704 may influ- ence transcription factor binding, while CALB2 rs8063760 may influence miRNA binding. In our study, CALB2 rs889704 was associated with lower serum calretinin levels in all subjects and subjects without asbestos-related diseases, while there was no association in patients with MM. None of the selected CALB2 SNPs or haplotypes were sig- nificantly associated with MM susceptibility. To the best of our knowledge, the functional role of CALB2 SNPs and their association with asbestos- related diseases was not investigated yet. However, one intronic SNP in CALB2 was previously associ- ated with calretinin expression in tumor cell lines and the development of colon cancer, but no asso- ciation with lung cancer was observed.30 Data on CALB2 genetic variability are therefore lacking and further studies are needed to evaluate its role in MM and serum calretinin levels. Three important transcription factors were pre- viously associated with regulation of calretinin.23,24 E2F2 is a transcription factor that binds to CALB2 promoter and was associated with calretinin ex- Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases 483 pression in mesothelioma cell lines.23 In our study, E2F2 rs2075995 was associated with decreased MM risk. When comparing MM patients to only subjects without disease, the association remained significant even after taking into account the age of the subjects. E2F2 rs2075995 was also associated with higher serum calretinin level among subjects with asbestosis. E2F2 has an important role in the regulation of cell cycle, but also affects other impor- tant processes such as cell proliferation, apoptosis and inflammation.42 In cancer, it was mostly asso- ciated with promoting tumor progression in vari- ous malignancies, including lung cancer.42 E2F2 could also contribute to the cell cycle-dependent differences observed for calretinin expression.23 E2F2 rs2075995 is a nonsynonymous SNP and may influence splicing. So far, E2F2 rs2075995 was only evaluated in patients with colorectal cancer, where no association with cancer risk was observed.43,44 However, no studies evaluated the association of E2F2 rs2075995 with MM. Still, the E2F gene fami- ly was often associated with different types of can- cer. Several other E2F2 polymorphisms were asso- ciated with oral and oropharyngeal squamous cell carcinoma risk and might also affect the course of the disease.45 Combinations of different E2F2 gene SNPs were proposed as a risk factor for squa- mous cell carcinoma of the head and neck.46 The E2F2 gene was also associated with ovarian cancer risk.47 Additionally, E2F2 genetic variability was proposed as recurrence biomarker in squamous cell carcinoma of the oropharynx.48 Among other E2F2 SNPs, rs3218211 was in very high LD with rs2075995 investigated in our study. E2F2 rs3218211 was associated with T stage in oral and oropharyn- geal squamous cell carcinoma and decreased head and neck squamous cell carcinoma risk, consistent with our results.45,46 Taken together, this suggests further studies regarding the role of E2F2 genetic variability in asbestos-related diseases and its as- sociation with calretinin are needed. The second important calretinin-related tran- scription factor is NRF-1. It binds to CALB2 pro- moter and might be important for the transcrip- tional control of calretinin expression in MM.23 In our study, NRF1 rs13241028 was associated with lower serum calretinin level in subjects without MM, but it was not associated with MM suscepti- bility. NRF-1 regulates expression of various genes involved in oxidative phosphorylation, mitochon- drial biogenesis and other mitochondrial pro- cesses, including transcription of mitochondrial DNA.49 Additionally, NRF-1 can modify different aspects of carcinogenesis, including proliferation, invasion, and apoptosis.50 NRF1 rs13241028 may influence miRNA binding.51 So far, NRF1 genetic variability has been associated primarily with increased susceptibility to diabetes.52,53 NRF1 has also been associated with epithelial ovarian cancer risk.54 Further studies are needed to better evalu- ate the role of NRF-1 and its genetic variability in asbestos-related diseases. Septin 7 has also been identified as a factor that binds to the CALB2 promoter region, resulting in decreased calretinin expression in mesothelioma cell lines.24 Septin 7 is a GTP-binding protein that is involved in cytokinesis, cytoskeleton organization and other cellular processes.24,55 It was also impli- cated in calcium homeostasis.56 Several studies al- so reported that septin 7 plays an important role in cancer development, especially glioma.55,56 In our study, SEPTIN7 rs3801339 was not significantly associated with MM susceptibility or with serum calretinin levels. The functional role of SEPTIN7 rs3801339 is not yet understood: it was previously classified as a non-synonymous variant, while it is now described as a genic downstream transcript variant. Interestingly, SEPTIN7 rs1143149 in mod- erate LD with rs3801339 was proposed as a risk factor for the development of non-small cell lung cancer and was associated with shorter survival in long-term smokers.55 SEPTIN7 was often mutated in breast ductal carcinoma in situ cell lines and these mutations might participate in the progres- sion of breast ductal carcinoma.57 Recent studies therefore suggest that SEPTIN7 variability may play a role in some cancers, but it was not an im- portant risk factor in asbestos-related diseases in our study. MiRNAs affect gene expression on the post- transcriptional level and are often deregulated in cancer.58 Among miRNAs predicted to modify cal- retinin expression, common polymorphisms were only described for miR-335. In our study, carriers of two polymorphic MIR335 rs3807348 alleles were more likely to develop MM compared to subjects with asbestosis, even after adjustment for age. MIR335 rs3807348 was also associated with serum calretinin level in subjects without MM. MiR-335 can modulate cell proliferation, apoptosis, migra- tion and invasion through various signaling path- ways. It mostly acts as a tumor suppressor and is downregulated in different cancer types.58 MIR335 rs3807348 may influence transcription factor bind- ing, but its role has not been experimentally con- firmed. To date, no research has been done on the association of rs3807348 with MM. MIR335 rs3807348 was not associated with breast cancer Radiol Oncol 2023; 57(4): 473-486. Zupanc C et al. / Genetic factors and serum calretinin in asbestos diseases484 risk in a previous study59, but more studies would be needed in this field. As several genetic factors were associated with calretinin, we also evaluated how these factors in- fluence serum calretinin cut off values. We found that four SNPs, CALB2 rs889704, E2F2 rs2075995, MIR335 rs3807348, and NRF1 rs13241028 could be used to fine tune serum calretinin cut off values predicting MM. Calretinin as a biomarker could thus have higher sensitivity and specificity in indi- viduals with known genetic variability. Similar re- sults were observed for mesothelin, where predic- tive value was improved when taking into account polymorphisms located in 5’ UTR and 3’ UTR of the MSLN gene.27-29 In the future, combination of clinical and genetic factors could thus help guide calretinin cut-off values and decrease false nega- tive or positive results. This is the first study to show that genetic fac- tors can affect serum calretinin levels and that accounting for these genetic factors may improve the predictive value of serum calretinin. We have also shown that genetic factors associated with calretinin may play a role in the development of mesothelioma. A limitation of our study is that we only had serum calretinin concentrations avail- able for a subgroup of participants included in the study. On the other hand, we performed a com- prehensive analysis of the factors that could affect calretinin expression using literature review and detailed bioinformatics analysis. Genetic variabil- ity was evaluated in a large cohort, which gives additional power to the study. However, other pol- ymorphisms in the investigated genes could also affect calretinin concentration and other factors could affect calretinin regulation. In the future, further studies in this field and validation of these results in an independent population are needed. Conclusions The present study showed that genetic variability in CALB2 gene and genes coding for transcrip- tion factors and miRNAs that regulate calretinin expression could contribute to interindividual dif- ferences in serum calretinin levels in MM patients or asbestos-exposed subjects. These results could contribute to a better understanding of calretinin regulation and could potentially contribute to an earlier diagnosis of MM. Acknowledgments This study was supported by the Slovenian Research Agency (ARRS), research grants P1-0170, L3-8203 and L3-2622. References 1. Chapman SJ, Cookson WO, Musk AW, Lee YC. Benign asbestos pleural diseases. Curr Opin Pulm Med 2003; 9: 266-71. doi: 10.1097/00063198- 200307000-00004 2. IARC monographs on the evaluation of the carcinogenic risk of chemicals to man: asbestos. IARC Monogr Eval Carcinog Risk Chem Man 1977; 14: 1-106. PMID: 863456 3. Weiner SJ, Neragi-Miandoab S. Pathogenesis of malignant pleural mesothe- lioma and the role of environmental and genetic factors. J Cancer Res Clin Oncol 2009; 135: 15-27. doi: 10.1007/s00432-008-0444-9 4. Melaiu O, Gemignani F, Landi S. The genetic susceptibility in the develop- ment of malignant pleural mesothelioma. J Thorac Dis 2018; 10: S246-52. doi: 10.21037/jtd.2017.10.41 5. 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Received 4 April 2023 Accepted 31 May 2023 Correspondence to: Assist. Prof. Lučka Boltežar M.D., Ph.D., Division of Medical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: lboltezar@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. t(14;18)(q32;q21) translocation is an important genetic feature of follicular lymphoma resulting in an- tiapoptotic B-cell lymphoma 2 (BCL2) protein overexpression. On chromosome 18 breakpoint-site variation is high but does not affect BCL2. Breakpoint most commonly occurs at major breakpoint region (MBR) but may happen at minor cluster region (mcr) and between MBR and mcr at 3’MBR and 5’mcr. The aim of this study was to analyze the correla- tion of t(14;18)(q32;q21) breakpoint site with clinical characteristics in follicular lymphoma. Patients and methods. We included patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment and had the t(14;18)(q32;q21) translocation detected by polymerase chain reaction (PCR) at MBR, mcr or 3`MBR prior to first treatment. Among patients with different breakpoints, sex, age, disease grade, stage, B-symptoms, follicular lymphoma international prognostic index (FLIPI), presence of bulky disease, progression free survival and overall survival were compared. Results. Of 84 patients, 63 had breakpoint at MBR, 17 at mcr and 4 at 3`MBR. At diagnosis, the MBR group had a significantly lower disease stage than the mcr group. Although not significant, in the MBR group we found a higher progression-free survival (PFS) and overall survival (OS), lower grade, age, FLIPI, and less B-symptoms. Conclusions. Compared to patients with mcr breakpoint, those with MBR breakpoint seem to be characterised by more favourable clinical characteristics. However, a larger study would be required to support our observation. Key words: follicular lymphoma; t(14;18) translocation; breakpoint region; clinical characteristics Introduction Follicular lymphoma is a low grade B-cell lym- phoma, derived from germinal center. In Europe and USA, it is the second most common type of lymphoma. Follicular lymphoma is considered an incurable disease. It is characterized by an indo- lent clinical course though it may transform into a more malignant diffuse large B-cell lymphoma.1 An important genetic feature of follicular lympho- ma is the translocation between the chromosomes 14 and 18, which is present in up to 90% of folli- cular lymphoma.2 The clinical significance of the translocation remains unclear as conflicting re- sults have been reported regarding its correlation with outcome.3,4 Although not limited to follicular lymphoma5, the translocation helps in follicular lymphoma diagnosing, as well as response evalu- ation through minimal disease detection.6 Radiol Oncol 2023; 57(4): 487-492. Panjan M et al. / Follicular lymphoma and translocation breakpoint site488 The translocation places the antiapoptotic B-cell lymphoma (BCL2) gene next to the transcriptional enhancer of the immunoglobulin heavy chain gene (IGH), resulting in BCL2 protein overexpres- sion.7 BCL2 protein is a member of the BCL2 family which consists of pro- and antiapoptotic proteins as well as of proteins not linked to apoptosis. It is lo- calized in the outer mitochondrial membrane and exerts its antiapoptotic function by binding proap- optotic BCL2 family proteins such as BAX and BAC to prevent the release of cytochrome c from mito- chondria in the intrinsic apoptosis pathway.8 The translocation is an early event in lymphomagen- esis and although on its own likely insufficient, it plays an important role in follicular lymphoma pathogenesis. It results in extended survival of the tumor cells which may cause the accumulation of additional oncogenic genetic aberrations. Follicular lymphoma bears many chromosomal aberrations that vary in number, mostly of unknown or ques- tionable contribution to pathogenesis.9,10 The t(14;18)(q32;q21) translocation was first de- tected by karyotypic analysis, which is at present not used for this purpose.11 A commonly used method for translocation detection is Flourescence In Situ Hybridisation (FISH). FISH probes bind to the entire IgH and BCL2 genes thereby indiscrimi- nately detecting translocations at various sites across the BCL2 gene. It has close to 100% sensitiv- ity in the t(14;18)(q32;q21) detection.12 Unlike with FISH, with PCR it is possible to detect the exact breakpoint site, making it indispensable for a study of clinical implications of different breakpoints. PCR is also less expensive and time consuming. However, it does have lower sensitivity of 60-70% as PCR primers identify only short DNA sections.13 Alternatively, multiple primers may be used to am- plify and detect different breakpoints. This method has a higher sensitivity of up to 88%.14,15 In the t(14;18)(q32;q21) translocation, the break- point location on chromosome 14 is almost invari- able in one of the six JH gene segments, whereas on chromosome 18 different breakpoints occur relatively often. Since the breakpoint is usually located outside of the protein coding part of the BCL2 gene, variations in the breakpoint region do not affect the BCL2 protein. In 50% to 65% of cases the breakpoint occurs at the major break- point region (MBR) located at the 3’-untranslated region of the BCL2 exon 3. In about 10-20% of cases the breakpoint occurs at the minor cluster region (mcr) located 20 kilobases (kb) from 3’ of the MBR. Additionally, the breakpoint may also be located between the MBR and the mcr, at 3′MBR and 5′mcr subclusters, commonly called the intermediate cluster region (icr).15,16 The 3’MBR subcluster is po- sitioned 4 kb downstream of the MBR, while the 5’mcr subcluster is positioned 10 kb upstream of the mcr (Figure 1).16 The aim of this study was to analyze the corre- lation of t(14;18)(q32;q21) breakpoint site with clini- cal characteristics in follicular lymphoma. Patients and methods In this clinical retrospective study, we included 84 patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treat- ment between 2013 and 2020 at the Institute of Oncology, Ljubljana and had t(14;18)(q32;q21) de- tected by PCR prior to systemic treatment. PCR was performed on bone marrow samples as a part of the diagnostic procedure. All patients included in the study signed an informed consent allow- ing treatment and use of their clinical information and biological material for scientific purposes. The study was approved by the Committee for FIGURE 1. Diagram of the BCL2/JH t(14;18) translocation breakpoints. Relative positions of major breakpoint region (MBR), 3’MBR subcluster, 5’mcr subcluster and minor cluster region (mcr) are shown according to the report of van Dongen JJM et al.16 Radiol Oncol 2023; 57(4): 487-492. Panjan M et al. / Follicular lymphoma and translocation breakpoint site 489 Medical Ethics of Institute of Oncology Ljubljana (ERIDNPVO-0064/2022). Data regarding treatment protocol and patients’ clinical information were collected from the clinical information system. The following characteristics observed at the time of diagnosis were gathered: gender, age, Ann Arbor stage, grade, presence of B symptoms, FLIPI score, presence of bulky disease (largest lymphoma deposit > 10 cm or mediastinal mass > 1/3 of the thoracic diameter on posterior- anterior chest x-ray), and breakpoint region of the t(14;18)(q32;q21) translocation. Progression-free sur- vival (PFS) was defined as time from the end of the systemic treatment until relapse or end of observa- tion, overall survival (OS) as time from diagnosis until death or end of observation and lymphoma specific OS as time from diagnosis until lympho- ma-related death or end of observation. The data were collected on December 20, 2022. DNA was isolated from bone marrow speci- mens using the QIAamp DNA Blood mini kit (Qiagen GmbH, Hilden, Germany). The concentra- tion and the purity of DNA were determined using the Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). PCR was performed using IdentiClone™ BCL2/JH Translocation Assay (InVivo Scribe Technologies, San Diego, CA, USA). This assay amplifies genomic DNA between prim- ers targeting the BCL2 gene and conserved joining regions of the IGH gene. Master mixes for MBR, 3`MBR and mcr detection each contained prim- ers targeting the J region of the IGH gene (JH) and those targeting MBR, 3’MBR and mcr, respectively. The MBR master mix contained two MBR prim- ers (MBR1 and MBR2) and consensus JH primer; the 3’MBR master mix contained four 3’MBR primers (3’MBR1-4) and consensus JH primer; the mcr mas- ter mix contained three mcr primers (5’mcr, mcr1 and mcr2) and consensus JH primer. Primers de- sign and validation has been described by JJM van Dongen with colleagues.16 Primer sequences with National Center for Biotechnology Information (NCBI) accession numbers are shown in Table 1. PCR products were detected by polyacryla- mide gel electrophoresis (10% non-denaturing polyacrylamide TBE gel, 0.5X TBE running buffer) and visualized by UV illumination of gels stained with ethidium bromide (0.5 µg/ml). Tested samples were determined as positive for the presence of the t(14;18)(q32;q21) translocation if one or two of TABLE 1. Sequences of primers used for detection of the t(14;18)(q32;q21) translocation. Relative positions of primers are indicated downstream of the first nucleotide of corresponding reference sequence t(14;18) MBR primers primer name NCBI accession no. position primer sequence MBR1 AY220759.1 (+193443) 5’-GACCAGCAGATTCAAATCTATGG-3’ MBR2 AY220759.1 (+192940) 5’-ACTCTGTGGCATTATTGCATTATAT-3’ t(14;18) 3’MBR primers primer name NCBI accession no. position primer sequence 3’MBR1 AH010747.2 (+717) 5’-GCACCTGCTGGATACAACACTG-3’ 3’MBR2 AH010747.2 (+1530) 5’-GGTGACAGAGCAAAACATGAACA-3’ 3’MBR3 AH010747.2 (+1787) 5’-GTAATGACTGGGGAGCAAATCTT-3’ 3’MBR4 AH010747.2 (+2718) 5’-ACTGGTTGGCGTGGTTTAGAGA-3’ t(14;18) mcr primers primer name NCBI accession no. position primer sequence mcr1 AF275873.1 (+1961) 5’-TAGAGCAAGCGCCCAATAAATA-3’ mcr2 AF275873.1 (+2407) 5’-TGAATGCCATCTCAAATCCAA-3’ 5’mcr AH010747.2 (+15849) 5’-CCTTCTGAAAGAAACGAAAGCA-3’ Consensus JH primer primer name NCBI accession no. position primer sequence JH OL807663.1 (+239) 3’-CCAGTGGCAGAGGAGTCCATTC-5’ AF275873.1 = homo sapiens BCL2 gene, exon 3 and breakpoint region; AH010747.2 = homo sapiens genomic sequence downstream of BCL2; AY220759.1 = homo sapiens B-cell CLL/lymphoma 2 (BCL2) gene, complete coding sequence; MBR = major breakpoint region; mcr = minor cluster region; NCBI = National Center for Biotechnology Information; OL807663.1 = homo sapiens clone J6 immunoglobulin heavy chain variable region gene, partial coding sequence Radiol Oncol 2023; 57(4): 487-492. Panjan M et al. / Follicular lymphoma and translocation breakpoint site490 the amplified products (bands) within 100-2500 bp range were present. The quality of the input DNA was tested with Specimen Control Size Ladder Master Mix which targets multiple house-keeping genes and generates a series of amplicons approxi- mately 100, 200, 300, 400, and 600 bp long to ensure control of the quality and quantity of the input DNA. Clinical characteristics were compared among the groups defined by the breakpoint region us- ing 1way Analysis of Variance (ANOVA) test and Independent-Samples T-test for numerical and Fisher̀ s exact test for nominal variables. To com- pare OS and PFS between the groups Log Rank (Mantel-Cox) analysis was performed. p < 0.05 was defined as statistically significant. Results Among 84 included patients, the group with MBR breakpoint was the most numerous with 63 pa- tients, followed by mcr with 17 and 3`MBR with 4. Female predominance was present in all break- point-site groups. Overall, the median age was 61 years, with the mcr group being the oldest. Half of the 3`MBR group and up to one quarter of the 2 larger groups had grade 3 follicular lymphoma. FLIPI score was predominantly 2 or 3 and was low- est in the MBR group. B-symptoms were present in approximately half of the patients in the 3`MBR and mcr group whereas they were less common in the MBR group. Disease stage was highest in the mcr group although stage 4 was predominant in all 3 groups. Bulky disease was mostly absent in all groups with the mcr group having the lowest proportion (Tables 2,3). Comparing clinical characteristics at diagno- sis, a statistically significant difference in stage was found between the MBR and mcr groups (p = 0.023). No other significant correlation was established comparing the MBR, mcr and 3’MBR groups or the 2 larger groups only (Tables 2,3). All patients were treated with RCHOP (rituxi- mab, cyclophosphamide, doxorubicin, vincristine, prednisolone) or RCHOP-like chemoimmuno- therapy, followed by irradiation in case of residual disease. Treatment response was defined as com- plete remission, partial remission, stable or pro- gressive disease, based on the positron emission tomography-computerized tomography (PetCT) 3-5 weeks after the end of systemic treatment. In case of irradiation of residual disease, additional computerized tomography (CT) was performed 3 months after irradiation and was included in final response evaluation. After systemic treatment, pa- tients received maintenance rituximab for 2 years and were subject to a regular follow-up. TABLE 2. Comparison of clinical features at diagnosis between the breakpoint-site groups (MBR, 3’MBR, mcr) using Fisher`s exact test MBR (N = 63) 3`MBR (N = 4) mcr (N = 17) p1 p2 Male sex 24 (38%) 1 (25%) 4 (24%) 0.571 0.391 Grade* 3 11 (20%) 2 (50%) 4 (25%) 0.303 0.729 B-symptoms 23 (37%) 2 (50%) 8 (47%) 0.641 0.576 Bulky disease** 17 (27%) 1 (25%) 2 (12%) 0.497 0.335 MBR = major breakpoint region; mcr = minor cluster region; p1 = significance comparing all 3 groups; p2 = significance comparing the MBR and mcr groups only; * = Disease grade was determined in 76 cases only; ** = Defined as largest lymphoma deposit > 10 cm or mediastinal mass > 1/3 of the thoracic diameter on posterior-anterior chest x-ray TABLE 3. Comparison of clinical features at diagnosis between the breakpoint-site groups (major breakpoint region [MBR], 3`MBR, mcr) MBR (N = 63) 3`MBR (N = 4) mcr (N = 17) p1 p2 Median (mean) stage 4 (3.70) 4 (3.75) 4 (3.94) 0.361 0.023 Median (mean) FLIPI 2 (2.51) 3 (2.75) 3 (3.00) 0.226 0.094 Median (mean) age 61 (60.25) 62 (63.25) 64 (63.71) 0.423 0.218 p1 = significance comparing all 3 groups using 1way Analysis of Variance (ANOVA) (df = 2); p2 = significance comparing the major breakpoint region (MBR) and minor cluster region (mcr) groups using Independent-Samples T-test Radiol Oncol 2023; 57(4): 487-492. Panjan M et al. / Follicular lymphoma and translocation breakpoint site 491 During observation, 23 patients in the MBR and 9 patients in the mcr group relapsed and none in the 3`MBR group. The Log Rank PFS comparison found no significant difference in PFS between the 3 groups (p = 0.157) or between the 2 larger groups (p = 0.235). Though statistically insignificant, PFS was longer in the MBR group (Figure 2). In the MBR group, 11 patients died, whereas in the mcr group the number of deceased was 5 and no patients died in the 3`MBR group. No signifi- cant difference in OS between the 3 breakpoint- site groups (p = 0.426) or the MBR and mcr group (p = 0.351) was observed (Figure 3). Lymphoma specific survival analysis yielded similar results (Figure 4). Discussion It is supposed that translocation site in t(14;18) (q32;q21) translocation bears no prognostic or pre- dictive value as it does not alter the protein-coding part of the antiapoptotic BCL2 gene, nor does it af- fect BCL2 expression level.17 Nevertheless, a differ- ence in stage between the 2 common breakpoint sites mcr and MBR transpired in our routine clini- cal data at diagnosis, prompting this study. Among 84 included patients, we found MBR breakpoint to be by far the most common with 63 patients, followed by mcr with 17 patients. Only 4 patients had the 3`MBR breakpoint site, making a characterisation of this group difficult. We only found a few studies treating the sub- ject of this article. In one of them, Weinberg et al. studied clinical characteristics of 236 follicular lymphoma patients with the t(14;18)(q32;q21) trans- location, determining five different breakpoint regions, including MBR and mcr. MBR breakpoint was found in 118 and mcr in 11 patients.18 In an- other study, López-Guillermo et al. determined the BCL2 breakpoint site in 247 patients with indolent follicular lymphoma. They determined break- points at the MBR and mcr regions only. MBR breakpoint was found in 175 cases and mcr in 27.19 Compared to the two studies, our mcr group was proportionally the largest with mcr/MBR ratio at 0.27, compared to 0.09 in Weinberg̀ s and 0.15 in Guillermò s study. Comparing the groups with different break- point region, PFS, OS and lymphoma specific OS were found to be higher in patients with MBR breakpoint site compared to mcr, though the re- sults did not reach statistical significance. Apart from a higher proportion of bulky disease, the MBR group was indeed characterized by a more fa- vorable disease presentation, namely lower grade, smaller proportion of patients with B-symptoms, lower FLIPI score and younger age at diagnosis. Remarkably, the MBR group also had a signifi- cantly lower clinical stage compared to the mcr (p = 0.023). In the studies of Weinberg and López- Guillermo, no similar findings seemed to tran- FIGURE 2. Comparison of progression-free survival between the 3`MBR (blue), MBR (red) and mcr (green) groups. Censored cases are marked as vertical lines on their respective curves. Log Rank (Mantel-Cox) significance: 0.157. Log Rank (Mantel-Cox) significance comparing the MBR and mcr group: 0.235. MBR = major breakpoint region; mcr = minor cluster region FIGURE 3. Comparison of overall survival between the 3’MBR (blue), MBR (red) and mcr (green) groups. Censored cases are marked as vertical lines on their respective curves. Log Rank (Mantel-Cox) significance: 0.426. Log Rank (Mantel- Cox) significance comparing the MBR and mcr group: 0.351. MBR = major breakpoint region; mcr = minor cluster region Radiol Oncol 2023; 57(4): 487-492. Panjan M et al. / Follicular lymphoma and translocation breakpoint site492 spire. Weinberg compared MBR and “minor breakpoints” group where along with mcr, other breakpoints were included. No significant differ- ence was found in stage, nor in age, B symptoms, FLIPI score. Furthermore, no significant difference was observed comparing PFS and OS between the two groups.18 López-Guillermo compared the MBR and mcr group only and found no significant difference in stage, age, gender, and B symptoms. In contrast to our finding however, he observed a significantly longer PFS in the mcr compared to the MBR group. There was only 1 relapse among 27 patients with mcr breakpoint and 42 among 175 patients with MBR breakpoint. The study of López-Guillermo was indeed performed in the setting of the low-grade follicular lymphoma, with only 3% of patients having follicular lymphoma grade 3 compared to our 22%.19 To obtain more relevant results for this comparison, we conducted the same comparison on our grade 1 and 2 follicu- lar lymphoma, only to find similar results. Taken together, no clear conclusions can be drawn as to correlation between PFS and the t(14;18)(q32;q21) breakpoint region. In conclusion, we found follicular lymphoma patients with MBR breakpoint to exhibit a more favorable clinical presentation including a higher PFS and OS. Due to our limited sample size and some incongruity in the literature, a larger study would be required to confirm our observation. References 1. 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Buckstein R, Pennell N, Berinstein NL. What is remission in follicular lym- phoma and what is its relevance? Best Pract Res Clin Haematol 2005; 18: 27-56. doi: 10.1016/j.beha.2004.08.019 7. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chro- mosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 1984; 226: 1097-9. doi: 10.1126/science.6093263 8. Marie Hardwick J, Soane L. Multiple functions of BCL-2 family proteins. Cold Spring Harb Perspect Biol 2013; 5: a008722. doi: 10.1101/cshperspect. a008722 9. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 1988; 335: 440-2. doi: 10.1038/335440a0 10. Horsman DE, Connors JM, Pantzar T, Gascoyne RD. Analysis of secondary chromosomal alterations in 165 cases of follicular lymphoma with t(14;18). Genes Chromosomes Cancer 2001, 30: 375-82. doi: 10.1002/gcc.1103 11. Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR, Theologides A. Distinctive chromosomal abnormalities in histologic subtypes of non- Hodgkin’s lymphoma. N Engl J Med 1982; 307: 1231-6. doi: 10.1056/ NEJM198211113072002 12. Vaandrager JW, Schuuring E, Raap T, Philippo K, Kleiverda K, Kluin P. Interphase FISH detection of BCL2 rearrangement in follicular lymphoma us- ing breakpoint-flanking probes. Genes Chromosomes Cancer 2000; 27: 85- 94. doi: 10.1002/(SICI)1098-2264(200001)27:1<85::AID-GCC11>3.0.CO;2-9 13. Horsman DE, Gascoyne RD, Coupland RW, Coldman AJ, Adomat SA. Comparison of cytogenetic analysis, southern analysis, and polymerase chain reaction for the detection of t(14; 18) in follicular lymphoma. Am J Clin Pathol 1995; 103: 472-8. doi: 10.1093/ajcp/103.4.472 14. Barrans SL, Evans PAS, O’Connor SJM, Owen RG, Morgan GJ, Jack AS. The de- tection of t(14;18) in archival lymph nodes : development of a fluorescence in situ hybridization (FISH)-based method and evaluation by comparison with polymerase chain reaction. J Mol Diagn 2003; 5: 168. doi: 10.1016/ S1525-1578(10)60469-2 15. Gu K, Chan WC, Hawley RC. Practical detection of t(14;18)(IgH/BCL2) in follicular lymphoma. Arch Pathol Lab Med 2008; 132: 1355-61. doi: 10.5858/2008-132-1355-PDOBIF 16. van Dongen JJM, Langerak AW, Brüggemann M, Evans P a. S, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombina- tions in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257-317. doi: 10.1038/ sj.leu.2403202 17. Galteland E, Sivertsen EA, Svendsrud DH, Smedshammer L, Kresse SH, Meza-Zepeda LA, et al. Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin’s lymphomas. Leukemia 2005; 19: 2313-23. doi: 10.1038/sj.leu.2403954 18. Weinberg OK, Ai ZW, Mariappan MR, Shum C, Levy R, Arber DA. ″Minor″ BCL2 breakpoints in follicular lymphoma: frequency and correlation with grade and disease presentation in 236 cases. J Mol Diagn 2007; 9: 530. doi: 10.2353/jmoldx.2007.070038 19. López-Guillermo A, Cabanillas F, McDonnell TI, McLaughlin P, Smith T, Pugh W, et al. Correlation of Bcl-2 rearrangement with clinical characteristics and outcome in indolent follicular lymphoma. Blood 1999; 93: 3081-7. doi: 10.1182/blood.V93.12.4365 FIGURE 4. Comparison of lymphoma-specific overall survival between the 3’MBR (blue), MBR (red) and mcr (green) groups. Censored cases are marked as vertical lines on their respective curves. Log Rank (Mantel-Cox) significance: 0.409. Log Rank (Mantel-Cox) significance comparing the MBR and mcr groups: 0.301. MBR = major breakpoint region; mcr = minor cluster region Radiol Oncol 2023; 57(4): 493-506. doi: 10.2478/raon-2023-0046 493 research article The prognostic significance of tumor-immune microenvironment in ascites of patients with high-grade serous carcinoma Simona Miceska1,2, Erik Skof2,3, Simon Bucek1,2, Cvetka Grasic Kuhar2,3, Gorana Gasljevic4, Spela Smrkolj2,5, Veronika Kloboves Prevodnik1,6 1 Department of Cytopathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Department of Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Division of Gynaecology and Obstetrics, University Medical Centre, Ljubljana, Slovenia 6 Faculty of Medicine, University of Maribor, Maribor, Slovenia Radiol Oncol 2023; 57(4): 493-506. Received 27 June 2023 Accepted 13 August 2023 Correspondence to: Prof. Veronika Kloboves Prevodnik, M.D, Ph.D., Department of Cytopathology, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: vkloboves@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. High-grade serous carcinoma (HGSC) is often associated with ascites at presentation. Our objective was to quantify immune cells (ICs) in ascites prior to any treatment was given and evaluate their impact on progres- sion-free survival (PFS) and overall survival (OS). Patients and methods. Forty-seven patients with primary HGSC and ascites were included. Flow-cytometric analysis was performed to detect percentages of CD3+ T cells (CD4+, CD8+, Tregs, and NKT cells), B cells, NK cells (CD56brightCD16- and CD56dimCD16+ subsets), macrophages and dendritic cells (DCs). Furthermore, CD103 expression was analyzed on T cells and their subsets, while PD-1 and PD-L1 expression on all ICs. Cut-off of low and high percent- ages of ICs was determined by the median of variables, and correlation with PFS and OS was calculated. Results. CD3+ cells were the predominant ICs (median 51%), while the presence of other ICs was much lower (me- dian ≤10%). CD103+ expression was mostly present on CD8+, and not CD4+ cells. PD-1 was mainly expressed on CD3+ T cells (median 20%), lower expression was observed on other ICs (median ≤10%). PD-L1 expression was not detected. High percentages of CD103+CD3+ T cells, PD-1+ Tregs, CD56brightCD16- NK cells, and DCs correlated with prolonged PFS and OS, while high percentages of CD8+ cells, macrophages, and PD-1+CD56brightCD16- NK cells, along with low percentages of CD4+ cells, correlated with better OS only. DCs were the only independent prognostic marker among all ICs. Conclusions. Our results highlight the potential of ascites tumor-immune microenvironment to provide additional prognostic information for HGSC patients. However, a larger patient cohort and longer follow-up are needed to confirm our findings. Key words: ascites; immune cells; high-grade serous carcinoma; PD-1; PD-L1; prognostic markers. Introduction Ovarian carcinoma is a gynecological malignancy with the highest mortality rate in Western coun- tries and the sixth leading cause of cancer-related deaths among women.1 High-grade serous car- cinoma (HGSC) is the most common and aggres- sive histological type. Lack of symptoms and ad- Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma494 equate screening methods usually result in de- layed diagnosis and advanced stage with less than 40% of a 5-year survival rate for HGSC patients.2 Cytoreductive surgery combined with carboplatin/ paclitaxel chemotherapy (with or without bevaci- zumab) is still the standard treatment approach.3 Despite the good overall response, 70% of the pa- tients experience relapse or develop metastatic disease and resistance. Unfortunately, no signifi- cant improvement has been achieved in the last three decades, except for BRCA1/2 mutated tumors where poly-ADP-ribose polymerase (PARP) inhibi- tors slightly shifted the care paradigm for ovarian carcinoma.4 However, accumulating evidence is showing that tumor-immune microenvironment (TME) in ovarian carcinoma can open the door for the discovery of new prognostic markers and the development of immunotherapeutic treatment approaches. For instance, the presence of tumor- infiltrating CD3+ T cells in the primary tumor positively correlates with progression-free survival (PFS) and overall survival (OS) of ovarian carcino- ma patients, as does high CD8/CD4 ratio.5 Strong association with better OS was also seen on CD3+ T cells expressing CD103 tissue resident marker,6 while infiltration of regulatory T cells has an oppo- site impact, and the contribution of B cells remains undefined.7 Cells from innate immunity, such as natural killer (NK) cells, dendritic cells (DCs), and macrophages contribute to improved outcomes, except for macrophage subsets that polarize from tumor-inhibiting (M1) to tumor-promoting (M2) phenotype and are associated with disease pro- gression.8 Moreover, increased expression of PD-1 and PD-L1 is one of the inhibition mechanisms of anti-tumor response by induction of peripheral tol- erance, and TME has a significant role in its activa- tion.9 Several studies have examined the feasibility of using PD-1 and PD-L1 to serve as prognostic bio- markers for ovarian carcinoma, although their role is still controversial. Ascites is the most common sign of advanced ovarian carcinoma. Over the last years, studies have demonstrated that ascites contains almost the same immune cells (ICs) and extracellular compo- nents as the primary tumor.2,10,11 However, there is a lack of quantitative data about the percentages of ICs in ascites and data on their clinical importance. Moreover, the role of immune checkpoints is also poorly described. Our objective was to quantitate ICs in HGSC ascites at disease presentation, assess the expression of PD-1 and PD-L1 on ICs, and inves- tigate their prognostic significance for PFS and OS. Patients and methods Patients Patients diagnosed with primary HGSC be- tween January 2019 and May 2021 at the Institute of Oncology Ljubljana (IOL) and/or University Medical Centre Ljubljana were included in the study. The inclusion criteria were as follows: age > 18 years, WHO performance status from 0–1, histologically confirmed HGSC, International Federation of Gynecology and Obstetrics (FIGO) stage ≥ IIIB, presence of malignant ascites, and in- dication for first-line systemic treatment with plati- num agents. All patients received standard chemo- therapy treatment. The study was approved by the National Ethics Committee in Ljubljana, Slovenia (0120-33/303/2018/3 and 0120-33/303/2018/6). All pa- tients signed informed consent before inclusion in the study. The study was conducted in accordance with the Helsinki Declaration and Good Clinical Practice. Study design Ascites samples were collected at disease presen- tation, specifically during laparoscopy or laparot- omy before the tumor biopsy was performed and any treatment was initiated, and were immediate- ly sent to the Department of Cytopathology, IOL, where were processed as previously described by our group.12 Aliquots of ascites were prepared for flow-cytometric analysis. Percentages of T cells, B cells, NK cells, macrophages, and DCs, and expression of CD103, PD-1, and PD-L1 were analyzed. Their correlation with patient’s PFS and OS was calculated. Survival analysis was based on a 3-year patient follow-up. Clinical data were obtained from patient’s electronic medical record. Treatment characteristics such as type of surgery, residual disease after surgery, chemo- therapy, treatment with bevacizumab or olapa- rib, and quantity of ICs in the ascites were also analyzed and further correlated with PFS and OS. Surgery was defined as primary (first treatment procedure), secondary (interval surgery after neo- adjuvant chemotherapy was possible), or no sur- gery (interval surgery after neoadjuvant therapy was not possible because the tumor was still in- operable). Residual disease after primary or in- terval surgery was defined as no residual tumor, residual tumor ≤ 1 cm, or residual tumor > 1 cm. Chemotherapy was defined neoadjuvant (before surgery), or adjuvant (after surgery). Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma 495 Flow-cytometric analysis Sample preparation for flow-cytometric measure- ment was carried out as previously described by our group.13 Antibodies (Supplementary Table 1) were divided into 5 test tubes according to the ana- lyzed ICs (Supplementary Figure 1) and half a mil- lion cells per 100 μl were put in each tube. Flow- cytometric data was acquired with a 10-color BD FACSCanto™ II Flow Cytometer and FACSDiva 8.0.2 software (BD Bioscience, USA). FSC files were analyzed using FlowJo v10.8 1 (BD Biosciences, USA). Different ICs were gated according to their immunophenotype (Figure 1): T cells (CD3+), helper T cell subset (CD4+), cytotoxic T cell subset (CD8+), regulatory T cell subset (Tregs; CD4+CD25+CD127±), NKT cell subset (CD3+CD56+), B cells (CD19+), NK cells (CD3-CD56dimCD16+ and CD3-CD56brightCD16- subsets), macrophages (CD11b+CD14+CD68+) and their M1-like (CD206-) and M2-like (CD206+) sub- sets, and DCs (lineage-(CD3-CD11b-CD14-CD16- CD19-CD20-CD34-CD56-)HLADR+CD123+CD11c+). Expression of CD103 was analyzed on CD3+, CD4+, and CD8+ T cells. Percentages of T cells, B cells, NK cells, macrophages, and DCs were given as a ratio per CD45+. Percentages of CD4+, CD8+, Tregs, and NKT cells were given as a percentage of CD3+, while M1-like and M2-like macrophages were given as a percentage per all macrophages. Expression of PD-1 and PD-L1 was analyzed on each IC population/subset separately. Statistical analysis Descriptive statistics was used to describe the ba- sic features of the data. The median (range) was calculated for each IC population/subset. Mann– Whitney U and Kruskal-Wallis nonparametric tests were used to compare if there were differenc- es in the percentages of ICs, and PD-1 and PD-L1 expression levels among IC subsets and within dif- ferent treatment characteristics. A cut-off value of low and high percentages of ICs was determined by the median of the variables. Kaplan Maier method (with log-rank test) was used to evaluate PFS and OS for treatment characteristics, as well as PFS and OS for low and high percentages of ICs. PFS was calculated as the time from diagnosis un- til disease progression or death, and OS was calcu- lated as the time from diagnosis to death. Hazard A B C D E G H I J K K F FIGURE 1. Gating strategy for immune cells in ascites. (A) CD3+ T cells were gated on CD45+. (B) The depicted gate shows CD4+ vs. CD8+ subsets gated on CD3+ T cells and (C) CD103 expression on CD8+. The same gating strategy was applied for CD103 expression on CD4+ (not shown). (D) Gating representative for Tregs. The dot plot depicts CD4+ cells discriminated according to CD127 and CD25 positivity. (E) NKT cells were gated according to CD3 and CD56 positivity. (F) NK cells were gated according to CD16 and CD56 positivity. Two subsets were defined: CD56brightCD16- and CD56dimCD16+. (G) The dot plot depicts macrophages according to CD14 and CD68 positivity (pre-gated on CD11b+CD45+ cells). (H) M1-like macrophages were defined as CD206- macrophages, and M2-like as CD206+ macrophages. (I) DCs were gated per exclusion − as lineage- negative cells (no expression of CD3/CD11b/CD14/CD16/CD19/CD20/CD34/CD56) and further discriminated by CD123 and HLA-DR positivity. (J) B cells were gated as CD19+ cells per CD45+. (K) PD-1 and PD-L1 positivity was detected on each cell population/subset. Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma496 ratio (HR) and 95% confidence interval (CI) were calculated for both univariate and multivariate analysis. Parameters that proved to be significant in the univariate analysis were included in the multivariate analysis. Median survival was ex- pressed in months. P < 0.05 was considered signifi- cant. Statistical analysis was performed with IBM SPSS v 28.0.1.0 (142) and GraphPad Prism 9 statistic software. Results Patients and treatment characteristics Forty-seven patients with histologically confirmed HGSC and ascites were included in the study. Ascites was collected at disease presentation and prior to any treatment. The mean age of the pa- tients was 64 years (range 41−84 years). Eleven patients underwent primary surgery, resulting in no residual tumor after surgery in 6/11 patients and residual tumor ≤ 1 cm in 5/11 patients. All 11 patients were then treated with adjuvant chemo- therapy. Twenty-three patients underwent neoad- juvant chemotherapy followed by interval surgery and adjuvant chemotherapy. No residual tumor was achieved in 13/23 patients, while residual tu- mor ≤ 1 cm and residual tumor > 1 cm were pre- sent in 7/23 and 3/23 patients, respectively. The remaining 13 patients were inoperable and were treated only with chemotherapy. Furthermore, 16/47 patients were post-surgery treated with bev- acizumab maintenance for 15 months or until dis- ease progression, while 12/47 patients with known BRCA1/2 mutation with olaparib maintenance for 24 months or until disease progression. In our 3-year follow-up analysis, 36/47 patients had dis- ease progression, and 23/47 patients died (Table 1). Patients, diagnosed with HGSC at age ≤ 65 years had significantly better PFS and OS than older patients (PFS: p = 0.022, 22.6 vs. 13.1 months; OS: p = 0.002, 74.2 vs. 54.8 months). As expected, patients diagnosed at FIGO stage III demonstrated significantly better OS outcomes when compared to patients diagnosed at FIGO stage IV (p = 0.026, 23.0 vs. 13.0 months). Furthermore, patients who underwent surgery (primary or interval) and ad- juvant chemotherapy had significantly better PFS and OS than those with no surgery and neoadju- vant chemotherapy (PFS: p < 0.001 and p = 0.022; OS: p < 0.001 and p = 0.039, respectively). There was no difference in PFS and OS among the pa- tients who underwent primary surgery and those who underwent interval surgery. Treatment with TABLE 1. Clinical characteristics of the patients included in the study Age at diagnosis (years) Mean 64 Range 41−84 FIGO stage (N, %) IIIB 1 (2) IIIC 34 (72) IVA 7 (15) IVB 5 (11) Surgery (N, %) Primary 11 (23) Interval 23 (49) No surgery (remained inoperable) 13 (28) Residual disease after surgery (N, %) No residual tumor 19 (40) Residual tumor ≤ 1 cm 12 (26) Residual tumor > 1 cm 3 (6) Chemotherapy (N, %) Adjuvant 11 (23) Neoadjuvant* 36 (77) Bevacizumab (N, %) No 31 (66) Yes 16 (34) Positive family history (N, %) No 30 (64) Yes 17 (36) BRCA1/2 mutation (N, %) No 31 (66) Yes 12 (26) Unknown 4 (9) Olaparib (N, %) No 35 (74) Yes 12 (26) Disease progression No 11 (23) Yes 36 (47) Death No 24 (51) Yes 23 (49) *13 of these patients were inoperable and received only chemotherapy, while the other 23 patients were operable and received adjuvant chemotherapy as well Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma 497 FIGURE 2. Kaplan-Meier curves for (A) progression-free survival (PFS) and (B) overall survival (OS) of high-grade serous carcinoma (HGSC) patients based on treatment characteristics. ns = non-significant A B Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma498 bevacizumab did not affect PFS (p = 0.950, medi- an 20.4 vs. 14.2 months), but OS was prolonged (p = 0.003, median 33 vs. 16 months). Treatment with olaparib for patients with known BRCA1/2 muta- tion indicated significantly better PFS (p = 0.005, median 19.75 vs. 16.13 months) and OS (p = 0.044, median 21.5 vs. 23 months) (Table 2, Figure 2). TABLE 2. Univariate analysis of patient’s survival based on treatment characteristics Progression-free survival Overall survival HR (95% Cl) P-value HR (95% Cl) P-value Age at diagnosis (≤ 65 vs. > 65 years) 0.504 0.266−0.954 0.022 0.271 0.117−0.627 0.002 FIGO stage (III vs. IV) 1.331 0.691−2.566 0.381 0.312 0.112−0.872 0.026 Surgery < 0.001 < 0.001 no vs. primary 3.365 1.248−9.072 0.002 9.907 3.045–32.23 < 0.001 no vs. interval 2.981 1.133–7.845 0.001 8.529 2.270–32.05 < 0.001 primary vs. interval 0.570 0.253–1.282 0.192 0.605 0.186–1.973 0.239 Residual disease after surgery < 0.0001 < 0.001 no residual tumor vs. ≤ 1cm 0.764 0.314–1.858 0.538 0.189 0.045 –0.799 0.020 no residual tumor vs. > 1cm 0.121 0.006–2.256 < 0.001 0.066 0.003–1.408 < 0.001 residual tumor ≤ 1cm vs. > 1cm 0.131 0.008−2.209 < 0.001 0.131 0.009−1.988 < 0.001 Chemotherapy (adjuvant vs. neoadjuvant) 0.358 0.177–0.725 0.022 0.307 0.129–0.732 0.039 Bevacizumab (no/yes) 0.979 0.500–1.917 0.950 4.280 1.888–9.703 0.003 BRCA1/2 mutation (no vs. yes) 1.721 0.827–3.584 0.186 1.734 0.698–4.307 0.274 Olaparib (no vs. yes) 3.486 1.765–6.884 0.005 3.148 1.329–7.629 0.044 FIGURE 3. Box plots showing median (range) and quartiles for (A) T cells, NK cells, macrophages, DCs, B cells, and their subsets in the ascites of HGSC patients, and (B) the expression of PD-1 for each immune population/subset at disease presentation. CD3+ T cells, NKT cells, CD56brightCD16- and CD56dimCD16+ NK cells, macrophages, DCs, and B cells are given as a percentage per all CD45+ cells, while each subset is given as a percentage per its main population. A B Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma 499 Immune cells, and PD-1 and PD-L1 expression in the ascites at disease presentation CD3+, CD4+, and CD8+ T cells were measured in as- cites samples of all 47 patients. Due to the limited number of cells in some samples, Tregs, NKT cells, NK cells, B cells, macrophages, DCs, and CD103 ex- pression were measured in 39 samples, and due to the later inclusion of CD206 antibody in our study, M1-like and M2-like macrophages were analyzed in only 15 samples. PD-1 and PD-L1 expression was measured in 39 out of 47 samples (Supplementary Table 2). The results of ICs (Figure 3 A) showed a predominance of CD3+ T cells in ascites, with a me- dian percentage of 51% (range 6−86). In fact, the me- dians of CD4+ subsets and CD8+ subsets were 52% (range 30−83%) and 39% (range 14−63%), respective- ly, with CD4+ being significantly more abundant (p < 0.001). The median frequency for Tregs was 6% (range 2−17%). CD103 was expressed on CD3+ T cells (median 3%, range 1−34%). The majority of CD3+ T cells that expressed CD103+ were CD8+ (median 9%, range 2−49%), while only a small minority of CD4+ showed expression of CD103 (p < 0.001, median 2%, range 1−9%). We also examined the frequency of NKT cells and NK cells. The median percentage of NKT cells was 7% (range 1−39%) and of NK cells 6% (range 1−16). More precisely, 2% (range 1−16) of the NK cells were CD56dimCD16+, and 4% (range 1−8) were CD56brightCD16-. The median percentage of macrophages was 5% (1−24%), 61% of them were M1-type (range 17-90%), and 24% (range 1−52%) were M2-type. M1-like macrophages were signifi- cantly more abundant than M2-like macrophages (p < 0.001). We also identified the presence of DCs with a median frequency of 1% (range 1−7%) and B cells with 5% (range 1−19%). Furthermore, PD-1 was mainly expressed on T cells, without signifi- cant differences among CD4+, CD8+, and Treg sub- sets. The median expression for all T cell subsets was roughly 20%. Similar results were found for CD103+ positive T cells. Macrophages and DCs had slightly lower PD-1 expression than T cells (median < 10%). Significantly higher PD-1 expression was observed on M2-like macrophages (median 24%, range 1−52%) compared to M1-like macrophages (p = 0.049; median 1%, range < 1−26%). NK cells and B cells had the lowest expression of PD-1 (median < 2%) (Figure 3 B). We did not detect an expression of PD-L1 in any of the analyzed ICs. Immune cells and their association with treatment characteristics Furthermore, we aimed to determine if there are differences in the percentages of ICs at disease presentation that could be associated with treat- ment characteristics of HGSC such as primary operability (ability to perform primary surgery) FIGURE 4. Box plots showing the median (range) and quartiles for the percentages of different immune cells at presentation and their association with surgery type (no surgery vs. primary vs. interval) the patients were later assigned with. Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma500 and residual disease after surgery. According to the Kruskal-Wallis overall comparison, significant differences among the three surgery subgroups (no surgery, primary, and interval surgery) were observed for NK cells (p = 0.014) and DCs (p = 0.003) with lower percentages of NK cells and DCs in inoperable patients. However, according to the pairwise comparison within the subgroups, in ad- dition to the association with lower NK cells (p = 0.006) and DCs (p = 0.001), lower percentages of CD103+CD3+ T cells (p = 0.018), CD8+ (p = 0.048), and higher percentages of CD4+ (p = 0.046) and Treg (p = 0.032) were observed in the inoperable patient group vs. primary surgery group (Figure 4). When we compared the residual disease with per- centages of ICs we observed an association with significantly lower percentages of CD103+ CD3+ T cells and DCs in the patients with more residual tumor (Figure 5). We also wanted to see if high and low percentag- es of ICs correlate with PFS and OS. Patients strati- fied by having high percentages of CD103+CD3+ T cells (p = 0.017, median 18.6 vs. 10.6 months), CD56brightCD16- NK cells (p = 0.044, median 17.2 vs. 12.8 months) and DCs (p = 0.042, median 17.8 vs. 9.5 months) were associated with significantly better PFS compared to patients having low per- centages of these ICs (Table 3, Figure 5 A). Also, a trend towards longer PFS was observed in patients stratified by having low percentages of CD4+, high percentages of CD8+, and all macrophages (Table 2, Supplementary Figure 2 A). For Tregs, B cells, M1-like and M2-like macrophage subsets, CD56dimCD16+ NK subsets, and CD8/CD4 index, no differences in PFS for patients stratified by having high and low percentages were seen. We also ob- served a significant association with better PFS for patients with high PD-1 expression on Tregs (p = 0.044, median 18.2 vs. 12.0 months) (Figure 5 B), and a trend towards better PFS for patients strati- fied by having a low expression of PD-1 on NKT (median 17.8 vs. 15.1 months) and high expression of PD-1 on M2-like macrophages (median 17.2 vs. 14.2 months) (Supplementary Figure 1 B). PD-1 ex- pression on the other ICs showed no difference be- tween patients with low and high PD-1 expression rates in the ascites. We observed significantly longer OS in pa- tients stratified by having high percentages of CD103+CD3+ T cells (p = 0.009, median 22.7 vs. 15.8 months), CD8+ T cells (p = 0.008, median 27.3 vs. 15.3 months), CD56brightCD16- NK cells (p = 0.016, median 22.2 vs. 17.2 months), macrophages (p = 0.008, median 22.7 vs. 17.7 months) and DCs (p = 0.007, median 26.5 vs. 16.4 months), and low per- centages of CD4+ T cells (p = 0.038, median 27.3 TABLE 3. Univariate analysis of patient’s survival based on the low/high percentages of immune cells in the ascites at disease presentation Progression-free survival Overall survival HR (95% Cl) P-value HR (95% Cl) P-value CD3+ (low vs. high) 1.098 0.563–2.142 0.777 0.670 0.301–1.493 0.324 CD4+ (low vs. high) 0.547 0.260–1.150 0.110 0.401 0.174–0.928 0.038 CD8+ (low vs. high) 1.918 0.887–4.148 0.066 2.854 1.182–6.889 0.008 CD8/CD4 index (low vs. high) 1.076 0.560–2.069 0.820 2.973 1.304–6.780 0.010 Tregs (low vs. high) 0.856 0.412–1.778 0.677 0.807 0.324–2.006 0.649 CD103+CD3+ (low vs. high) 2.152 1.050–4.408 0.017 3.234 1.365–7.661 0.009 CD103+CD4+ (low vs. high) 0.515 0.232–1.147 0.108 0.816 0.328–2.031 0.661 CD103+CD8+ (low vs. high) 1.984 0.899–4.379 0.085 1.744 0.706–4.309 0.245 NKT cells (low vs. high) 0.520 0.246–1.101 0.060 1.208 0.507–2.878 0.661 CD56brightCD16– NK cells (low vs. high) 2.111 1.013–4.396 0.044 2.903 1.304–6.464 0.016 CD56dim16+ NK cells (low vs. high) 1.399 0.694–2.820 0.362 1.851 0.756–4.533 0.157 Macrophages (low vs. high) 0.601 0.245–1.478 0.275 1.943 0.754–5.006 0.008 M1-like macrophages (low vs. high) 0.868 0.224–3.370 0.835 1.562 0.321–7.604 0.533 M2-like macrophages (low vs. high) 2.142 0.553–8.297 0.224 2.854 1.182–6.889 0.080 B cells (low vs. high) 1.161 0.560–2.450 0.686 0.464 0.188–1.141 0.102 DCs (low vs. high) 2.245 0.799–6.310 0.042 3.307 0.939–11.65 0.007 Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma 501 vs. 16.5 months) compared to their counterparts. Furthermore, patients with high CD8/CD4 in- dex were associated with significantly longer OS compared to patients with low CD8/CD4 index (p = 0.010, median 30.0 vs. 16.5 months) (Table 3, Figure 4 C). For other ICs, no significant correla- tions with OS were observed. Furthermore, high expression of PD-1 on Tregs (p = 0.003, median 29.4 vs. 14.6 months) and CD56brightCD16- cells (p = 0.044, median 22.2 vs. 10.6 months) showed signifi- cantly better OS (Table 3, Figure 5 D). In addition, a trend towards better OS was seen for patients stratified by having low PD-1 expression on NKT and high PD-1 expression on CD56dimCD16+ NK cells and B cells (Supplementary Figure 3 B). We also performed a multivariate analysis of significant parameters among treatment charac- teristics and IC populations affecting patient’s survival. Considering the low number of patients and presence of multiple subgroups in the clini- cal parameters, multivariant analysis required re-categorizing surgery type as either no vs. pri- mary surgery, and residual disease after surgery as either no and ≤ 1 cm of residual tumor vs. re- sidual tumor > 1 cm and inoperable tumor, and no more than five significant variables were chosen. According to the results of multivariate analysis, only residual tumor after surgery was identified as an independent prognostic marker for both PFS (p = 0.046) and OS (p < 0.001) among treatment characteristics and DCs (low vs. high) as an inde- pendent prognostic marker among ICs for PFS (p = 0.049) only (Table 4). Discussion HGSC is the most aggressive gynecological ma- lignancy which is usually diagnosed at advanced stages when the disease has already spread in the peritoneum.14 Ascites is therefore often the first sign of the disease.15 We hypothesized that ICs in ascites might be a promising source of novel prog- nostic markers for HGSC. We assessed the pres- ence of different ICs together with CD103, PD-1, and PD-L1 expression levels and showed that per- centages of cytotoxic ICs (CD8+, CD56brightCD16- NK cells) as well as macrophages, might affect patient’s survival. We also showed that DCs are independ- ent prognostic marker for PFS of HGSC patients. As expected, our results on clinical and treat- ment characteristics of HGSC patients included in the study aligned with the already published data on the impact of age at diagnosis, FIGO stage, surgery, residual disease, and chemotherapy and maintenance therapy.2,3,4 This data confirms the adequacy of our analyzed patient cohort. According to the evaluation of ICs in ascites, our findings demonstrated that CD3+ T cells (me- dian 51%) are the predominant population in the FIGURE 5. Box plots showing the median (range) and quartiles for the percentages of different immune cells at presentation and their association with residual disease after surgery (less (no and ≤ 1cm) residual tumor vs. more (< 1 cm and inoperable) residual tumor). Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma502 ascites of HGSC patients (FIGO stage ≥ III, ascites collected before initiation of treatment), with CD4+ significantly more abundant than CD8+, while the amount of Tregs was low as well as the other ICs investigated. Auer et al. reported higher percentag- es of CD3+ (median 80%) compared to our results, with an equal ratio of CD4+ and CD8+ subsets, and higher percentages of NKT cells (median 15%).1 They did not specify patient’s FIGO stage and when ascites was collected, which could explain the difference in the results of our and their study. However, the percentages of Tregs, NK cells, and B cells in their study were in concordance with our findings.1 Similar percentages of CD4+ and Tregs, T cells, and DCs as in our study were reported in other studies.2,16 We detected low percentages of macrophages (median 5%) in our series of ascites samples; the majority of them were M1-like. On the contrary, Steitz et al. described roughly 70% of macrophages, with equal ratios of both M1- like and M2-like subsets. Unfortunately, they did not describe disease progression by FIGO stage and ascites collection time either.17 Therefore, we speculate that lower percentages of macrophages in our study might be related to the inclusion of ascites samples from HGSC patients at the time of diagnosis, and not later at disease progression. In the literature, we have found some data about percentages of ICs in HGSC ascites, but to our knowledge, there was no data providing in- formation about the association of ICs at disease presentation and treatment assigned to the pa- tients. Interestingly, in our study, we observed higher percentages of CD103+, CD8+, Tregs, NK cells, and DCs, and lower percentages of CD4+ cells in the ascites of patients with less tumor bur- den that underwent primary surgery compared to inoperable patients, which due to the size of the tumor were no eligible for surgery. These results might indicate an association of the amount and cell type of ICs in ascites at disease presentation with the extent of the tumor burden. Furthermore, we observed higher percentages of CD103+ T cells and DCs in patients who underwent interval sur- gery compared to the inoperable group. This data suggests the possibility of using these ICs to help us predict which patients, after receiving neoad- juvant chemotherapy, are likely to be eligible for surgery later on and have a lower amount of re- sidual tumor. However, a much larger patient co- hort is needed to confirm these findings. And as mentioned above, we have not found any similar studies to compare our results with. Furthermore, most of the research on the influ- ence of ICs on HGSC patient’s survival is carried out on primary tumor tissues, and very little is known about the role of ICs in ascites. For instance, it has been reported that T cells in primary tumors improve the survival of HGSC patients. In fact, CD8+ cells correlated with improved survival, and Tregs, as well as CD4+ cells were seen as an indica- tor of poor prognosis.18,19 On the other hand, stud- ies on T cells in ascites have failed to confirm this correlation, even though a trend towards improved survival in patients with low CD4+ T cells was re- ported.2 However, the ratio between CD8+ and CD4+ T cells or even Tregs has been reported as a more appropriate indicator of better OS.20-23 In our study, similarly, we observed an association with signifi- cantly longer OS for patients stratified by having low CD4+ and high CD8+ T cells, and high CD8/ CD4 index compared with the patients stratified by having high CD4+ and low CD8+ T cells, and also a low CD8/CD4 index. Regarding NKT cells, data TABLE 4. Multivariate analysis of the treatment characteristics and immune cells Variables included in the multivariate analysis Progression-free survival Overall survival HR (95% Cl) P-value HR (95% Cl) P-value Primary surgery (no vs. yes) 0.640 0.194−2.114 0.509 0.592 0.101−3.454 0.560 Residual disease after surgery (no residual tumor and ≤ 1 cm of residual tumor vs. > 1 cm residual tumor) 0.408 0.169−0.983 0.046 0.009 0.001−0.092 < 0.001 CD103+CD3+ (low vs. high) 0.605 0.266−1.374 0.230 0.632 0.182−1.307 0.470 CD56brightCD16– NK cells (low vs. high) 1.707 0.683−4.265 0.252 NA NA NA DCs (no vs. yes) 0.394 0.155−0.998 0.049 0.419 0.135−1.307 0.134 Macrophages (low vs. high) NA NA NA 0.592 0.101−3.545 0.560 NA = no available Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma 503 A B C D FIGURE 6. Kaplan-Meier curves for progression-free survival (PFS) and overall survival (OS) of high-grade serous carcinoma (HGSC) patients. PFS curves show significant differences for patients stratified as having (A) low or high percentages of immune cells, (B) low or high expression of PD-1 on immune cells, OS curves show significant differences for patients stratified as having (C) low or high percentages of immune cells, (D) low or high expression of PD-1 on immune cells. Cut-off values were based on the population median. P-values for significant differences are given. Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma504 on their role in ovarian carcinoma survival is gen- erally limited. According to our results, there was not a significant correlation between NKT cells and survival rate for patients stratified as having high percentages of NKT cells. We also investigated the role of CD103 on T cells in HGSC ascites. CD103 is a subunit of the αE/β7 integrin that helps to retain expressing cells on the epithelium.24 CD103 has been proposed as a marker of activated and tumor- reactive CD8+ T cells in ascites HGSC25,26 but no da- ta correlated with survival was given. We showed that CD103 was mostly expressed on CD8+, and not on CD4+ subsets, which was shown by us and by two other studies.26,27 Furthermore, patients strati- fied by having high percentages of CD103+CD3+ T cells in HGSC ascites were associated with better PFS and OS. CD103+CD3+ T cells in ascites seem to have the same potential of prognostic informa- tion as reported for the CD103+ tumor-infiltrated T cells in the primary tumor27 and we speculate that these cells in the ascites might be involved in the improvement of the antitumor response in the peritoneum. Of course, a larger patient cohort and additional tests are needed to gain a more compre- hensive understanding of the significance and role of CD103+CD3+ T cells in HGSC ascites. The role of B cells regarding their contribution to impaired antitumor immunity in HGSC has not been investigated as much as the role of T cells. However, there are few reports showing a trend towards worse OS in patients with high infiltration of B cells in ascites. These findings are consistent with ours.28,29 Interestingly, opposite findings have been reported in primary tumors where a high percentage of B cells correlated with favorable sur- vival, indicating that more studies are needed to estimate the role of B cells in ovarian tumors.30,31 DCs in ascites have been poorly investigated. Only one study described a trend of high per- centages of DCs in HGSC ascites toward a better survival outcome.2 Similarly, we confirmed a sig- nificant association between DCs and patient’s survival. Consistently, patients stratified by hav- ing high percentages of DCs were associated with improved prognosis in the primary tumors as well.31,32 NK cells have attracted attention due to their ability to kill tumor cells without prior sensitiza- tion. There is limited data on the contribution of NK cell immunity to the clinical outcome of ovar- ian carcinoma. Infiltration of NK cells in primary tumors has shown a contradictory impact on sur- vival outcomes in HGSC.33 However, recently, one study showed an association of high percentages of CD56+ NK cells in ascites with better PFS and OS.14 Similarly, we showed the same association of both CD56brightCD16– and CD56dimCD16+ NK cells with the survival outcome in our patient cohort. It is generally thought that CD56brightCD16– NK cells have a higher capacity for cytokine production and have mainly proliferative potential, and on the contrary, CD56dimCD16+ NK cells have weak cyto- toxic activity,34,35 which explains why we observed significant results for CD56brightCD16– and only a trend towards CD56dimCD16+. Macrophages in ovarian ascites are gaining a lot of attention in recent years, due to their plas- ticity to switch from antitumor M1 to protumor M2 phenotypes.8 M2-type macrophages have been characterized by the expression of markers such as the scavenger receptors CD206 or CD163.36 Published data is speculating that M2-type mac- rophages are taking the main role in immune suppression and angiogenesis promotion to sus- tain tumor growth.37,38 Even though we identified lower percentages of macrophages than report- ed, we showed that patients stratified by having higher percentages of M1-like macrophages than M2-like macrophages were associated with better survival. We speculate that when the diagnosis is given, even though the total macrophage count is low, M1-like macrophages are predominant and are probably the ones contributing to a better out- come. However, during disease progression, M2- like macrophages outnumbered the M1 subset and most probably contributed to tumor progression and poor outcome of the disease.39 Immune tolerance is defined by the inability of ICs to express immune checkpoints such as PD-1 and PD-L1. PD-1 receptor is an inhibitor of both adaptive and innate immune responses and can be expressed on CD8+ T cells, CD4+ T cells, and Tregs in ovarian tumors, whereas PD-L1 is expressed on activated T cells, tumor-infiltrating macrophages or fibroblasts, contributing to tumor immune es- cape.40 However, the expression of PD-1 and PD-L1 in HGSC ascites and its correlation with survival has not yet been fully investigated. In the present study, we showed that PD-1 expression is present in almost all ICs, except on NK cells and B cells (less than 1%). We detected roughly 20% PD-1 ex- pression on CD4+, CD8+ T cells and Tregs. However, Imai et al. reported 2x higher level of PD-1+CD4+ and PD-1+CD8+ cells in the ascites.41 Possible reasons for this discrepancy might be the different PD-1 clone selections for the analysis, as well as the diversi- ty of the patient cohort. Imai et al. performed the analysis on different types of malignant epithelial Radiol Oncol 2023; 57(4): 493-506. Miceska S et al. / Immune cells in the ascites of high-grade serous carcinoma 505 ovarian carcinomas, and a few cases on borderline and benign tumors. Similar to us, they did not find a correlation between survival and PD-1 expres- sion on CD4+ and CD8+ T cells. However, Sato et al. found an association between CD8+ cells and PD-1 expression in advanced epithelial ovarian carci- noma.20 On the contrary, Pawłowska et al. demon- strated an association of high percentages of both PD-1+CD4+ and PD-1+CD8+ cells in ascites with worse outcomes, indicating a negative regulation of the anticancer immune response and exhaus- tion of T cells in the ascites.42 Results on primary ovarian tumors have also reported a correlation of higher PD-1 expression on T cells with shorter survival and worse prognosis.43,44 For the other ICs in ovarian carcinoma ascites, data on PD-1 expres- sion and survival correlation is also missing. We showed that high expression of PD-1 on Tregs and CD56brightCD16- NK cells is associated with bet- ter survival. We do not know how to interpret the correlation of high PD-1 expression with better instead of worse survival as expected. Additional studies are necessary to clarify if PD-1 expression on Tregs and CD56brightCD16- NK cells could be a positive prognostic marker for patient survival. We are also the first to confirm that PD-L1 is not expressed on ICs in HGSC ascites, since we did not find data on PD-L1 expression on ICs in ascites. One study on primary tumors has found that al- most 2/3 of the tumors had a low level of PD-L1 expression, mainly on ICs rather than tumor cells, and the expression of PD-L1 was associated with significantly worse prognosis,45 indicating loca- tion-dependent loss of expression of PD-L1 on ICs in ascites. Nevertheless, it is worth mentioning that all published data on ovarian carcinoma also reports results on low and high percentages of ICs in as- cites in correlation with patient survival without stratifying patients in subgroups according to their treatment characteristics. This is due to the low number of HGSC patients2,16,26,27,28,32 each re- search group confronts, and also the reason why we did not conduct that kind of analysis within the treatment subgroups either. Yet, the multivariate analysis indicated that residual tumor is the only independent prognostic marker for PFS and OS, and DCs are an independent prognostic marker for PFS only. We believe that multicentric studies on large patient cohorts could give more accurate in- formation on the prognostic meaning of DCs and other ICs in the ascites. In conclusion, we found that CD3+ were the pre- dominant cells in HGSC ascites at disease presen- tation and showed that high levels of CD103+CD3+ T cells, CD56brightCD16- NK cells and DCs improve both PFS and OS, whereas high levels of CD8+, CD8/CD4 index, macrophages, PD-1+ Tregs and PD-1+CD56brightCD16- NK cells, and low levels of CD4+ improve OS only. We also confirmed that the residual disease is the only clinical independent prognostic marker for PFS and OS, and we showed that DCs are the only ICs that might become an independent prognostic marker for PFS. Data ob- tained highlight the potential of ascites as a source to provide additional prognostic information for HGSC patients. However, a larger patient cohort and longer follow-up are necessary to assess the independent prognostic significance of ICs togeth- er within different treatment characteristics. Acknowledgments The study was supported by Slovenian Research Agency (research program P3-0289). Sincere ap- preciation is extended to Sara Glinšek, M.S. and Zala Ciglar, M.S. for their valuable contribution in sample preparation and flow cytometry acquisi- tion. References 1. Auer K, Bachmayr-Heyda A, Sukhbaatar N, Aust S, Schmetterer KG, Meier SM, et al. Role of the immune system in the peritoneal tumor spread of high grade serous ovarian cancer. 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Onco Targets Ther 2019; 12: 8437-45. doi: 10.2147/OTT.S221340 Radiol Oncol 2023; 57(4): 507-515. doi: 10.2478/raon-2023-0056 507 research article Management of tumor volume changes during preoperative radiotherapy for extremity soft tissue sarcoma: a new strategy of adaptive radiotherapy Marion Geneau De Lamarliere1, Amélie Lusque2, Justine Attal Khalifa1, Vincent Esteyrie3, Christine Chevreau4, Thibaud Valentin4, Dimitri Gangloff5, Thomas Meresse5, Louis Courtot6, Philippe Rochaix7, Bérénice Boulet8, Eliane Graulieres9, Anne Ducassou1 1 Department of Radiation Oncology, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse - Oncopole, Toulouse, France 2 Statistics department, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse - Oncopole, Toulouse, France 3 Department of Radiation Oncology, Rodez, France 4 Department of Medical Oncology, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse - Oncopole, Toulouse, France 5 Department of Surgery, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse - Oncopole, Toulouse, France 6 Department of Surgery, Pierre Paul Riquet Hospital, Toulouse, France 7 Department of Pathology, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse - Oncopole, Toulouse, France 8 Department of Imagery, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse - Oncopole, Toulouse, France 9 Department of Engineering and Medical Physics, Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse – Oncopole. Toulouse, France Radiol Oncol 2023; 57(4): 507-515. Received 13 April 2023 Accepted 25 September 2023 Correspondence to: Anne Ducassou, M.D., Departement of Radiation Oncology, Institut Claudius Regaud, Institut universitaire du cancer de Toulouse – Oncopole, 1 avenue Irène Joliot-Curie; IUCT-O; 31059 Toulouse Cedex 9, France. E-mail: ducassou.anne@iuct-oncopole.fr 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. Using adaptive radiotherapy (ART), to determine objective clinical criteria that identify extremity soft tissue sarcoma (ESTS) patients requiring adaptation of their preoperative radiotherapy (RT) plan. Patients and methods. We included 17 patients with a lower extremity ESTS treated between 2019 and 2021 with preoperative RT, using helicoidal intensity-modulated RT (IMRT) tomotherapy, before surgical resection. We collected clinical, tumor parameters and treatment data. Repositioning was ascertained by daily Megavoltage computed tomography (MVCT) imaging. Using the PreciseART technology we retrospectively manually delineated at least one MVCT for each patient per week and recorded volume and dosimetric parameters. A greater than 5% change be- tween target volume and planned target volume (PTV) dosimetric coverage from the initial planning CT scan to at least one MVCT was defined as clinically significant. Results. All 17 patients experienced significant tumor volume changes during treatment; 7 tumors grew (41%) and 10 shrank (59%). Three patients (18%), all undifferentiated pleomorphic sarcomas (UPS) with increased volume changes, experienced significant reductions in tumor dose coverage. Seven patients required a plan adaptation, as determined by practical criteria applied in our departmental practice. Among these patients, only one ultimately experienced a significant change in PTV coverage. Three patients had a PTV decrease of coverage. Among them, 2 did not receive plan adaptation according our criteria. None of the patients with decreased tumor volumes had reduced target volume coverage. Monitoring volume variations by estimating gross tumor volume (GTV) on MVCT, in addition to axial and sagittal linear tumor dimensions, appeared to be most effective for detecting reductions in PTV coverage throughout treatment. Conclusions. Variations in ESTS volume are evident during preoperative RT, but significant dosimetric variations are rare. Specific attention should be paid to grade 2-3 UPSs during the first 2 weeks of treatment. In the absence of dedi- cated software in routine clinical practice, monitoring of tumor volume changes by estimating GTV may represent a useful strategy for identifying patients whose treatment needs to be replanned. Key words: soft tissue sarcoma; preoperative radiotherapy; adaptive radiotherapy; image guided radiotherapy; volumes changes Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas508 Introduction Preoperative RT is one of the current standard of care for the management of extremity soft tissue sarcoma (ESTS), mainly in large tumors, requiring reconstructive surgery or possible R1 resection.1,2 This strategy allows to reduce the irradiation field, uses lower doses, reduces late toxicities3 and facili- tates the delineation.4,5 Preoperative RT specifically confronts physi- cians with variations in tumor volume, enhanced particularly since the advent of image guided ra- diotherapy (IGRT), the development of volume repositioning systems such as cone-beam com- puted tomography scan (CBCT) and megavoltage computed tomography (MVCT). Approximately half of the patients treated with neoadjuvant ra- diotherapy, reported in the literature, present with a significant variation in tumor volume during the course of treatment.6 These variations in volume require the modification of planned treatment in approximately 8 to 30% of cases.6-11 Offline adap- tive radiotherapy (ART) is currently the most ap- propriate approach to address gradual sarcoma anatomy changes, which may otherwise introduce interfractional errors. Several authors have defined a tumor size varia- tion of >1cm in any direction or recurrent incorrect and unacceptable repositioning, to trigger a plan adaptation.10 Others have opted to define tumor volume criteria.11 But there is currently no con- sensus on any objective cutoff thresholds which would prompt an adaptation of the previously planned treatment. The aim of this retrospective study was to de- termine objective criteria to identify patients re- quiring plan adaptation in the clinical setting. We initially considered the threshold of a 5% decrease in planned target volume (PTV) coverage to be un- acceptable for optimal treatment, with the ration- ale of ultimately finding an objective criterion that could be monitored throughout RT treatment. Patients and methods This retrospective monocentric observational study was approved by and conducted in accord- ance with local ethic committee requirements (# F20210208164425). All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. We reviewed data from computer file records of patients treated between August 2019 and January 2021. Patient inclusion criteria were defined as: (i) a localized lower extremity ESTS, (ii) age of >18 years, (iii) preoperative radiothera- py or concomitant radio-chemotherapy (RTCT) with helicoidal intensity-modulated radiotherapy (IMRT) on tomotherapy. Patient treatments were planned on the Accuray® precision treatment planning system. FIGURE 1. Percentage of maximal GTV changes during treatment: patients with significant gross tumor volume (GTV) increase (A), patients with significant GTV decrease (B)*. Day 0 corresponds to CTs1. Each color represents a different histology subtype; replanned patients appear as dotted line. DLS = dedifferentiated liposarcomas; MLS = myxoid liposarcomas; PRS = pleomorphic rhabdomyosarcoma; UPS = undifferentiated pleomorphic sarcomas * Only the largest variation is shown for patients who presented with both an increase and a decrease in tumor size during the course of treatment. A B Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas 509 All dosimetric plans complied with our institu- tional optimal coverage criteria: 95% of PTV had to be covered by 95% of the prescribed dose, whilst respecting healthy organ dose limits. Daily high-energy 3D IGRT (MVCT) image guidance was performed for all patients. Data was retrospectively uploaded into the tomography in- tegrated PreciseART® adaptive radiation therapy software. The same radiation oncologist manually contoured each individual patient’s gross tumor volume (GTV), clinical target volume (CTV) and PTV on at least one MVCT per week. According to our departmental practice, the technicians reported any significant change in tu- mor volume (specifically a linear variation of >1cm in any direction), weight loss or any other incon- gruous parameter, compared to the initial plan- ning CT (CTs1), to the radiation oncologist who then decided whether or not to adapt the plan. If a new plan was initiated, this involved performing a new CT simulation scan (CTs2), determining the GTV/CTV/PTVs, delineating the critical structures and recalculating the dosimetry from scratch. This new tailored plan was then applied to the patient in the next few days, and patients continued the treatment with the old plan waiting the new one. The co-recording and fusion of the CT simula- tion scan (CTs1) with the contoured MVCT allowed us to determine axial and sagittal linear tumor di- mensions, tumor volume, and dose coverage of the GTV and PTV over time. Since tumor volume in clinical practice cannot be obtained without re- delineation, we estimated it from axial and sagittal linear tumor dimensions using the formula ת x r2 x h (where r: axial diameter/2 and h: sagittal height), which assumes that the tumor is a cylinder. A change in target volume of 5% at any time during treatment was defined as significant. We defined any change in dosimetric coverage of the PTV of 5% between the initial planning CT and the last MVCT as unacceptable. Statistical analysis Continuous variables are represented as medians or means with a range (minimum–maximum), and categorical variables as frequencies and percent- ages. For each patient, the percentage change of indi- vidual parameters was plotted over the six weeks of radiotherapy and the plan adaptation (if appli- cable). Percentage changes were calculated from the initial planning CT (CTs1) at each MCVT. If a plan adaptation was performed, the percentage TABLE 1. Patients, tumor and treatment characteristics Characteristics: n (%) Sex Male 12 (71) Female 5 (29.4) Age at initial diagnosis, years Median (range) 69 (43-90) Dimension, cm (diagnostic MRI) Median (range) 12.8 (6-30) Pathology Undifferentiated pleomorphic sarcoma (UPS) 8 (47) Myxoid liposarcoma (MLS) 6 (35) Dedifferentiated liposarcoma (DLS) 2 (12) Pleomorphic rhabdomyosarcoma (PRS) 1 (6) Grade 1 6 (35) 2 7 (41) 3 4 (24) RT schedule (total dose, dose fraction) 50.4 Gy, 1.8Gy 5 (29) 50 Gy, 2Gy 10 (59) 45 Gy, 3 Gy 1 (6) 70 Gy, 2 Gy* 1 (6) GTV on CTs1 (ml) Median (range) 381 (84-2908) PTV on CTs1 (ml) Median (range) 1373 (587-5793) D95%PTV on CTs1 (%) Median (range) 97.1 (96-99.1) Interval between CTs1 and MVCT 1 (days) Median (range) 13 (9-17) Neoadjuvant CT** No CT 10 (59) Adriamycin ifosfamide ((doxorubicin 20 mg/m2 and ifosfamide 2500 mg/m2 day 1, 2 and 3 for 4 cycles (21-day cycle)) 6 (35) Adriamycin (doxorubicin 75 mg/m2 day 1 for 4 cycles (21-day cycle)) 1 (6) CTs1 = CT simulation scan; D95%PTV = dose received by 95% of the PTV volume; MRI = Magnetic Resonance Imaging; MVCT1 = first fraction of RT GTV = gross tumor volume; MVCT = megavoltage computed tomography;PTV = planned target volume; RT = radiotherapy * Patient initially scheduled to have preoperative 50 Gy in 25 fractions but deemed inoperable, leading to a modification of the prescription. ** 6 patients received 3 cycles, 1 patient had 4 cycles Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas510 change at each MVCT post adaptation was cal- culated from the new CT simulation scan (CTs2) for dosimetric data. The percentage change from CT1 at each MVCT in GTV was plotted over the six weeks of radiotherapy according to tumor histol- ogy in patients with increased or decreased GTV. Statistical analysis was carried out using Stata ver- sion 16 (StataCorp LLC, College Station, TX). Results Patients, tumor, and treatment characteristics are presented in Table 1. All patients had a lower limb sarcoma. There were 8 undifferentiated pleomorphic sarcomas (UPS), 6 myxoid liposarcomas (MLS), 2 dediffer- entiated liposarcomas (DLS) and 1 pleomorphic rhabdomyosarcoma (PRS). Majority of patients re- ceived 50 to 50.4 Gy delivered in 25 to 28 fractions. The median interval between the end of RT and surgery was 57 days (range 32-131 days), all resections except one were R0 (adequate margins). Seven patients among the 14 patients for whom post-surgical data was available developed an acute surgical complication: 3 scar disunions, 2 infections, 1 deep vein thrombosis and 1 lympho- cele. The 3 scar disunions all occurred in patients with shrinking tumors, and despite a skin flap reconstruction in 2 of the patients, during the sar- coma surgery. Volumetric and dosimetric changes are present- ed in Table 2 All patients experienced a GTV volume varia- tion of >+/-5% during treatment, a decrease in GTV in 10 patients (59%) and an increase in 7 patients (41%). Among these latter 7 patients, GTV initially increased in 3 patients before significantly de- creasing. Mean maximal tumor volume changes were +24% (range +8% to +36%) for the GTV in- creases and -35% (range -7% to -52%) for GTV de- creases. GTVs predominantly increased during the 1st week (78%) and decreased during the 3rd week (55%) of treatment. Significant PTV volume changes, although less substantial (+16% (range +5% to +29%) for increases and -20% (range -7% to -37%) for decreases), fol- lowed the same trends as GTV changes, except for TABLE 2. Gross tumor volume (GTV) and planned target volume (PTV) volumes variations, the largest GTV and D95% PTV changes during the course of treatment M ax im al G TV v ar ia tio n du rin g th e co ur se o f t re at m en t Pathology Vol GTV CTs1 (ml) Vol PTV CTs1 (ml) Vol GTV last MVCT Vol PTV last MVCT Largest GTV vol change % (week) Largest D95%PTV change % (week) UPS 674 1373 560 1150 +36.4 (2) -12 (2)* PRS 239 1037 238 966 +33.7 (2) -3.1 (1) UPS 381 1432 486 1706 +32.3 (5) -20.1 (6)* UPS 2546 5292 3243 6263 +27.4 (6) + 1 (4) DLS 2908 5793 3444 6628 +18.9 (4) -4.6 (3) UPS 1356 3534 1319 3446 +11 (1) -2.1 (1) UPS 234 999 246 1086 +7.9 (2) -14.1 (4)* UPS 140 587 144 598 -7.2 (5) -3.4 (2) UPS 826 2073 781 2031 -9.2 (1) -7.6 (4) DLS 531 1679 461 1509 -13.2 (6) -1.3 (3) MLS 258 952 184 805 -28.9 (6) -1.3 (2) MLS 415 1514 233 1098 -43.8 (6) + 1.6 (6) MLS 192 816 104 630 -46.2 (6) -2 (6) MLS 228 629 122 398 -46.4 (6) +0.4 (4) MLS 1062 3084 568 2158 -46.5 (6) + 1.14 (5) UPS 208 961 96 602 -52 (6) -0.9 (1) MLS 84 606 44 548 -52 (5) -7 (3) DLS = dedifferentiated liposarcoma; MLS = myxoid liposarcoma; PRS = pleomorphic rhabdomyosarcoma; UPS = undifferentiated pleomorphic sarcoma Re-planned patients are shown in gray, patients with PTV under-coverage are marked with a star (*) Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas 511 one patient whose PTV was very close to the skin, which presumably prevented its expansion. Axial and sagittal GTV dimensions varied less significantly and remained stable in 24% and 29% of patients respectively. Variations based on the calculated estimate of the GTV (GTVr = ת x r2 x h) were overall well correlated with those of the ac- tual GTV: 7 tumors increased (47%) and 9 tumors decreased (53%), 1 remained stable, at the time of maximal GTV changes. Volume variations and the most substantial GTV changes are shown in Table 2. Figure 1 shows GTV volume changes be- tween CTs1 and the end of treatment in patients who presented a significant GTV increase (a), and in those who presented a significant GTV decrease (b), according to tumor histology subtypes. MLSs decreased in size during RT, whereas 63% of UPS increased. Similarly, all grade 1 tumors decreased whilst 63% of grade 2 and 3 sarcomas increased. We evaluated whether GTV variations correlat- ed with changes in axial and sagittal linear tumor dimensions (Table 3). The axial and sagittal tumor dimensions were stable when the GTV variation was at its maxi- mum but varied significantly during subsequent MVCTs. A lag between significant GTV variation and significant changes in axial or sagittal linear tumor dimension was observed in 59% of cases. The estimated GTV also correlated well with the GTV over time. The dose received by 95% of the PTV (D95%PTV) remained satisfactory for most patients. The larg- est D95%PTV changes are presented in Table 2. Three patients (18%) exhibited a significant drop in PTV coverage (mean maximal D95%PTV change of -15.3% (range -14.1% to -20.1%)). All occurred within the first 2 weeks of treatment at a mean of 4 fractions, following an average increase in GTV and PTV volumes of +17% and +11% respectively. All 3 patients had UPS, only one had a plan adap- tation, because of an increase in axial length (+1.3 cm). The plan adaptation was performed in week 4 and restored an appropriate dosimetric plan. The remaining two patients sustained PTV under- dosage until the end of treatment. Volumetric and dosimetric data for these 3 patients are presented in Table 4. All 3 patients experienced significant increases in GTV, estimated GTV and PTV vol- umes. Conversely, sagittal and axial linear tumor dimensions were less informative. In patient 16, GTV and PTV did not immediately increase when the reduction in PTV coverage became apparent (Table 4). No quantitative variation in volume or dimen- sion could precisely identify which of the 7 pa- tients with growing tumors were going to have insufficient PTV coverage. Among the 10 patients with shrinking tumors, the bone near-maximum absorbed dose (D2%) in- creased by an average of 1.6% (range 0.4 to 2.9%) in 9 patients. The D2% delivered to the joint increased by an average of 17% (range 1 to 65%) in 7 patients. TABLE 3. Correlations between GTV volume variations and dosimetric and clinical characteristics Maximal GTV change Decrease (n=10) Increase (n=7) GTV largest axial axis* Decrease 7 (70%) 0 Stable 3 (30%) 3 (43%) Increase 0 4 (57%) GTV largest sagittal axis* Decrease 8 (80%) 0 Stable 2 (20%) 3 (43%) Increase 0 4 (57%) Estimated GTV volume Decrease 9 (90%) 0 Stable 1 (10%) 0 Increase 0 7 (100%) PTV volume Decrease 8 (80%) 0 Stable 2 (20%) 0 Increase 0 7 (100%) D95% PTV Decrease 0 3 (43%) Stable 10 (100%) 4 (57%) Surgical Yes 3 (33%) 4 (80%) complications** No 6 (67%) 1 (20%) Histology subtypes UPS 3 (30%) 5 (71%) DLS 1 (10%) 1 (14%) MLS 6 (60%) 0 PRS 0 1 (14%) Histology grade 1 6 (60%) 0 2 2 (20%) 5 (71%) 3 2 (20%) 2 (29%) Neoadjuvant CT Yes 3 (30%) 4 (57%) No 7(70%) 3 (43%) * Axial and sagittal linear tumor dimensions were recovered from the same slice for each MVCT, the slice that contained the largest tumor axial and sagittal axis on the CTs1. **Data available for 14 patients: one patient had not been operated; the two others had not been operated when the analysis was performed. GTV = gross tumor volume; MVCT = megavoltage computed tomography; PTV = planned target volume Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas512 Adaptive radiotherapy Seven patients had a plan adaptation, based on em- pirical clinical criteria defined by the department: 4 tumor size increases, 2 tumor shrinkages1 loss of weight. Dosimetric and clinical data of patients with a plan adaptation are presented in Table 5. The decision to adapt a plan was predominant- ly made the 3rd week, at a mean dose of 21.6 Gy (range 0 Gy to 42 Gy). Retrospectively, PTV cover- age remained adequate for 6 out of the 7 patients. In the two patients with tumor shrinkage, PTV coverage was stable and OAR constraints were re- spected. On the CTs2, an average increase of 17.2% (range -41 to 42%) in tumor volume was observed. The new plans resulted in a 4.7% gain of PTV cov- erage, all OAR constraints were improved by mak- ing a new treatment planning. Clinical considerations Six MLS were included in our study. All tumors shrank during treatment, by 44% on average. Their dose coverage remained relatively stable with a -1.2% mean maximal D95% PTV change during the treatment. Only one of the cases presented a sig- nificant decrease in PTV coverage (-7% at week 3). Graphs for each of the 17 patients showing the evo- lution of tumor dimensions and volumes through- out treatment are included in the appendix. Discussion The current trend in the treatment of patients with locally advanced ESTS is the use of neoadjuvant RT. This practice forces physicians to consider the TABLE 4. Dosimetric and clinical data of patients with significant reductions in PTV coverage Dosimetric data at first MVCT showing a variation of volume Concomitant volume changes Clinical dimension change* D95% PTV on last MVCTD95% PTV on CTs1 Fraction n° Variation of D95 coverage (%) New D95% PTV D95% GTV GTV (%) PTV (%) Estimated GTV(%) 99%* 6* -11.8* 87.22%* 72.96%* +36.4* +22.8* +27.6* + 1.3 cm (ax)* 98.71%* 96.1% 6 -10.4 86.49% 98.03% +10.7 +6.2 +12.2 + 1 cm (sag) 77.07% 98.2% 1 -5.9 92.39% 98.91% +2.8 +3.3 +2 No 94.29% Significant reductions in coverage are indicated in bold. The patient who had a plan adaptation is marked with a star (*). *The “clinical dimension change” corresponds to the largest visible variation, all slices combined. GTV = gross tumor volume; MVCT = megavoltage computed tomography; PTV = planned target volume TABLE 5. Dosimetric and clinical data of patients with a plan adaptation Dosimetric data just before plan adaptation Evolution of D95%PTV Clinical Reason* Fr n° % GTV volchange %PTV vol change OAR constraints At CTs1 Before re- planning At CTs2 Ax. increase (+ 1.3 cm) 13 + 33 + 22 Better 99.1% 88% 97.5% Ax. increase (+ 0.9 cm) 4* + 9 + 9 Better 98.1% 93.5% 96% Sag. increase (+ 1.2 cm) 1 + 23 + 10 Better 96.8% 93.7% 95.9% Sag. increase (+ 1 cm) 11 + 17 + 12 NA 96.9% 97.6% 97.6% Ax. decrease (- 1.8 cm) 13 - 36 - 21 Bone Dm+ 1.8% 96.9% 97.2% 98.7% Sag. decrease (- 2 cm) 17 - 28 - 17 Bone Dm+ 1% 96.4% 97.5% 96.5% Weight loss 21** - 13 - 10 Bone Dm+ 30.7% 96.4% 97.1% 96.9% Ax = axial; Bone Dm = average dose to the bone; CTs1 = CT computed tomography simulation scan; CTs2 = new Fr n° = fraction number; GTV = gross tumor volume; NA = result not available; OAR = organ at risk; PTV = planned target volume; Sag = sagittal * The “clinical reason” data of axial and sagittal linear tumor dimensions of patients who had a plan adaptation corresponds to the largest visible variation, all slices combined. Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas 513 management of tumor volume changes during ra- diotherapy. The 17 patients included in our study and treated by helical IMRT on tomotherapy, all presented a significant volume change at some point during their treatments. We observed more tumor size reductions (59%) than increases (41%). ESTS volume changes during preoperative radi- otherapy are obvious and widely described in the literature. However, the magnitude and frequency of changes vary substantially due to variations in the criteria adopted, the histology subtype and the nature of the neoadjuvant treatment administered. Abu-Hijlih et al. reported 61% of tumor volume variations, the majority decreases 57%. The com- parison was only based on the last CBCT and there- fore excluded any short-term fluctuations.9 Under similar conditions, 18% of our patients would have been considered stable (with 59% reductions but only 23% increases). Conversely, Dickie et al. who only considered patients with a plan adaptation, noted more tumor parameter increases than de- creases (64% vs 36%).10 Betgen et al. only found 52% volume variations between the start and the end of treatment although they applied the same varia- tion criteria as our study. Surprisingly, 60% of their tumors were MLS, which are known to significant- ly decrease in size during treatment. Betgen et al. observed a 33% tumor shrinkage among the MLSs. Furthermore, their 5 tumors that increased in size, exhibited a mean GTV change that was lower than ours (+14% vs +22%).11 Histology subtype was a risk factor for tumor volume changes. In our study, UPSs tended to increase in size, whilst MLSs con- sistently decreased. The high frequency of myxoid liposarcomas (6 patients, 35%) in our cohort, which are known to decrease in size after treatment, may explain the higher frequency of tumor shrinkages that we observed. Authors such as Dickie and al., who considered that the cohort of tumors that in- creased in size predominantly consisted of UPS, have previously reported the increase in UPS size during treatment. Pitson and Magierowski et al. also compared MLS to UPS behavior.12,13 They ana- lyzed volume changes of 16 MLS and 16 UPS. The mean pretreatment and post treatment volume of the MLS was 415 and 199 cm3, respectively (P = <0.0001). The mean pretreatment and post treat- ment volume of the UPS was 264 and 273 cm3, respectively (p = 0.804). These studies confirmed that MLS decrease during RT, meanwhile UPS are stable or grow. Concurrent neoadjuvant chemotherapy also increased tumor volume changes. To the best of our knowledge, our study is the first to include patients treated with concurrent radio-chemother- apy RTCT. However, we did not observe a signifi- cant correlation between GTV changes and the ad- ministration of neoadjuvant CT. Most of our adaptive interventions occurred during the 3rd week of treatment, at a mean of 11 fractions, which is consistent with Abu-Hijlih and Dickie et al. In 2013, Betgen et al. proposed an optimal time point for adaptive intervention: af- ter week 1 for non-MLS patients and after week 3 for MLS patients.11 This is totally consistent with both the clinical and dosimetric variations that we observed over the whole treatment period, since we noted that significant volume changes occurred predominantly during the 3rd week for tumors that were shrinking whilst significant tu- mor volume increases appeared earlier, during the 1st week. Grade 2 or 3 UPS behavior seemed to be more difficult to anticipate, requiring specific at- tention during the first week of treatment. Despite all of these tumor volume variations, a reduction in PTV coverage was ultimately not a very frequent occurrence. Over the whole treat- ment period, only 3 patients presented with a de- crease in D95%PTV >5%, all were growing UPS tu- mors and these changes occurred during the first 2 weeks of treatment. In shrinking tumors, we did not observe any changes in PTV coverage >5%, or significant over- dosing of OARs. Our results are consistent with those of Dickie et al. Among their 8 patients who had a 2nd CT scan because of tumor shrinkage, no significant change in the mean dose to the PTV and no significant increase in the dose to the adja- cent bone were observed. In the 7 patients (41%) who were replanned ac- cording to our departmental protocol, only one patient presented with a significant decrease of D95%PTV. Our plan adaptation rate is higher than that reported in the literature: Abu-Hijlih et al. adapted the plan of 17% of their 23 patients, O’Sullivan et al. 15% of patients in the phase II IG-IMRT trial and Rick L. Hass et al. only 8%.6,14 There are several reasons for this. Firstly, some authors only re-planned tumors that were grow- ing whereas we also included the 3 tumors that shrank. Secondly, as already highlighted, we ob- served more volume variations and overall, more pronounced amplitudes in our cohort compared to previous studies, perhaps due to the chemo- therapy. Our institutional practice also required patients to be replanned based on clinical data, of- ten when axial or sagittal linear tumor dimensions changed by >1cm. In these cases, patients directly Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas514 benefited from a new planning CT with complete treatment plan adaptation, potentially resulting in more replanifications than in other centers where plan adaptation was performed only in cases of ef- fective PTV under-coverage.10 As plan adaptation is time-consuming, we tried to evaluate better clinical tools to identify patients that would benefit from plan adaptation. Since variations in axial and sagittal linear tumor di- mensions occurred later compared to variations in GTV, PTV and estimated GTV volumes, we found estimated GTV volume (which considers the tu- mor as a cylinder and is estimated based on only 2 dimensions; axial diameter and sagittal height) to be a sensitive clinical parameter, that is easy to calculate, allowing weekly volume changes to be evaluated on MVCT or CBCT. Results from our small series of patients may be useful to modify ART practices. We observed that tumors at high risk of volume increases (spe- cifically UPSs) might be expected to have a more substantial reduction in PTV coverage than others. For this type of tumor histology, we therefore sug- gest a detailed follow-up during the first 2 weeks of treatment, measuring axial and sagittal linear tumor dimensions, calculating an estimated GTV on repositioned imaging, and comparing this with the initial GTV estimate. Plan adaptation does not seem useful in shrink- ing tumors, due to the apparent absence of conse- quences on PTV or OAR coverage. However, the occurrence of 2 scar disunions in our cohort calls for caution. The lack of specific data on healthy soft tissue or skin doses prevented us from pursu- ing this analysis. Our study had several limitations, mainly its small series of patients. Then, limitations come from the ART procedure itself. Firstly, dedicated ART software cannot be easily accessed in the practice, due in part to its substantial cost. With the recent development of this technique, many more software options to address this shortfall are now emerging. Some of these use CBCT with appropri- ate contrasts for the delineation, most include elas- tic registration and automatic contour delineation. An innovative alternative, MRlinac®, uses 4D-MR imaging with minimal latency times allowing bet- ter visualization of soft tissue and on-line ART. Deep learning-based dose prediction, as recom- mended for offline plan adaptation, is considered an appropriate solution for real-time dose recon- structions.15,16 We used the PreciseART software (Accuray®), which allows ART to be performed on MVCT. Although the software has elastic registra- tion and automatic delineation capabilities, these techniques have not yet been validated and we opt- ed for rigid registration and predominantly manu- al delineation. Under these conditions, the practice of ART is time-consuming and rigid registration is known to be suboptimal for dose reconstruction studies. These limitations were recently described in the POP-ART study in which 177 centers from 40 countries responded to a questionnaire about their ART practices. ART was used by 61% of re- spondents; the plan adaptation decision was made “ad-hoc” (without protocol) in the vast majority of cases (69%) and was predominantly performed of- fline. Only 10% used MR imaging, which allowed daily online plan adaptation. Nineteen percent of respondents used their in-house software because commercially available software lacked func- tionality. In addition, only 4 centers in this study specifically adapted treatment to sarcomas, all of them, like us, by ad hoc off-line plan adaptation.17 Conclusions Variations in tumor volume are apparent dur- ing preoperative ESTS-RT, but their dosimetric consequences are rare and mostly affect patients with tumor volume increases. To identify patients at risk of significant variations in PTV coverage, special attention should be paid to grade 2 and 3 UPS patients during the first 2 weeks of treatment. Monitoring volume changes by calculating an esti- mated GTV volume in addition to monitoring axial and sagittal linear tumor dimensions throughout radiation therapy may prove to be a good ap- proach for detecting any significant reductions in PTV coverage. References 1. Beane JD, Yang JC, White D, Steinberg SM, Rosenberg SA, Rudloff U. Efficacy of adjuvant radiation therapy in the treatment of STS of the extremity: 20-year follow-up of a randomized prospective trial. Ann Surg Oncol 2014; 21: 2484-9. doi: 10.1245/s10434-014-3732-4 2. O’Sullivan B, Davis AM, Turcotte R, Bell R, Catton C, Chabot P, et al. Preoperative versus postoperative radiotherapy in STS of the limbs: a randomised trial. Lancet 2002; 359: 2235-41. doi: 10.1016/S0140- 6736(02)09292-9 3. Davis AM, O’Sullivan B, Turcotte R, Bell R, Catton C, Chabot P, et al. Late radiation morbidity following randomization to preoperative versus post- operative radiotherapy in extremity soft tissue sarcoma. Radiother Oncol 2005; 75: 48-53. doi: 10.1016/j.radonc.2004.12.020 4. Le Péchoux C, Moureau-Zabotto L, Llacer C, Ducassou A, Sargos P, Sunyach MP, et al. RECORAD 2016: Radiothérapie des sarcomes des tissus mous de l’adulte. Cancer Radiother 2016; 20S: S235-43. doi: 10.1016/j.can- rad.2016.07.028 Radiol Oncol 2023; 57(4): 507-515. De Lamarliere MG et al. / Strategy of adaptive radiotherapy for extremity sarcomas 515 5. Wang D, Bosch W, Roberge D, Finkelstein SE, Petersen I, Haddock M, et al. RTOG Sarcoma Working Group Consensus on the GTV and CTV for pre- operative RT. Int J Radiation Oncology Biol Phys 2011; 81: e525–e528. doi: 10.1016/j.ijrobp.2011.04.038 6. Haas RL, van Beek S, Betgen A, Ali S, Schneider CJ, Diddens FH, et al. Substantial volume changes and plan adaptations during preoperative ra- diotherapy in extremity soft tissue sarcoma patients. Practical Radiat Oncol 2019; 9: 115-122. doi: 10.1016/j.prro.2018.11.001 7. Mc Nair H, Buijs M. Image guided radiotherapy moving towards real time adaptive radiotherapy; global positioning system for radiotherapy? Tech Innov Patient Support Radiat Oncol 2019; 12: 1-2. doi: 10.1016/j. tipsro.2019.10.006 8. Stankiewicz M, Li W, Rosewall T, Tadic T, Dickie C, Velec M. Patterns of practice of adaptive re-planning for anatomic variances during cone-beam CT guided radiotherapy. Tech Innov Patient Support Radiat Oncol 2019; 12: 50-5. doi: 10.1016/j.tipsro.2019.10.003 9. Abu-Hijlih R, Mheid S, Abuhijla F, Asha W, Mohamad I, Alrashdan A, et al. Adaptive radiotherapy in patients receiving neoadjuvant radiation for soft tissue sarcoma. Rep Pract Oncol Radiother 2019; 24: 263-8. doi: 10.1016/j. rpor.2019.02.007 10. Dickie C, Parent A, GriffinAM, Wunder J, Ferguson P, Chung PW, et al. The value of adaptive preoperative radiotherapy in management of soft tissue sarcoma. Radiother Oncol 2017; 122: 458-63. doi: 10.1016/j.ra- donc.2016.12.028 11. Betgen A, Haas RLM, Sonke JJ. Volume changes in soft tissue sarcomas dur- ing preoperative radiotherapy of extremities evaluated using cone-beam CT. J Radiat Oncol 2013; 2: 55-62. doi: 10.1007/s13566-012-0085-0 12. Pitson G, Robinson P, Wilke D, Kandel RA, White L, Griffin AM, et al. Radiation response: an additional unique signature of myxoide lipo- sarcoma. Int J Radiat Oncol Biol Phys 2004; 60: 522-6. doi: 10.1016/j. ijrobp.2004.03.009 13. Magierowski K, Moseley J, Lockwood G, Parent A, Euler C, Sharpe M, et al. Retrospective study of volume changes in two pathological subtypes of sarcomas using deformation image registration. Int J Radiat Oncol Biol Phys 2008; 72: S504. doi: https://doi.org/10.1016/j.ijrobp.2008.06.1471 14. O’Sullivan B, Griffin AM, Dickie CI, Sharpe MB, Chung PWM, Catton CN, et al. Phase 2 study of preoperative image-guided intensity-modulated radia- tion therapy to reduce wound and combined modality morbidities in lower extremity soft tissue sarcoma. Cancer 2013; 119: 1878-84. doi: 10.1002/ cncr.27951 15. Thorwarth D, Low DA. Technical challenges of real-time adaptive MR- guided radiotherapy. Front Oncol 2021; 11: 634507. doi: 10.3389/ fonc.2021.634507 16. Chen X, Men K, Li Y, Yi J, Dai J. A feasibility study on an automated method to generate patient-specific dose distributions for radiotherapy using deep learning. Med Phys 2019; 46: 56-64. doi: 10.1002/mp.13262 17. Bertholet J, Anastasi G, Noble D, Bel A, van Leeuven R, Roggen T, et al. Patterns of practice for adaptive and real-time radiation therapy (POP-ART RT) part II: Offline and online plan adaption for interfractional changes. Radiother Oncol 2020; 153: 88-96. doi: 10.1016/j.radonc.2020.06.017 Radiol Oncol 2023; 57(4): 516-523. doi: 10.2478/raon-2023-0050 516 research article Dosimetric comparison of postoperative interstitial high-dose-rate brachytherapy and modern external beam radiotherapy modalities in tongue and floor of the mouth tumours in terms of doses to critical organs Örs Ferenczi1, Tibor Major1,2,3, Georgina Fröhlich1,4, Dalma Béla1, Szabolcs Tódor1, Csaba Polgár1,2,3, Hironori Akiyama5, Botond Bukovszky1,2,6, Zoltán Takácsi-Nagy1,2,3 1 Centre of Radiotherapy, National Institute of Oncology, Budapest, Hungary 2 Department of Oncology, Semmelweis University, Budapest, Hungary 3 National Tumour Biology Laboratory, National Institute of Oncology, Budapest, Hungary 4 Eötvös Loránd University, Faculty of Science, Budapest, Hungary 5 Department of Oral Radiology, Osaka Dental University, Osaka, Japan 6 Department of Oral Diagnostics, Semmelweis University, Budapest, Hungary Radiol Oncol 2023; 57(4): 516-523. Received 19 May 2023 Accepted 23 August 2023 Correspondence to: Örs Ferenczi M.D., National Institute of Oncology, Centre of Radiotherapy, 1122 Budapest, Ráth György St. 7-9., Hungary. E-mail: ferenczi.ors@oncol.hu 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 dosimetrically compare interstitial high-dose-rate (HDR) brachytherapy (BT) and modern external beam radiotherapy modalities, as volumetric modulated arc therapy (VMAT) and stereotactic radiotherapy with Cyberknife (CK) of tumours of the tongue and floor of the mouth in terms of dose to the critical organs. Patients and methods. In National Institute of Oncology, Budapest, between March 2013 and August 2022 twenty patients (11 male/9 female) with stage T1–3N0M0 tongue (n = 14) and floor of mouth (n = 6) tumours received post- operative radiotherapy because of close/positive surgical margin and/or lymphovascular and/or perineural invasion. High-dose-rate interstitial brachytherapy applying flexible plastic catheters with a total dose of 15 x 3 Gy was used for treatment. In addition to BT plans VMAT and stereotactic CK plans were also made in all cases, using the same fractionation scheme and dose prescription. As for the organs at risk, the doses to the mandible, the ipsilateral and the contralateral salivary glands were compared. Results. The mean volume of the planning target volume (PTV) was 12.5 cm3, 26.5 cm3 and 17.5 cm3 in BT, VMAT and CK techniques, respectively, due to different safety margin protocols. The dose to the mandible was the most favour- able with BT, as for the salivary glands (parotid and submandibular) the CK technique resulted in the lowest dose. The highest dose to the critical organs was observed with the VMAT technique. The mean values of D2cm3 and D0.1cm3 for the critical organs were as follows for BT, VMAT and CK plans: 47.4% and 73.9%, 92.2% and 101.8%, 68.4% and 92.3% for the mandible, 4.8% and 6.7%, 7.3% and 13.8%, 2.3% and 5.1% for the ipsilateral parotid gland, 3.5% and 4.9%, 6.8% and 10.9%, 1.5% and 3.3% for the contralateral parotid gland, 7.3% and 9.4%, 9.0% and 14.3%, 3.6% and 5.6% for the contralateral submandibular gland. Conclusions. The present results confirm that BT, despite being an invasive technique, is dosimetrically clearly ben- eficial in the treatment of oral cavity tumours and is a modality worth considering when applying radiotherapy, not only as definitive treatment, but also postoperatively. The use of the CK in the head and neck region requires further investigation. Key words: floor of mouth tumour; tongue tumour; HDR; brachytherapy; VMAT; stereotactic; dosimetry; cyberknife Radiol Oncol 2023; 57(4): 516-523. Ferenczi Ö et al. / Dosimetry of tumours of the tongue and floor of the mouth Ferenczi Ö et al. 517 Introduction Surgery is usually the primary treatment for ad- vanced tumours of the oral cavity, including the tongue and the floor of the mouth, but smaller lesions can also be treated with laser resection, cryotherapy, external beam radiotherapy (EBRT) or brachytherapy (BT). The latter is particularly important in the treatment of early-stage oral ma- lignant lesions.1-4 If surgery is performed for small tumours (T1–2), postoperative irradiation may be necessary based on the surgical histology (close or positive surgical margins, lymphovascular or perineural invasion).5,6 If the neck staging shows no regional metastasis and the depth of tumour invasion is less than 5 mm, treatment of the neck with either dissection or RT is not necessary.7,8 In such early-stage lesions, postoperative radiothera- py can be performed using either percutaneous or interstitial RT. The aim of RT is to administer the maximum dose to the target volume (tumour bed). However, with external RT unnecessary radia- tion exposure to the surrounding critical organs (salivary glands, mandible, spinal cord, etc.) may result, thereby increasing the incidence of side ef- fects (xerostomia, osteoradionecrosis, fibrosis, tris- mus, etc.). Today, the state-of-the-art irradiation modal- ity routinely used is intensity modulated radia- tion therapy (IMRT), where a multileaf collimator (MLC) allows accurate tracking of the three-di- mensional (3D) shape of the target volume using a reference isodose surface, while significantly re- ducing exposure of critical organs.9,10 An improved version of this is rotating-arc intensity modulated radiotherapy or volumetric modulated arc therapy (VMAT), which allows even more precise irradia- tion of very complex target volumes (e.g. head and neck tumour regions) while further reducing the dose burden on the tissues to be protected.11,12 The current flagship of stereotactic RT is the Cyberknife (CK) technique. The treatment aims to deliver the highest possible dose to the tumour us- ing many non-coplanar beams. At the same time, the surrounding healthy tissue receives a relative- ly low dose and remains intact as the beams are scattered in a 3D geometry.13,14 The tumour bed can also be treated with inter- stitial BT for oral cavity tumours, if indicated.2,15,16 The treatment involves introducing radioactive isotope(s) into the tissue of the target volume by direct implantation (seed) or by applicators (rigid metal needles, flexible plastic catheters). BT al- lows a higher dose to be delivered locally and provides greater protection to surrounding intact tissue due to the rapid dose fall-off around the source. Alongside the long-established low dose rate (LDR) BT, the increasingly widely used high dose-rate (HDR) method can produce the same therapeutic results, but while the former requires better patient cooperation due to the need for isola- tion and longer treatment times, the latter method eliminates these problems.17,18 In the current model study, we compared HDR postoperative BT plans of 20 patients treated with tongue and floor of mouth cancer with VMAT and CK treatment plans in terms of dosimetry of the organs at risk (OARs). Patients and methods At National Institute of Oncology, Budapest, be- tween January 2016 and December 2021, 20 patients (T1–3N0) underwent tumour extirpation and uni- lateral (85%, 17/20) or bilateral (15%, 3/20), selective neck dissection for tongue or sublingual cancers following negative neck staging. Histology did not confirm metastatic lymph node. For local postop- erative BT to be justified, one of the following cri- teria had to be met: T3 tumour, surgical margin ≤ 2 mm, lymphovascular infiltration or perineural invasion. Based on the processing of histopathol- ogy, 20% had T3 size (TNM 8th)19, 85% had a sur- gical margin of ≤ 2 mm and 40% had perineural spread. The treatments were performed with an HDR afterloading device using Iridium-192 iso- tope (Flexitron, Elekta Brachytherapy, Veenendaal, The Netherlands) after implantation of flexible catheters (median 6, range 6–8) into the tumour bed. The insertion was performed via submental penetration by the help of trocars, in the operating room, under general anaesthesia. The mean time between interstitial BT (implantation) and surgery was 54 days (range: 42–81 days). Brachytherapy planning After catheter implantation, all patients under- went CT imaging with 3 mm slice thickness cover- ing the whole head including the tumour bed, the parotids and submandibular gland. In all cases, BT planning with Oncentra Brachy v4.5.3 (Elekta Brachytherapy, Veenendaal, The Netherlands) was performed. The total dose of BT was 45 Gy. 3 Gy per fraction was delivered twice daily, 6 hours apart. Imaging of the primary tumour (CT, mag- netic resonance imaging [MRI]) and palpation of Radiol Oncol 2023; 57(4): 516-523. Ferenczi Ö et al. / Dosimetry of tumours of the tongue and floor of the mouth518 the surgical site helped to determine the target volume (clinical target volume [CTV]: tumour bed [gross tumor volume, GTV] + 0.5 cm safety mar- gin). There was no safety margin around the CTV, so the planning target volume (PTV) was equal to the CTV. The ipsilateral (il.) and contralateral (cl.) parotids and cl. submandibular salivary gland, the skin and the mandible were contoured as organs to be protected. Skin was defined as a layer of 0.5 cm below the outer body surface. Source dwell positions and dose reference points were deter- mined individually for each implant. Geometric and graphical dose optimization was performed. The isodose line for dose prescription was chosen to achieve 90% dose coverage of the PTV (V100 = 90%). The BT planning was based on the recom- mendations of GEC-ESTRO (Groupe Européen de Curiethérapie and the European Society for Radiotherapy and Oncology) Head and Neck Working Group. VMAT planning To prepare the external beam RT plan, the CT im- ages of the patients were exported to the external planning system (Eclipse v11, Varian, USA) com- plying with the DICOM (Digital Imaging and Communications in Medicine) RT protocol to- gether with the structure set defined in BT plans, and subsequently the IMAT plans were prepared. This method ensured that the target volume and the organs to be protected were always exactly the same in the two planning systems, thus eliminat- ing inaccuracies due to contouring. From this it also follows that the differences obtained in the comparison were due solely to the differences be- tween the two irradiation techniques and were not influenced by other factors. For the IMAT plans, the CTV was extended by 3 mm in each direction to create the PTV. The VMAT plans were created using 6 MV photon energy. VMAT plans were op- timized using the Varian RapidArc progressive resolution optimization algorithm (PRO) and the dose was calculated using the analytical aniso- tropic algorithm (AAA). After dose normalization the coverage of the PTV by the prescribed dose (PD) was 90% (V100 = 90%). Cyberknife planning In order to prepare the stereotactic plans, the CT images and the RT structures (Radiotherapy Structure Set) were transferred from the Eclipse system to the Accuray Precision (Accuray, Sunnyvale, CA, USA) version 3.1.0.0. planning sys- tem. The PTV used for stereotaxic plans was creat- ed by extending the brachytherapy CTV symmet- rically with 2 mm. The Cyberknife plans were gen- erated using the multileaf collimator system, 6MV FFF photon energy using the VOLO optimizer for dose optimization and the FSPB (Finite Size Pencil Beam) for dose calculation. The dose prescription was chosen to achieve V100 = 90% for the PTV. Comparison of the plans The same dose prescription and fractionation (15 x 3 Gy) were used for all three techniques. Parameters calculated from dose volume histo- gram were used to compare the plans. To describe the target coverage, the volume of the PTV irra- diated by the PD was used (V100). The objective comparison was based on the same target cover- age, V100 = 90%, for all three techniques. It follows from this that any differences found between the plans were only due to the characteristics of the irradiation techniques. The conformity of dose distributions was quantified using the conformal index (COIN), which takes into account both the target coverage and the unnecessary irradiation of normal tissues.20 Its maximum value is 1, and the higher the value, the more conformal the dose distribution. Dose homogeneity was characterized with the dose nonuniformity ratio (DNR) in BT plans, and homogeneity index (HI) in the VMAT and CK plans. DNR is the ratio of volume irradi- ated by 1.5 times the PD to volume irradiated by the PD. The HI was calculated according to recom- mendation of ICRU (International Commission on Radiation Units and Measurements) Report 83.21 By definition, HI = (D2%-D98%)/D50%. To charac- terize the unintended irradiation of OARs, small volumes of high dose were used. Dxcm3 represents the minimum dose to the most exposed x cm3 of an organ (mandible, parotid). For all OARs mean D2cm3 and D0.1cm3 were calculated and compared. Friedman ANOVA and Fisher-LSD (Least Significant Difference) post-hoc tests were used (Statistica 12.5, StatSoft, Tulsa, OK, USA) to com- pare dose volume parameters of VMAT, CK and HDR BT techniques. The level of significance was 0.05. Results Due to the same dose prescription (V100 = 90%) the mean target volume dose coverage in all modali- Radiol Oncol 2023; 57(4): 516-523. Ferenczi Ö et al. / Dosimetry of tumours of the tongue and floor of the mouth Ferenczi Ö et al. 519 ties was 90.0%. Figure 1 shows representative dose distributions for the three investigated techniques. It can be seen that the target was irradiated prop- erly in each case, but notable differences can be observed for the volumes irradiated by doses cor- responding to middle and lower isodose values (< 70%). In the BT plan, these volumes are the small- est, especially in regions near the target volume. Table 1 shows the dosimetric data for PTV. Due to the safety margins used in VMAT and CK plans, the largest volume was for VMAT and the small- est for BT. The plans were more conformal with EBRT compared to BT. The most conformal plans occurred with CK, probably due to the lots of non- coplanar beams. However, the VMAT plans were more homogeneous than the CK plans (HI: 0.09 vs. 0.20). It is obvious, that with BT the homogeneity is much worse, and the comparison with EBRT is meaningless. Table 2 shows the quantitative dosi- metric parameters for the OARs. The dose to the mandible was the lowest with the use of BT (mean D2cm3: 47.4% p < 0,001) compared to the other mo- dalities: VMAT (92.2%) and CK (68.4%). Regarding the salivary glands, the CK technique resulted in the lowest dose on both the ipsilateral and con- tralateral sides (il. parotid gland, cl. parotid gland, and cl. submandibular gland - CK mean D2cm3: 2.3% (p < 0,001), 1.5% (p < 0,001), 3.6% (p < 0,001) vs. BT: 4.8%, 3.5%, 7.3% vs. VMAT: 7.3%, 6.8%, 9.0%) (Table 1). Similar results were obtained by compar- ing the values of D0.1cm3. The data in Table 2 clearly show that out of the three techniques VMAT re- sulted in the highest doses to the protected organs. Figures 2 and 3 graphically show the comparisons of D2cm3 for the mandible and for the il. parotid gland. Discussion The comparison of new radiotherapy technologies in the head and neck region has recently become a very interesting area of research. In this study, we performed a dosimetric analysis of 20 cases re- quiring exclusive postoperative irradiation of the tumour bed. The analysis allowed us to compare our BT planning with VMAT and CK techniques for the same target volume, with special attention to the doses to OARs. In a recent review paper BT was dosimetrically compared to modern EBRT techniques for various cancer types.22 Although other author used more fractions with the same dose per fraction (18x3 Gy), we have been using 15x3 Gy fractionation in exclusive postoperative BT since 2014, in line with international recom- mendations, and our experience so far is that it is well tolerated by patients, with no grade 4 toxic- ity.2,15,23,24 It was shown that from a dosimetric point of view, BT can compete with even the most ad- vanced EBRT techniques, in respect of a higher dose centrally within the target volume and spar- ing adjacent OARs. However, only a few publica- tions are available in the literature that compare dose-volume parameters of critical organs using BT or other RT modalities. Sresty et al.25 compared plans of image guided HDR-BT and IMRT for mobile tongue cancer and FIGURE 1. Representative dose distributions in a brachytherapy (BT), a volumetric modulated arc therapy (VMAT) and a Cyberknife (CK) plan. Radiol Oncol 2023; 57(4): 516-523. Ferenczi Ö et al. / Dosimetry of tumours of the tongue and floor of the mouth520 found a very good dose conformity in image guided BT (IGBT), which was almost the same as in IMRT, but the dose to the critical structures was lower in BT in all of the cases. Yoshida et al.26 were the first, who reported dose volume histogram analysis of HDR BT for mobile tongue cancer in 2014. In their five patients - applying image-based planning - the mean V100(CTV), the mean D0.1cm3(mandible) and D2cm3(mandible) were 98.1%, 80.1% and 55.7%, respectively. Yoshida’s results were supported by the work of Akiyama et al.27 published in 2018. This study is considered to include the largest number of patients in this respect. The study was designed to present dosimetric comparison of IGBT with VMAT for head and neck cancer regarding con- formity of dose distribution to PTV and doses to the OARs. Thirty-eight consecutive patients with T1-4 mobile tongue, floor of mouth and base of tongue cancer treated with IGBT were selected. For these patients additional VMAT treatment plans were also prepared using identical CT data. V100 was superior with IGBT (89.0% vs. 76.7%, p < 0.05). Significantly lower values were obtained with IGBT to OARs compared with VMAT (man- dible: D0.1cm3 77.0 vs. 85.4, D2cm3 48.4 vs. 68.4, p < 0.05; il. parotid gland: D0.1cm3 9.1% vs. 13.8%, D2cm3 7%, vs. 10.5%, p < 0.05; cl. parotid gland: D0.1cm3 8.9% vs. 15.3%, D2cm3 4.9% vs. 9.1%, p < 0.05; cl. subman- dibular gland: D0.1cm3 13.4% vs. 29.7%, D2cm3 8.1% vs. 18.3%, p < 0.05). The results prove the superiority of IGBT in the protection of OARs and the impor- tant role of this invasive method in the era of new external beam techniques. Similarly, we have cur- rently achieved favourable results with IGBT com- pared to VMAT in respect of the protection of criti- cal organs. Akiyama and colleagues used the same PTV for BT and VMAT, but we used the extension usually applied for external irradiation (CTV + 3 mm), so the volume of mean PTV for BT was small- er (VPTV 12.5 cm3 vs. 26.5 cm3, p < 0.001), which is also an advantage of this technique, as it is more suitable for protecting the surrounding intact tis- sue. Of the three techniques, the most conformal dose distributions were obtained with CK (COIN = 0.86), but in contrast, homogeneity was better with VMAT (HI = 0.09). For BT, the conformality was in- ferior compared to the EBRT, but its advantage was in lower doses to mandible. Osteoradionecrosis (ORN) of the mandible is one of the most dreaded complications of head and neck irradiation. The incidence of ORN has decreased in recent times, from approximately 20% (several decades ago) to 4–8% (in the mod- ern era). This tendency might be attributed to im- provements in RT techniques, such as the IMRT currently used.28 Peterson et al.29 clarified the im- pact of cancer therapies on the prevalence of ORN FIGURE 2. Mean dose in % to the most exposed 2 cm3 volume of the mandible. BT = brachytherapy; CK = Cyberknife; VMAT = volumetric modulated arc therapy TABLE 1. Mean dosimetric parameters of planning target volume (PTV) with ranges BT VMAT CK p-value* BT vs. VMAT** BT vs. CK** VMAT vs. CK** VPTV (cm3) 12.5 (2.6-21.5) 26.5 (7.7-42.6) 17.5 (5.6-33.6) < 0.001 < 0.001 0.5553 0.0043 Conformal index 0.62 (0.48-0.80) 0.84 (0.78-0.87) 0.86 (0.79-0.93) < 0.001 < 0.001 < 0.001 0.5480 Homogenity index DNR = 0.38(0.30-0.50) 0.09 (0.05 - 0.10) 0.20 (0.17-0.20) NA NA NA < 0.001 * = Friedman ANOVA test; ** = LSD post hoc test BT = brachytherapy; COIN = conformal index; CK = Cyberknife; DNR = dose non-uniformity ratio; NA = not available; PTV = planning target volume; VMAT = volumetric modulated arc therapy; VPTV = volume of the planning target volume Radiol Oncol 2023; 57(4): 516-523. Ferenczi Ö et al. / Dosimetry of tumours of the tongue and floor of the mouth Ferenczi Ö et al. 521 TABLE 2. Mean dosimetric parameters of organs at risk (OARs) with ranges BT VMAT CK p-value* BT vs. VMAT** BT vs. CK** VMAT vs. CK** Mandible D2 (%) 47.4(29.2–73.4) 92.2 (73.1–100.4) 68.4 (39.3–87.3) < 0.001 < 0.001 < 0.001 < 0.001 D0.1 (%) 73.9(1.7–93.9) 101.8 (97.1–103.9) 92.3 (72.7–100.7) < 0.001 < 0.001 < 0.001 NS Ipsilateral parotid gland D2 (%) 4.8(2.5–11.9) 7.3 (0.9–13.9) 2.3 (0.3–6.7) < 0.001 0.0011 NS < 0.001 D0.1 (%) 6.7(3.5–19.0) 13.8 (3.7–25.0) 5.1 (0.3–12.3) < 0.001 < 0.001 NS < 0.001 Contralateral parotid gland D2 (%) 3.(0.0–7.6) 6.8 (0.6–15.8) 1.5 (0.0–4.7) < 0.001 0.0018 NS < 0.001 D0.1 (%) 4.9(0.0–11.9) 10.9 (0.9–20.2) 3.3 (0.3–14.0) < 0.001 0.0105 NS 0.0020 Contralateral submandibular gland D2 (%) 7.3(3.9–16.3) 9.0 (0.8–17.7) 3.6 (2.0–6.0) 0.0098 NS 0.0198 0.0016 D0.1 (%) 9.4(6.2–21.4) 14.3 (2.1–23.1) 5.6 (3.0-11.3) 0.0098 NS 0.0146 < 0.001 * = Friedman ANOVA test; ** = LSD post hoc test BT = brachytherapy; CK = Cyberknife; DX = dose to the most exposed X cm3 volume; NS = non-significant; VMAT = volumetric modulated arc therapy based on 43 articles published between 1990 and 2008. The weighted prevalence for ORN were 7.4%, 5.1%, 6.8% and 5.3% with conventional RT, IMRT, chemoradiotherapy and BT, respectively. Our re- sults show that the mandible is better protected with BT than with VMAT. Stereotactic radiotherapy with Cyberknife is an attractive option because it delivers a highly con- formal dose in a small number of fractions (like BT), with steep dose gradients resulting in reduced normal tissue irradiation and with a short overall treatment time. It can be an efficacious treatment option for recurrent previously irradiated head and neck carcinoma, especially for nonresectable tumours, or in elderly and medically unfit patients. However, in head and neck (oral cavity) tumours with negative lymph node status where definitive local RT is recommended, or in postoperative care where neck RT is not necessary, it has been consid- ered as a therapeutic option, but currently only in the form of clinical trial.30,31 The STEREO POSTOP GORTEC 2017-03 trial is a non-randomised phase II trial, the first prospective study to investigate postoperative stereotactic body radiation therapy (SBRT) for head and neck cancers in early-stage oropharyngeal and oral cancers with high-risk surgical margins. In SBRT a total dose of 36 Gy is delivered in 6 fractions over 2 weeks. The primary endpoint is severe late toxicity, with secondary endpoints including acute toxicity, local and lo- coregional control, disease-free and overall sur- vival, and quality of life, with a planned end date of January 2024.32 Stereotactic contouring protocols are very heterogeneous, but generally 1-5 mm for GTV-CTV extension and 2-3 mm for CTV-PTV ex- tension in head and neck cancers.33 In our analysis 2 mm for CTV-PTV extension was used. Zhang Y et al.34 investigated the feasibility of lar- ynx SBRT therapy planning on a conventional gan- try-based linac and compared its plan quality with that made by the Cyberknife on an anthropomor- phic head and neck phantom. This study revealed that a gantry-based linear accelerator can achieve FIGURE 3. Mean dose to the most exposed 2 cm3 volume of the ipsilateral parotid. BT = brachytherapy; CK = Cyberknife; VMAT = volumetric modulated arc therapy Radiol Oncol 2023; 57(4): 516-523. Ferenczi Ö et al. / Dosimetry of tumours of the tongue and floor of the mouth522 similar dosimetric endpoints as the Cyberknife, by employing non-coplanar VMAT arcs. According to the current study, the CK technic was inferior to BT regarding mandibular Dx (p < 0.001), despite giving better results than VMAT for OARs, while it outperformed BT for the cl. submandibular salivary gland (D2cm3: p = 0.0198, D0.1cm3: p = 0.0146). Although the parotid glands are important for salivary secretion, as they provide 70% of the sa- liva, their relatively large distance from the target volume means that they are not affected by ra- diation exposure during oral cancer irradiation, which otherwise causes xerostomia.35 One of the limitations of our study is that while VMAT and BT techniques are routinely used in the treatment of oral tumours in our department, we have no experience with CK therapy in this set- ting, the other limitation being that this is a do- simetric comparison without discussing clinical consequences. In the future, it would be interest- ing to study the side effects and survival param- eters when these different radiotherapy modalities are used side by side in the postoperative treat- ment of oral cavity tumour beds. Conclusions All three irradiation techniques studied resulted in adequate dose distribution in postoperative RT for tongue and floor of mouth cancer. While the doses to small volumes of the mandible was less with BT, in terms of salivary glands, the CK technique resulted in the lowest dose. The high- est dose to the critical organs was observed using the VMAT technique. The above results confirm that BT, despite being an invasive technique, is dosimetrically clearly beneficial in the treatment of oral cavity tumours and is a modality worth considering when applying radiotherapy, not only definitively, but also postoperatively. The role of the CK technique for radiotherapy in the head and neck region appears promising, but requires fur- ther investigation. Acknowledgments The project was implemented with the sup- port from the National Research, Development and Innovation Fund of the Ministry of Culture and Innovation under the National Laboratories Program (National Tumour Biology Laboratory (2022-2.1.1-NL-2022-00010)) and the Hungarian Thematic Excellence Program (under project TKP2021-EGA-44) Grant Agreements with the National Research, Development and Innovation Office. References 1. Strnad V. Treatment of oral cavity and oropharyngeal cancer. Indications, technical aspects, and results of interstitial brachytherapy. Strahlenther Onkol 2004; 180: 710-17. doi: 10.1007/s00066-004-9196-x 2. Ferenczi Ö, Major T, Akiyama H, Fröhlich G, Oberna F, Révész M, et al. Results of postoperative interstitial brachytherapy of resectable floor of mouth tu- mors. Brachytherapy 2021; 20: 376-82. doi: 10.1016/j.brachy.2020.10.008 3. Kovács G, Martinez-Monge R, Budrukkar A, Jose Luis G, Bengt J, Vratislav S, et al. 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A multicenter prospective phase II study of postoperative hypofraction- ated stereotactic body radiotherapy (SBRT) in the treatment of early-stage oropharyngeal and oral cavity cancers with high risk margins: the STEREO POSTOP GORTEC 2017-03 trial. BMC Cancer 2020; 20: 730. doi: 10.1186/ s12885-020-07231-3 33. Kim MS, Malik NH, Chen H, Poon I, Husain Z, Eskander A, et al. Stereotactic radiotherapy as planned boost after definitive radiotherapy for head and neck cancers: Systematic review. Head and Neck 2022; 44: 770-82. doi: 10.1002/hed.26948 34. Zhang Y, Chiu T, Dubas J, Tian Z, Lee P, Gu X, et al. Benchmarking techniques for stereotactic body radiotherapy for early-stage glottic laryngeal cancer: LINAC-based non-coplanar VMAT vs. Cyberknife planning. Radiat Oncol 2019; 14: 193. doi: 10.1186/s13014-019-1404-z 35. Owosho AA, Thor M, Oh JH, Riaz N, Tsai CJ, Rosenberg H, et al. The role of parotid gland irradiation in the development of severe hyposalivation (xerostomia) after intensity-modulated radiation therapy for head and neck cancer: Temporal patterns, risk factors, and testing the QUANTEC guidelines. J Craniomaxillofac Surg 2017; 45: 595-600. doi: 10.1016/j.jcms.2017.01.020 Radiol Oncol 2023; 57(4): 524-529. doi: 10.2478/raon-2023-0060 524 research article Phase angle as a prognostic indicator of surgical outcomes in patients with gastrointestinal cancer Jana Gulin1, Ester Ipavic1, Denis Mlakar Mastnak2, Erik Brecelj2, Ibrahim Edhemovic2, Nada Rotovnik Kozjek2,3 1 Medical Centre Ljubljana, Ljubljana, Slovenia 2 Institute of Oncology Ljubljana, Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia Radiol Oncol 2023; 57(4): 524-529. Received 3 August 2023 Accepted 30 September 2023 Correspondence to: Asst. Prof. Nada Rotovnik Kozjek, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: nkozjek1@gmail.com. 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. In patients with gastrointestinal cancer with planned elective surgery, malnutrition increases the risk of adverse outcomes in the postoperative period. The phase angle, measured by the bioelectrical impedance analysis is an indicator of the metabolic and functional status of the patient. It may be an important prognostic indicator for the clinical outcome of post-surgical treatment in patients with gastrointestinal cancer. Patients and methods. In this prospective study, 70 patients with gastrointestinal cancer had their phase angles measured by the bioelectrical impedance analysis before the surgery. During the first month after the surgery, we documented the postoperative complications from the patient’s records and classified them according to the Clavien Dindo classification of surgical complications. The time of hospitalization was also recorded. The data was statistically analysed in SPSS. Results. We found a statistically significant difference (p = 0.036) in the average value of phase angles between the group of patients who had postoperative complications (phase angle 5.09°) and the group without postoperative complications (5.64°). We noted a correlating trend of decreasing phase angle values and increasing hospitalization time (Pe R = –0,40, p = 0,001). The phase angle cut-off value (5.5°) was calculated using the ROC curve method, pre- dicting a higher risk of the postoperative complications (p = 0,037) in patients with lower phase angle. Conclusions. Lower phase angle values before surgery were associated with more complications during the first month after surgery and longer hospitalization time. We found that a phase angle below than 5.5° could serve as a marker that predicts a greater risk of postoperative complications. Key words: phase angle; colorectal cancer; postoperative complications; malnutrition Introduction Bioelectric Impedance Analysis (BIA) is a tech- nique used to assess body composition and is be- coming increasingly established as a tool to assess nutritional status in patients due to its ease of use, low cost, and non-invasiveness.1,2 BIA does not di- rectly measure the body composition, but instead measures the drop in voltage of an alternating electric current, as it travels across the body. The phase angle is the quotient of measured resistance and reactance.1-4 It is interpreted as an indicator of membrane integrity and water distribution be- tween the intracellular and extracellular spaces. Radiol Oncol 2023; 57(4): 524-529. Gulin J et al. / Phase angle as a prognostic indicator of surgical outcome 525 Phase Angle is used to evaluate body cell mass, which serves as a tool to assess nutritional status in adults and children. Lower phase angle sug- gests cell degradation with a concomitant decrease in overall cell number with reduced integrity and functional capacity of cell membranes. On the oth- er hand, higher phase angle points to the presence of more functional, intact cell membranes.5-9 Malnutrition is a common manifestation of ad- vanced stage cancer and can severely affect the pa- tient morbidity and mortality.10 On a cellular level malnutrition is reflected by the diminished integ- rity of the cell membranes and by altered water distribution throughout the body.11 Body compo- sition analysis is therefore an essential tool in as- sessing nutritional status in cancer patients.12 The clinical role of measuring the patient’s phase angle is becoming progressively more important. BIA- derived phase angle can help establish guidelines for preventive and supportive measures in patients with malnutrition, as it allows for early identifica- tion of high-risk patients with inadequate physi- ological reserves. The method has previously been shown to have prognostic value in patients with liver cirrhosis, haemodialysis, cancer, HIV/AIDS infection, and lung disease.5,13-18 For example, pa- tients with stage III or IV colorectal cancer who had a phase angle less than or equal to 5.57° had a median survival of 8.6 months, while those who had a phase angle greater than 5.57° had a median survival of 40.4 months.14 The primary objective of this study was to de- termine if the phase angle can be useful as an inde- pendent predictor of post-surgical complications in gastrointestinal cancer patients. Patients and methods Setting and patients Our study was a prospective observational study that was performed at the Institute of Oncology Ljubljana, Slovenia, between November 2018 and February 2019. During the study period, BIA was performed on every gastrointestinal cancer patient over 18 years of age that was admitted for elective surgery at the Institute of Oncology Ljubljana and agreed to participate in the study. Exclusion criteria were pregnancy or an implanted pacemaker. The study protocol was approved by the Ministry of Health Medical Ethics Committee and the Institute of Oncology (No. 0120-518/2018/7). Every included patient was fully informed of the study design and signed a written informed consent form. Bioelectrical Impedance Analysis (BIA) and other measurements BIA was performed with a Bodystat (R) Quadscan 4000 machine (Douglas, UK). This phase-sensitive BIA device uses an alternating current of 0.8 mA with frequencies of 5, 50, 100, and 2000 kHz to measure the body impedance. The BIA-derived phase angle was calculated from the impedance at 100 kHz. Every patient had their phase angle measured the day before surgery and then a week and a month after the surgery. The patients were in- structed to abstain from eating and drinking for at least 4 hours prior to the measurement and to abstain from any physical exercise for 8 hours pri- or to the measurement as well. Two pairs of elec- trodes were placed on the dorsal side of their right hand and right foot, and they were instructed to lie still in a supine position with no parts of the body touching one another. Clinical data, includ- ing gender, age, the exact location of malignant disease, complications in the postoperative course, and hospitalization time were obtained from the hospital information system. Clavien Dindo Classification of surgical complications The patients were categorized in different grades of the Clavien Dindo classification of surgical complications (revised 2004).19 The classification uses the degree of most severe pharmacological or surgical treatment needed after surgery, to dis- tinguish between the grades of post-surgical com- plications. A normal postoperative course without any complications (use of analgesics is considered as normal postoperative course) is classified as de- gree 0. Degree 1 allows the use of antiemetics, an- tipyretics, potent analgesics, diuretics, physiother- apy, and treatment of wound infections. Degree 2 additionally includes the use of other drugs, blood transfusion, and total parenteral nutrition. Degree 3 allows surgical, endoscopic, or radiological inter- ventions. Degree 4 includes life-threatening com- plications that require treatment in an intensive care setting. Fatal complications are classified as a degree 5. Statistics Numeric variables are expressed in terms of their mean values and a 95% confidence interval. Discrete variables are expressed as percentages. Radiol Oncol 2023; 57(4): 524-529. Gulin J et al. / Phase angle as a prognostic indicator of surgical outcome526 The Shapiro-Wilk test was used to check wheth- er the data is normally distributed. The limit for statistical significance was set at p < 0.05. Logistic regression was used to test whether the initial pre- operative phase angle, as an independent variable, impacts the odds of post-surgical complications. The results are expressed as quotients. A cut-off value for the phase angle as a predictive factor for post-surgical complications was estimated with a ROC curve, and its sensitivity and specificity were calculated. The statistical analysis was done with the IBM SPSS 23.0 statistical program. Results During the study period (between November 2018 and February 2019), BIA analysis was performed on every gastrointestinal cancer patient planned for elective surgery at the Institute of Oncology Ljubljana that agreed to participate in the study. Seventy patients were recruited. Characteristics of the included patients (column 1), of patients with- out any complications (column 2) and patients with complications (column 3) are presented in Table 1. The mean age of all patients was 65.0 ± 10.7 years, and 71% were male. Most patients were admitted because of rectal cancer (64.3%). Mean phase angle of all patients was 5.23° ± 2.77°. In total, 52 (74,3%) patients had a complication (Clavien Dindo grade 1-5) after surgery (Table 1 and Table 2). Phase angle and the likelihood of complications The phase angle of patients without a complication was significantly higher than that of the patients with a complication (5.64° ± 0.72° vs. 5.09° ± 0.98°, p = 0.036). Univariate logistic regression analysis showed that the phase angle was associated with the likelihood of a complication (phase angle: odds ratio = 0.48). The odds of a complication in a patient with a phase angle of 5.0 was 3.83, whereas it was only 1.84 in a patient with a phase angle of 6.0. The probabilities of the occurrence of a complication at different phase angles were calculated and are shown in Table 3. The area under the curve (AUC) of the ROC curve for phase angle for the likeli- TABLE 1. Characteristics of patients Characteristics All patients Patients with nocomplications (G0) Patients with complications (G1–G5) p-value Gender Male 50 (71 %) Female 20 (29 %) Age (years)a 65.0 (10.7) [62.4–67.6] 61.7 (9.8) [56.9–66.6] 66.1 (10.9) [63.1–69.2] 0.137 Phase Angle (°)a 5.23 (2.77) [5.0–5.5] 5.64 (0.72) [5.3–6.0] 5.09 (0.98) [4.8–5.4] 0.036* Location of the primary tumor (%) Colon 17 (24,3) 4 (22.2) 13 (25.0) Rectosigmoid 2 (2,9) 1 (5.5) 1 (1.9) Rectum 45 (64,3) 11 (61.1) 34 (65.4) Anus 1 (1,4) 1 (5.5) 0 (0) Breast 1 (1,4) 0 (0) 1 (1.9) Ovary 4 (5,7) 1 (5.5) 3 (5.8) a Arithmetic mean (standard deviation) [confidence interval]. * Statistically significant p-value TABLE 2. Mean age and mean phase angle in each Clavien Dindo grade Number of patients (%) Age (years) a Phase angle (°) a Grade 0 18 (26) 61.7 [56.9–66.6] 5.64 [5.28–6.00] Grade I 5 (7) 56.0 [50.2–61.8] 6.22 [5.64–6.80] Grade II 38 (54) 67.1 [63.4–70.7] 4.99 [4.67–5.31] Grade III 4 (6) 62.2 [45.0–79.5] 5.15 [3.41–6.88] Grade IV 5 (7) 72.2 [60.5–83.9] 4.64 [3.77–5.51] Grade V 0 (0) a Arithmetic mean (standard deviation) [95% confidence interval]. Radiol Oncol 2023; 57(4): 524-529. Gulin J et al. / Phase angle as a prognostic indicator of surgical outcome 527 6.0°, the odds of a complication are reduced to 1.84, and the likelihood of a complication is reduced to 64.8%. We are not aware of any prospective studies in- vestigating the role of phase angle as a prognostic indicator of post-surgical complications in colorec- tal cancer patients. The retrospective data analysis from 210 elderly patients (aged ≥ 65 years) under- going gastrectomy showed that preoperative low phase angle predicts a higher risk of postoperative complications.20 Similar observations were made in a study among patients admitted to the ICU, where phase angle at admission was shown to be a predictor of 90-day mortality. The mean phase angle of survivors was significantly higher than that of the non-survivor group (5.0° ± 1.3° versus 4.1° ± 1.2°, p < 0.001).21 In a study that compared outcomes after cardiothoracic surgery between a group of low phase angle (< 5.38°) patients and a group of normal phase angle patients, the partici- pants from the first group had a higher number of post-operative infections, a larger percentage of them were ventilated for more than 12 hours, and had higher rates of mortality. However, after considering other parameters like sex, age, the type of operative procedure, risk assessment, in- flammation activity, hypoalbuminemia, heart fail- ure, time of cardiopulmonary bypass, and time of aortic cross-clamp, the phase angle was found not to be statistically significant in correlation with aforementioned complications. Still, the difference between the groups stayed statistically significant in regard to the hospitalization time and the time spent in the intensive care unit.22 FIGURE 1. The ROC curve for the phase angle. TABLE 3. Odds for developing a complication and probabilities of developing a complication at different values of phase angle Phase Angle (°) Odds of a complication Probability of a complication (%) 4.5 5.532 84.7 5.0 3.831 79.3 5.5 2.651 72.6 6.0 1.837 64.8 TABLE 4. Contingency Table with the sensitivity and a specificity of the phase angle Phase angle < 5,5 Phase angle ≥ 5,5 Complication 33 (true positive) 19 (false negative) 52 No complication 7 (false positive) 11 (true negative) 18 40 30 hood of a complication is 0.666 (CI: 0.529 - 0.803, p = 0.037), see Figure 1. The determination coefficient (Nagelkerke R square) is 0.104, which means that our model explains 10.4% of the complication like- lihood variance. The Hosmer and Lemeshow test value for the model is 0.766. The optimal cut-off value for the phase angle The cut-off value of phase angle that was derived from the ROC-curve was 5.5° (Figure 1). It yielded a sensitivity of 63% and a specificity of 61% (Table 4). The likelihood of a complication was higher in pa- tients with phase angle below 5.5° than in the pa- tients with phase angle above that value (82.5% vs. 63.3%, Figure 1). Discussion The patients with a lower initial phase angle were shown to have a statistically significant higher likelihood of post-surgical complications. The mean phase angle in the group with complications (5,64°) was statistically higher than the mean phase angle in the group without complications (5,09°). We used univariate logistic regression to cal- culate the likelihood of a complication at different initial phase angle values. At a phase angle of 5.0°, the odds of a complication are 3.83, and the likeli- hood of a complication is 79.3%. At a phase angle of Radiol Oncol 2023; 57(4): 524-529. Gulin J et al. / Phase angle as a prognostic indicator of surgical outcome528 In addition, we aimed to find a cut-off phase an- gle value that can be used to identify high-risk pa- tients that are more likely to have a severe compli- cation after gastrointestinal cancer surgery. Using 5.5° as a cut-off value, we were able to successfully identify these patients in in 67% of the cases with a sensitivity of 63% and a specificity of 61%. Different phase angle cut-off values have ap- peared in literature to identify patients with lower physiological reserves who are at risk for increased morbidity and mortality. The proposed PA values in literature are 5.5° for patients newly admitted to the hospital23, 4.73° for patients newly diagnosed with head and neck cancer24, and 5.2° for patients with various cancers before starting radiother- apy25. The phase angle values put forward as a predictor of survival were 4.5° for patients with a non-small cell lung cancer15, 5.0° for patients with advanced pancreatic cancer26, 5.4° for patients with liver cirrhosis5, and 5.57° for patients with colon and rectum cancer14. One of the limitations of this study is a relative- ly heterogeneous group of patients. The patients included in our study had cancer in different loca- tions and stages in abdomen and they were under- going different treatment protocols. Additionally, our analysis only took into account the pre-opera- tive value of the phase angle. The patients’ phase angle might have changed in the peri-operative period, especially on account of nutritional sup- port or further medical interventions. Therefore, the value might not have been representative of the patients overall physical state within the entire observed period. The obtained cut-off phase angle value of 5.5° needs to be considered as a tentative value as it was calculated using a specific sample in one population. Further research is needed to identify the cut-off value for different subtypes of colorectal cancer and to evaluate the validity of our obtained cut-off value in distinct clinical set- tings. The primary clinical implication of this study is that the phase angle measurement can assist in identifying GI cancer patients with a higher risk of post-operative complications. This could benefit patients that would otherwise not have been iden- tified. Further research is needed to investigate if nutritional or other medical interventions can significantly alter the phase angle and thus affect surgical outcomes. References 1. Maggiore Q, Nigrelli S, Ciccarelli C, Grimaldi C, Rossi GA, Michelassi C. Nutritional and prognostic correlates of bioimpedance indexes in hemodi- alysis patients. Kidney Int 1996; 50: 2103-8. doi: 10.1038/ki.1996.535 2. Baumgartner RN, Chumlea WC, Roche AF. Bioelectric impedance phase angle and body composition. Am J Clin Nutr 1988; 48: 16-23. doi: 10.1093/ ajcn/48.1.16 3. Kyle UG, Bosaeus I, De Lorenzo AD, Deurenberg P, Elia M, Gómez JM, et al. Bioelectrical impedance analysis - Part I: review of principles and methods. 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The bioelectrical impedance phase angle as an indi- cator of undernutrition and adverse clinical outcome in cardiac surgical patients. Clin Nutr 2012; 31: 981-6. doi: 10.1016/j.clnu.2012.05.002 23. Kyle UG, Soundar EP, Genton L, Pichard C. Can phase angle determined by bioelectrical impedance analysis assess nutritional risk? A comparison between healthy and hospitalized subjects. Clin Nutr 2012; 31: 875-81. doi: 10.1016/j.clnu.2012.04.002 24. Małecka-Massalska T, Mlak R, Smolen A, Morshed K. Bioelectrical imped- ance phase angle and subjective global assessment in detecting malnutri- tion among newly diagnosed head and neck cancer patients. Eur Arch Otorhinolaryngol 2016; 273: 1299-305. doi: 10.1007/s00405-015-3626-5 25. Motta RST, Castanho IA, Velarde LGC. Cutoff point of the phase angle in pre- radiotherapy cancer patients. Nutr Hosp 2015; 32: 2253-60. doi: 10.3305/ nh.2015.32.5.9626 26. Gupta D, Lis CG, Dahlk SL, Vashi PG, Grutsch JF, Lammersfeld CA. Bioelectrical impedance phase angle as a prognostic indicator in advanced pancreatic cancer. Br J Nutr 2004; 92: 957-62. doi: 10.1079/BJN20041292 Radiol Oncol 2023; 57(4): 530-537. doi: 10.2478/raon-2023-0053 530 research article Correlation between maximum heart distance and thoracic diameter changes and diaphragmatic descent in left-sided breast cancer patients during deep inspiration breath- hold (DIBH) He-Gou Wu, Guang-Wei Zhang, Jian-Feng Liu, Jun-Guo Yang, Xiao-Hui Su Department of Radiation Oncology, Shenzhen People’s Hospital, The Second Clinical Medical College of Jinan University, Shenzhen, Guangdong, People’s Republic of China Radiol Oncol 2023; 57(4): 530-537. Received 13 April 2023 Accepted 28 August 2023 Correspondence to: Dr. He-Gou Wu, Department of Radiation Oncology, Shenzhen People’s Hospital, The Second Clinical Medical College of Jinan University, Dongmenbei Road 1017, Shenzhen, 518000, Guangdong, People’s Republic of China. E-mail: 55143240@qq.com Data availability statement: The data that support the findings of this study are available from the corresponding author, [He-Gou Wu, ORCID: 0000-0002-7050-0209], upon reasonable request. Disclosure: No potential conflicts of interest were disclosed. The authors declare that no funds, grants, or other support were received dur- ing the preparation of this manuscript. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Cardioprotection is valued in radiotherapy for patients with left-sided breast cancer. Deep inspiration breath-hold (DIBH) technique can achieve cardioprotection well. However, during DIBH, the extent to which the heart enters the radiation field is affected by the movement of the thorax and diaphragm. The aim of this study was to analyze the correlation between the maximum distance of the heart entering the field (maximum heart distance, MHD) and thoracic diameter changes and diaphragmatic descent in left-sided breast cancer patients during DIBH. Patients and methods. Ninety-eight patients with left-sided breast cancer were included in this retrospective study. They performed simulation in Sentinel-guided DIBH, and two sets of CT images were collected under both free breath- ing (FB) and DIBH, and diaphragm positions, anteroposterior thoracic diameter (ATD), transverse thoracic diameter (TTD), gating window level (GWL), and MHD were measured, and the change (Δ) of each parameter in DIBH relative to that in FB were calculated. Pearson or Spearman test were used to analyze the correlation between ΔMHD and the changes in other parameters. Results. For all patients with DIBH, the average of ΔMHD was -8.3 mm, and the average of ΔATD and ΔTTD were 11.0 and 8.6 mm, and the median of both left diaphragmatic descent (LDD) and right diaphragmatic descent (RDD) were 35.0 mm, and the median of GWL was 11.1 mm. The correlation coefficients between MHD decrease (ΔMHD) and LDD, RDD, and ΔTTD were -0.430 (p = 0.000), -0.592 (p = 0.000) and 0.208 (p = 0.040), respectively, but not significantly correlated with ΔATD or GWL. Conclusions. The MHD decrease showed a moderate correlation with diaphragmatic descent In Sentinel-guided DIBH for patients with left-sided breast cancer, while there was a weak or no correlation with thoracic diameter changes or GWL. Abdominal breathing can lower diaphragm more and may be more beneficial to the heart stay away from tangential field. Key words: left-sided breast cancer; maximum heart distance; thoracic diameter; diaphragmatic descent; deep inspiration breath-hold Radiol Oncol 2023; 57(4): 530-537. Wu HG et al. / Deep inspiration breath-hold and cardioprotection 531 Background In radiotherapy for left-sided breast cancer, it is necessary to face issues such as heart protection, lung protection, and the influence of respiratory motion, etc. As an important organ adjacent to the breast, the heart has received special attention in radiotherapy for left-sided breast cancer. Studies have shown that the increase in the dose to the heart has a linear relationship with the increase in major coronary events1. Therefore, in clinical practice of radiotherapy for left-sided breast can- cer, cardioprotection has increasingly become the focus of attention. Deep inspiration breath-hold (DIBH) technology can increase the distance be- tween the heart and the chest wall, keep the heart away from the center of the radiation field, and reduce the volume of the heart entering the ra- diation field2, thereby achieving the purpose of protecting the heart. Many studies have shown that the use of DIBH technology in radiotherapy for left-sided breast cancer can effectively reduce the dose to the heart3-7. Other studies have shown a strong correlation between the maximum dis- tance of the heart into the tangential field (maxi- mum heart distance, MHD) and cardiac dose re- duction8-10. Therefore, to a certain extent, MHD can reflect the level of cardiac exposure, and can even be used as a predictor of the average cardiac exposure dose10, so it has a positive effect on clini- cal work to find out which factors affect MHD. However, there are few reports on the relation- ship between the extent of the heart entering the radiation field (MHD size) and the movement of the thorax and diaphragm during DIBH. In this article, we will study the correlation between MHD change and thoracic diameter changes and diaphragm descent, and reveal the main factors affecting MHD, so as to provide reference for the clinical practice of radiotherapy in DIBH for left- sided breast cancer. Patients and methods Ninety-eight patients with left-sided breast cancer (age range 26 to 64 years, median age 44.0 years) were included in this retrospective study. They received radiotherapy in DIBH in our center from January 2020 to May 2022. This study was per- formed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Shenzhen People’s Hospital (Date 2021-2-24/No LL-KY-2021047). Written informed consent was waved because this was a retrospec- tive study. CT simulation All patients in this study were immobilized by vac- uum bags in supine position with both arms lifted up. CT simulation was performed under the guid- ing of Sentinel which is a CT-end of the CatalystTM system--an optical surface guided system (C-RAD AB, Uppsala, Sweden)11. Before CT simulation, all patients underwent Sentinel-guided DIBH breath- ing training under the guidance of doctors. No uniform requirement was made for breathing pattern during DIBH, and each patient chose his most comfortable breathing pattern, which can be thoracic breathing, abdominal breathing or mixed breathing. The cone-beam CT (CBCT) scan before delivered usually takes more than 40 seconds, in order to meet the scanning duration of CBCT, the patient’s DIBH duration was required to be ≥ 40 s. Sentinel detects patient’s breathing through a gat- ing point, which is a region with a diameter of 4 cm located on the patient’s skin surface, and Sentinel monitors the spatial position of the gating point by emitting a laser (wavelength: 635-690 nm, fre- quency: 15 Hz) to monitor the breath-holding state. In this study, the gating point was placed at 1-2 cm superior to the xiphoid process. Sentinel uses the gating window to set the allowable range of chest rise and fall during breath holding, the lower limit of the allowable range is “LOW”, the upper limit of the allowable range is “HIGH”, and the distance between “HIGH” and “LOW” is the width of the gating window. In this study, the width of the gat- ing window was set to 3 mm, and the gating win- dow level (distance from Baseline to “LOW”, GWL) is not uniformly required, but the GWL should ex- ceed the amplitude of free breathing (FB). Sentinel is equipped with goggles, which provide visual feedback for patients during breathing training or CT scanning. Two sets of CT images (CTfb, CTdibh) were acquired for each patient using SOMATOM Definition AS (SIEMENS Healthcare, Germany) under FB and DIBH. Scanning parameters were 120 KV, 140 mAs (CARE Dose4D was selected), and the slice thickness was 5 mm. Patient coordinate system and respiratory characteristic parameters measurement In order to evaluate the size of the heart enter- ing the radiation field in patients with DIBH, we Radiol Oncol 2023; 57(4): 530-537. Wu HG et al. / Deep inspiration breath-hold and cardioprotection532 refer to the measurement method of Kenneth Wikström12, and take the maximum distance of the heart edge into the simulated tangential field (MHD) as an evaluation parameter. The position of MHD is usually at the level of the right diaphrag- matic top, so we chose the level of the right dia- phragmatic top in FB as the location for the simu- lated measurement. On the CTfb and CTdibh, we measured the thoracic diameters and MHD in the FB and DIBH, respectively (Figure 1). DICOM im- age viewing software CARESTREAM Vue PACS (Version 12.2.0.0314, Carestream Health Inc., USA) were used to measure the parameters. The win- dow width during measurement is 1600, and the window level is -599. First, on the CTfb images, we construct a line from the center of the sternum (A) to the center of the spinal cord cavity (B), which is the anteroposterior thoracic diameter (ATDfb). Draw a perpendicular to AB at the midpoint of AB, and the vertical line reaches the right edge of the lung (C) and the left edge of the lung (D). CD is the transverse thoracic diameter (TTDfb). Connect the two points A and D, and measure the maximum distance from the edge of the heart to AD as the maximum heart distance (MHDfb). Second, using the above method, on the CTdibh image, which is equivalent to the same level of vertebral body height corresponding to the right diaphragmatic top in FB, the anteroposterior tho- racic diameter (ATDdibh), transverse thoracic di- ameter (TTDdibh), and maximum heart distance (MHDdibh) were measured. Respiratory characteristic parameters calculation The change of each parameter reflected the change of DIBH state relative to FB state. Accordingly, this study uses the difference of parameters in the two states for evaluation. For each patient, the param- eter difference was calculated as follows: First, ATDfb was subtracted from ATDdibh to obtain the increase in ATD (ΔATD) in DIBH. Second, sub- tract TTDfb from TTDdibh to obtain the increase in TTD (ΔTTD) in DIBH. Third, MHDfb was sub- tracted from MHDdibh to obtain the reduction in MHD in DIBH (ΔMHD, It’s usually a negative num- ber). Fourth, measure the downward displacement of the diaphragm in DIBH: left diaphragmatic de- scent (LDD), right diaphragmatic descent (RDD). On CT images of FB and DIBH, take the position of the vertebral body corresponding to the level of the diaphragm dome in FB as the reference, and the vertical descending distance of the diaphragm dome in DIBH relative to the reference is LDD or RDD. Fifth, in Sentinel gating window, the “LOW” minus the “Baseline” is used as the gating window level (GWL). Dosimetric parameters According to the RTOG guidelines, target volumes and organs at risk (OARs) were contoured on Eclipse (version 13.6, Varian Medical Systems, Palo Alto, CA). 3D conformal radiotherapy (3DCRT) plans were generated on both the DIBH and FB simulation CT images for dosimetric analysis. Each patient prescribed 40.5 Gy in 15 fractions for whole breast and chest wall. Four dosimetric parameters were obtained, including mean heart dose (Dmean), heart volume in field (HVIF), which is the heart volume contained in the 50% isodose line, percentage volumes of the heart receiving doses ≥ 10Gy (V10), and percentage volumes of the heart receiving doses ≥ 5Gy (V5). Then, subtract the value of a parameter in FB from the value of this parameter in DIBH to obtain each parameter difference (ΔDmean, ΔHVIF, ΔV10, ΔV5). FIGURE 1. Parameter measurement diagram. For each patient, at the height of the vertebral body corresponding to the right diaphragm top during free breathing, anteroposterior thoracic diameter (ATD), transverse thoracic diameter (TTD) and maximum heart distance (MHD) were measured on free breathing CT images and deep inspiration breath-hold CT images respectively. Point A at the center of the sternum, and point B at the center of the medulla, the line AB was constructed as the ATD. Through the center of the line AB at M, a second line was constructed perpendicular to AB reaching the lateral edge of the lung at C and D, and the line CD was the TTD. The line AD approximates the direction of the tangential field, and the maximum distance from the left edge of the heart to AD is taken as the MHD. Radiol Oncol 2023; 57(4): 530-537. Wu HG et al. / Deep inspiration breath-hold and cardioprotection 533 Data analysis This study analyzed the correlation between change in MHD and changes in other parameters. After calculating geometric parameters (ΔMHD, ΔATD, ΔTTD, RDD, LDD, GWL) and dosimetric parameters (ΔDmean, ΔHVIF, ΔV10, ΔV5), statis- tical analysis was performed using SPSS 25 (IBM Corp.). For normal data, the mean (± standard deviation) was used to describe, and Pearson cor- relation coefficient test was used for correlation analysis; for non-normal data, median (range) was used to describe and Spearman correlation coeffi- cient test was used for correlation analysis. P < 0.05 means the difference is statistically significant. Result Respiratory characteristic parameters and GWL The measured values and calculation results of each respiratory characteristic parameter are shown in Table 1. Among the 98 patients, 91 pa- tients (92.9%) had MHD reduction to varying degrees, the largest reduction in MHD was -22.2 mm (ΔMHD), and 7 patients (7.1%) had increased MHD in DIBH (ΔMHD were 3.75, 1.29, 1.26, 0.93, 0.68, 0.49, 0.10 mm, respectively). Of these patients with increased MHD in DIBH, in addition to 2 who had pleural effusion and prosthesis implantation respectively, in the other 5 patients with increased MHD, the largest increase in MHD was 1.26 mm. Moreover, during DIBH, one patient had no change in left diaphragm (LDD 0 mm), and there was a patient who had no descending in the right diaphragm (RDD 0mm), and another one who had a decrease in the TTD (ΔTTD -2 mm). Correlation between ΔMHD and thoracic diameter changes and diaphragmatic descent In this study, ΔMHD showed a moderate correla- tion with RDD and LDD (Figure 2), the r-values were -0.592 (p = 0.000), -0.430 (p = 0.000). ΔMHD was weakly correlated with ΔTTD, the r-value was 0.208 (p = 0.040), while it was no significant correlation with ΔATD. Since there was only a weak correlation between ΔMHD and ΔTTD, we grouped ΔATD and ΔTTD according to the mean. For all patients, the mean of ΔATD was 11.0 mm, and the mean of ΔTTD was 8.6 mm. Patients were divided into high and low groups according to the means, which were ΔATDhigh (ΔATD > = 11.0 mm, 52 patients), ΔATDlow (ΔATD < 11.0 mm, 46 pa- tients), ΔTTDhigh (ΔTTD > = 8.6 mm, 47 patients), ΔTTDlow (ΔTTD < 8.6 mm, 51 patients), the mean of parameters in each group are shown in Table 2. Statistical analysis showed that there was a statis- tical difference in ΔMHD between the ΔATDhigh and ΔATDlow groups (t = 2.179, p = 0.032), and FIGURE 2. Correlation between ΔMHD and left diaphragmatic descent, right diaphragmatic descent [r: Spearman rank correlation coefficient; p: p-value (2-tailed)]. ΔMHD = maximum heart distance in deep inspiration breath-hold minus maximum heart distance in free breathing Radiol Oncol 2023; 57(4): 530-537. Wu HG et al. / Deep inspiration breath-hold and cardioprotection534 that ΔMHD was correlated with ΔATD in the ΔATDlow group (r = -0.372, p = 0.011), but not in the ΔATDhigh group. Correspondingly, the means of ΔMHD in ΔTTDhigh and ΔTTDlow were also statistically different (Z = 1.966, p = 0.049), while ΔMHD was not correlated with ΔTTD in the two groups. Correlation between MHD and GWL Using Sentinel to guide DIBH in patients with left- sided breast cancer, the degree of chest expansion is reflected by GWL. In this study, the patient’s breathing pattern was not specified, and each patient chose a comfortable breathing pattern to achieve DIBH. As a result, the GWL of the patients varied relatively widely, ranging from 4.6 mm to 20.7 mm. However, it was found that there was no significant correlation between ΔMHD and GWL. Furthermore, the patients were divided into three groups according to GWL: Group 1: < 9 mm (24 pa- tients), Group 2: ≥ 9 mm and < 15 mm (63 patients), Group 3: ≥ 15 mm (11 patients), no significant cor- relation between GWL and ΔMHD was found in each group, and no statistical difference was found in the ΔMHD among the three groups. Dosimetric parameters and their correlation with geometric parameters In 95 of the 98 patients (96.9%), Dmean, HVIF, V10, and V5 were all reduced to varying degrees in DIBH compared with FB, with a maximum reduction of 590.1 cGy, 15.7%, 19.7%, and 21.7%, respectively, and the median of Dmean, ΔHVIF, ΔV10, and ΔV5 were: -176.1 cGy, -3.981%, -4.764%, -5.444%, respectively. Meanwhile, the dosimetric parameters showed moderate correlation with the geometric parameters ΔMHD, RDD, and LDD, as shown in Table 3. Discussion In this article, the correlation between ΔMHD and the thoracic diameter changes and diaphragmatic descent was studied. Ninety-eight patients with left-sided breast cancer were simulated in Sentinel- guided DIBH. Their MHD, thoracic diameters, and diaphragmatic descent were measured on CTfb and CTdibh. It was found that the change of MHD were moderately correlated with the left and right diaphragmatic descent [–0.430 (p = 0.000), -0.592 (p = 0.000)], while the correlation with thoracic diame- ter changes was weak, only weakly correlated with ΔTTD (r = 0.208), and not correlated with ΔATD. This shows that in DIBH, the greater descending of the diaphragm, the smaller the MHD, and the change of thoracic diameters does not seem to have a significant effect on MHD. Meanwhile, it was also found that RDD and LDD were moder- ately correlated with ΔDmean, ΔHVIF, ΔV10 and ΔV5 through cardiac dose analysis (Table 3), which indicated that the greater the diaphragmatic de- scent, the greater the decrease in Dmean, HVIF, V10, V5. Several previous studies have shown that MHD is associated or strongly associated with car- diac dose8,9,10,13,14. Similar results were obtained in this study, ΔMHD and ΔDmean showed moderate correlation (Table 3). However, thoracic diameter changes were only weakly or not linearly associat- TABLE 1. The values of respiratory characteristic parameters in free breathing (FB) and/or deep inspiration breath holding (DIBH), and the differences in the values of some respiratory characteristic parameters between DIBH and FB (N = 98) Parameters FB DIBH Δ MHD (mm) 26.8 (±5.1) 18.4(±7.5) -8.3 (±5.9) ATD (mm) 127. 0(±11.6) 139.0(±11.6) 11.0 (±3.8) TTD (mm) 227.0 (±10.6) 235.6(±10.4) 8.6 (±4.4) RDD (mm) 35.0(0.0 – 60.0) LDD (mm) 35.0(0.0 – 55.0) GWL (mm) 11.1(4.6 – 20.7) ATD = anteroposterior thoracic diameter; GWL = gating-window level; LDD = left diaphragmatic descent; MHD = maximum heart distance; RDD = right diaphragmatic descent; TTD = transverse thoracic diameter; Δ = differences between DIBH and FB TABLE 2. Comparison of the mean of parameters in ΔATDlow (n = 46) and ΔATDhigh (n = 52) and in ΔTTDlow (n = 51) and ΔTTDhigh (n = 47) LDD RDD ΔATD ΔTTD ΔMHD ΔATDlow (mm) 32.2 34.0 7.7 7.5 -7.0 ΔATDhigh (mm) 34.2 35.4 13.8 9.6 -9.5 Increase of ΔATDhigh (mm) 2.0 1.4 6.1 2.1 -2.5 Increase of ΔATDhigh (%) 6.2% 4.1% 78.9% 27.9% 36.5% ΔTTDlow(mm) 35.3 37.1 10.5 5.4 -9.7 ΔTTDhigh (mm) 31.1 32.2 11.4 12.0 -6.9 Increase of ΔTTDhigh (mm) -4.2 -4.9 0.8 6.6 2.8 Increase of ΔTTDhigh (%) -12.0% -13.2% 7.9% 122.2% -29.3% ATD = anteroposterior thoracic diameter: ΔATDlow = group with ΔATD < 11 mm; ΔATDhigh = group with ΔATD > =11 mm; ΔTTDlow = group with ΔTTD < 8.6 mm; TTD = transverse thoracic diameter; ΔTTDhigh = group with ΔTTD > = 8.6 mm Radiol Oncol 2023; 57(4): 530-537. Wu HG et al. / Deep inspiration breath-hold and cardioprotection 535 ed with MHD, and were not associated with heart dosimetric parameters. For those patients with large thoracic diameter changes, is there a corre- lation between ΔMHD and ΔATD or ΔTTD? The result was also negative. In this study, 52 patients had ΔATD greater than the mean (11.0 mm), and 47 patients had ΔTTD greater than the mean (8.6 mm). However, there was still no significant correlation between their ΔMHD and ΔATD or ΔTTD. This may be because the expansion of the chest wall is anisotropic, it is difficult to expand uniformly in all directions, and it is difficult to show a linear change with the MHD even in the case of a greater degree of the chest wall expansion. In addition, in the case of further inspiration, the change of dia- phragm position was also greater, which may also have influenced the correlation of MHD with chest wall expansion. The reduction of MHD by DIBH is usually caused by the combination of two fac- tors. One is that diaphragm descending elongates the heart in the craniocaudal direction, reducing the transverse diameter of heart, thus keeping the heart away from the radiation field, and the other is that the lungs are more inflated in DIBH, keep- ing the chest wall away from the heart. The reason for the results of this study may be that the for- mer has a more lasting effect on reducing MHD than the latter, because the movement range of the diaphragm was larger than that of the chest wall (Table 1). In DIBH, after the chest wall has ex- panded to a certain extent, the diaphragm can still descend further and continue to have an effect on the reduction of MHD, so that a linear change may be formed between the diaphragmatic descent and the reduction of MHD. In addition, as mentioned earlier, the expansion of the chest cavity is aniso- tropic, which makes it difficult to form a relatively regular change in the distance between the heart and the chest wall, so the reduction of MHD is not easy to show a linear relationship with the thoracic diameters. Although there was weak or no linear correla- tion between MHD and thoracic diameters, differ- ent thoracic diameters would result in statistical differences in ΔMHD. According to the thoracic diameter grouping data, it was found that there were statistical differences in ΔMHD between the thoracic diameters high and low groups. MHD was smaller in ΔATDhigh group than in ΔATDlow group (ΔMHD mean -9.5 vs -7.0 mm), which meant that the hearts of patients in ΔATDhigh group were farther away from the tangent field, while the opposite was true in the grouping of ΔTTD, where MHD is greater in ΔTTDhigh than in ΔTTDlow (ΔMHD mean -6.9 vs -9.7 mm), thus it can be seen that the ΔMHD of patients with ΔATD greater than the mean benefited from the increase of ATD, but this was not the case for patients with ΔTTD greater than the mean, and the increase in TTD did not contribute further to the heart moving away from the radiation field. Further comparison of the diaphragmatic descent between the high and low groups, it was found that the diaphrag- matic descent of ΔATDhigh group was slightly greater than that of the ΔATDlow group (LDD 34.2 vs 32.2 mm, RDD 35.4 vs 34.0 mm), but that of the ΔTTDhigh group was smaller than that of the ΔTTDlow group (LDD 31.1 vs 35.3 mm, RDD 32.2 vs 37.1 mm), indicating that after the TTD in- creases to a certain extent, a continued increase in TTD will lead to the diaphragm rising, which is not conducive to the heart away from the radiation field. Therefore, during DIBH training, it is neces- sary to choose a relatively large but not excessive inspiratory volume. In this way, the thoracic di- ameters and the diaphragmatic descent increase, which will help to keep the heart away from the field. When inappropriate inspiratory volume or inspiratory pattern leads to excessive expansion of the chest, it will negatively affect the heart away from the field. Although the majority of patients had varying degrees of reduction in MHD in DIBH, but in this study, there were still 7 patients (7.1%) with in- creased MHD (range 0.10 to 3.75 mm), obviously, TABLE 3. Correlation between dosimetric parameters and geometric parameters ΔDmean ΔHVIF ΔV10 ΔV5 ΔMHD r 0.468 0.464 0.481 0.484 p < 0.001 < 0.001 < 0.001 < 0.001 LDD r - 0.340 - 0.365 - 0.382 - 0.373 p = 0.001 < 0.001 < 0.001 < 0.001 RDD r - 0.439 - 0.449 - 0.462 - 0.458 p < 0.001 < 0.001 < 0.001 < 0.001 ΔATD r 0.007 0.044 0.026 0.008 p 0.943 0.669 0.801 0.935 ΔTTD r 0.012 0.055 0.062 0.065 P 0.906 0.593 0.545 0.527 ATD = anteroposterior thoracic diameter; Dmean = heart mean dose; HVIF = heart volume in field, that’s the heart volume within the 50% isodose line; LDD = left diaphragmatic descent; MHD = maximum heart distance; RDD = right diaphragmatic descent; TTD = transverse thoracic diameter; V5 = percentage volumes of heart receiving doses ≥ 5Gy; V10 = percentage volumes of heart receiving doses ≥ 10 Gy; Δ the value of a parameter in deep inspiration breath holding (DIBH) minus the value of this parameter in free breathing (FB) Radiol Oncol 2023; 57(4): 530-537. Wu HG et al. / Deep inspiration breath-hold and cardioprotection536 the ΔMHD of these patients did not benefit from DIBH technique. In 4 of the 7 patients, there were several factors that could affect DIBH, including pleural effusion, prosthesis implantation, left dia- phragm not descending during DIBH, and the right diaphragm did not descend during DIBH, while no obvious factors affecting DIBH were found in the other 3 patients. In addition, one patient’s right dia- phragm did not descend (RDD 0mm) in DIBH, but her TTD increased significantly (ΔTTD 14.5 mm), and left diaphragm descended (LDD 10.0 mm), and her MHD decreased accordingly (ΔMHD -3.44 mm). Similarly, another patient’s TTD decreased rather than increased in DIBH (ΔTTD -2 mm) and her ATD also increased less (ΔATD 4 mm), but her left and right diaphragms descended significantly (LDD 45 mm, RDD 50 mm), and MHD was also reduced accordingly (ΔMHD -5.86 mm). It can be seen from the above that in DIBH, there are various factors affecting the reduction of MHD, including local lesions, prosthesis implantation, respiratory patterns, etc. Tanguturi et al. found that the mean heart dose was increased in 14 of 146 patients (10%) in DIBH15. It shows that these patients did not ben- efit from DIBH technology. Dell’Oro et al. observed that in DIBH, 1 of 20 patients (5%) had increased MHD16, which was similar to what was observed in this study. DIBH reduces MHD by keeping the heart away from the radiation field, but in order to achieve effective DIBH, in addition to adequate breathing training, it is necessary to consider pos- sible affecting factors and to screen patients by op- timizing the conditions. Different breathing patterns have different ef- fects on the geometric changes of thoracic anato- my, which may also affect the extent to which the heart enters the field. Breathing patterns are gen- erally divided into thoracic breathing, abdominal breathing and mixed breathing. According to the study of Kimiko Hirata et al., In DIBH-guided ra- diotherapy for left-sided breast cancer, there was no statistical difference between thoracic breath- ing and abdominal breathing on the heart doses and the displacement between the heart and the target17. Zhao et al. evaluated T-DIBH and A-DIBH with three-dimensional conformal radiotherapy (3DCRT) and intensity-modulated radiation ther- apy (IMRT) in 22 patients18, their conclusion is that abdominal breathing significantly reduces the heart doses in both 3DCRT and IMRT. In this study, when the patient performed DIBH, the breathing patterns was not limited, and the patient may per- form any of the three breathing patterns, but it can be inferred from the moderate correlation between the ΔMHD and the diaphragmatic descent that abdominal breathing may be more beneficial for keeping the heart away from the field. When Sentinel guides DIBH of left-sided breast cancer patients, Sentinel relies on the gating win- dow level (GWL) to monitor chest wall, which en- sures the reproducibility of the chest in DIBH19. However, we found that there was no significant correlation between ΔMHD and GWL, and there was no significant difference in ΔMHD among the three GWL groups (< 9 mm, ≥ 9 mm and < 15 mm, ≥15 mm). In a previous study, Leigh Conroy et al. also found no correlation between the breath- hold level and left anterior descending artery chest wall separation20. Tuomas Koivumäki divided the GWL into two groups, low (7–12 mm, 8 patients) and high (13–20 mm, 7 patients), and the results showed that the heart position in the low GWL group had greater variability during treatment21. Therefore, even though the GWL has little correla- tion with the extent to which the heart enters the radiation field, it is still recommended to optimize the GWL to make the patient’s DIBH state more stable. A limitation of this study is that the difference in MHD between thoracic breathing and abdomi- nal breathing was not analyzed because patients’ breathing pattern could not be assigned due to the retrospective data analysis. Overall, the MHD decrease showed a moder- ate correlation with the diaphragmatic descent In Sentinel-guided DIBH for patients with left-sided breast cancer, while there was a weak or no cor- relation with thoracic diameter changes or GWL. Abdominal breathing can lower the diaphragm more and may be more beneficial to the heart stay away from the tangential field in clinical practice. References 1. Darby SC, Ewertz M, McGale P, Bennet AM, Blom-Goldman U, Brønnum D, et al. Risk of ischemic heart disease in women after radiotherapy for breast cancer. N Engl J Med 2013; 368: 987-98. doi: 10.1056/NEJMoa1209825 2. Stranzl H, Zurl B. Postoperative irradiation of left-sided breast cancer patients and cardiac toxicity. 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Evaluation of target and cardiac position during visually monitored deep inspiration breath-hold for breast radiotherapy. J Appl Clin Med Phys 2016; 17: 25-36. doi: 10.1120/jacmp.v17i4.6188 21. Koivumäki T, Tujunen J, Virén T, Heikkilä J, Seppälä J. Geometrical un- certainty of heart position in deepinspiration breath-hold radiotherapy of left-sided breast cancer patients. Acta Oncol 2017: 56: 879-83. doi: 10.1080/0284186X.2017.1298836 Radiol Oncol 2023; 57(4): 538-549. doi: 10.2478/raon-2023-0052 538 study protocol Post-radiation xerostomia therapy with allogeneic mesenchymal stromal stem cells in patients with head and neck cancer: study protocol for phase I clinical trial Primoz Strojan1,2, Gaber Plavc1,2, Marko Kokalj1, Goran Mitrovic1, Olga Blatnik1, Luka Lezaic2,3, Aljaz Socan3, Aljosa Bavec2, Natasa Tesic4, Katrina Hartman4, Urban Svajger4,5 1 Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 3 University Medical Centre Ljubljana, Department of Nuclear Medicine, Ljubljana, Slovenia 4 Blood Transfusion Center of Slovenia, Ljubljana, Slovenia 5 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Radiol Oncol 2023; 57(4): 538-549. Received 4 September 2023 Accepted 25 September 2023 Correspondence to: Prof. Primož Strojan, M.D., Ph.D., Institute of Oncology Ljubljana, Department of Radiation Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: pstrojan@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. Xerostomia is a common side effect of radiotherapy in patients with head and neck tumors that nega- tively affects quality of life. There is no known effective standard treatment for xerostomia. Here, we present the study protocol used to evaluate the safety and preliminary efficacy of allogeneic mesenchymal stromal stem cells (MSCs) derived from umbilical cord tissue. Patients and methods. Ten oropharyngeal cancer patients with post-radiation xerostomia and no evidence of disease recurrence 2 or more years after (chemo)irradiation (intervention group) and 10 healthy volunteers (control group) will be enrolled in this nonrandomized, open-label, phase I exploratory study. MSCs from umbilical cord tissue will be inserted under ultrasound guidance into both parotid glands and both submandibular glands of the patients. Toxicity of the procedure will be assessed according to CTCAE v5.0 criteria at days 0, 1, 5, 28, and 120. Efficacy will be assessed by measuring salivary flow and analyzing its composition, scintigraphic evaluation of MSC grafting, retention, and migration, and questionnaires measuring subjective xerostomia and quality of life. In addition, the radiological, functional, and morphological characteristics of the salivary tissue will be assessed before, at 4 weeks, and at 4 months after the procedure. In the control group subjects, only salivary flow rate and salivary composition will be determined. Discussion. The use of allogeneic MSCs from umbilical cord tissue represents an innovative approach for the treat- ment of xerostomia after radiation. Due to the noninvasive collection procedure, flexibility of cryobanking, and bio- logical advantages, xerostomia therapy using allogeneic MSCs from umbilical cord tissue may have an advantage over other similar therapies. Key words: oropharyngeal cancer; xerostomia; mesenchymal stromal stem cells Introduction Xerostomia in patients with head and neck cancer Radiotherapy (RT) is one of the main therapeutic modalities for head and neck cancer (HNC): ap- proximately 80% of patients diagnosed with this cancer undergo RT.1 Unfortunately, RT is associ- ated with significant toxicity in the head and neck region. While the acute side effects of RT usually occur within 90 days of treatment initiation, the toxic late effects can occur months or even years Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy 539 after treatment completion. Acute side effects occur most frequently in tissues with high pro- liferation activity, and pathological mechanisms include radiation-induced death of stem cells with the subsequent inflammatory response. Healing and regeneration of the tissue depends on the survival of the remaining stem cells at the site of injury or on their migration from the surrounding tissue, which is less irradiated or not irradiated at all, to the damaged area.2 In the head and neck region, the most common RT -related acute side effects include radiodermatitis and radiomucosi- tis, affecting approximately 80% of patients. They also frequently suffer from dysgeusia, dysphagia, and xerostomia, i.e., a feeling of dry mouth due to saliva deficiency or changes in saliva composi- tion.3 The first symptoms of salivary deficiency may manifest after the first irradiation fractions and often progress to a chronic condition, especially when the average dose received by the major sali- vary glands, such as the parotid glands, reaches or exceeds 26 Gy or, in the case of the submandibu- lar glands, 35 Gy.4 Unfortunately, despite various improvements and advances, prevention of xeros- tomia remains a challenge. Modern radiation tech- niques, such as intensity-modulated radiotherapy, the use of proton or heavy ion beams, allow effi- cient, although not complete, sparing of the healthy tissue around the tumor.5,6 Careful oral hygiene, regular rinsing of the mouth, adequate hydration, and the use of intraoral stents that separate healthy tissue from the tumor are among the generally recommended preventive measures.7 The use of amifostine, a free radical scavenger, with its ques- tionable long-term efficacy and side effects occur- ring in nearly half of patients, remains the subject of professional debate.8 Surgical relocation of the submandibular gland contralateral to the tumor to the nonirradiated submental area is an invasive procedure and only suitable for a limited number of HNC patients.9 Various synthetic saliva substitutes based on methylcellulose are used to relieve the sensation of dry mouth, but with very variable effects in pa- tients. The same is true for acupuncture, electrical nerve stimulation, and hyperbaric oxygenation.10 If salivary gland function is partially preserved, cholinergic analogs (e.g., pilocarpine) can be used to stimulate saliva production, but they are rarely used in practice because of numerous side effects.10 Because they do not target regeneration of dam- aged glandular tissue, existing preventive and therapeutic approaches for newly diagnosed HNC patients or for patients with previously developed radiation-induced salivary gland hypofunction are limited and of questionable efficacy. As mentioned above, the pathophysiology of these complications is multifactorial and includes acute and late pro- cesses that may persist for several years after RT. They are characterized by chronic inflammation, development of fibrosis, loss of local stem and pro- genitor cells, and deterioration of survival condi- tions for the remaining acinar cells.4 Consequently, salivary gland hypofunction and resulting xeros- tomia remain the most common long-term side ef- fects of RT in HNC patients, which usually has a strong negative impact on their quality of life.11 Mesenchymal stromal stem cells Mesenchymal stromal stem cells (MSCs) originate from the mesodermal layer from which various mesenchymal tissues develop during embryonic development. In adults, MSCs represent a hetero- geneous cell population that includes multipotent MSCs that can differentiate into cartilage, bone, and fat cells. Because of their regenerative capabili- ties, they are often referred to as “adult stem cells” and are frequently studied in the field of regen- erative medicine.12 The most common biological sources for MSC production are bone marrow, adi- pose tissue, umbilical cord tissue, and others. The production and cultivation of MSCs is a relatively straightforward and safe process without signifi- cant ethical concerns. MSCs are characterized by low or no expression of major histocompatibility complex molecules, making them suitable for an allogeneic setting. Another phenotypic feature is the simultaneous expression of several markers, including CD73, CD90, and CD105, and the lack of expression of hematopoietic markers such as CD45.13 Although a specific subset of MSCs can differ- entiate into different cell types, there is increasing evidence that the main therapeutic effects of MSCs are mediated by paracrine mechanisms involv- ing the secretion of various active biomolecules. Factors secreted by MSCs may have angiogenic, anti-apoptotic, regenerative, and immunomodu- latory effects.14 Among all types of stem cells, MSCs possess a unique immunomodulatory ef- fect. Their activity is based on the expression of immunomodulatory enzymes such as indoleam- ine 2,3-dioxygenase (IDO) and inducible nitric oxide synthase (iNOS), immunosuppressive bio- molecules such as prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF), the production of Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy540 immunosuppressive cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β, or the expression of inhibitory surface molecules such as HLA-G and programmed death ligands PD -L1 and PD -L2. With such an extensive rep- ertoire of immunomodulatory mechanisms, MSCs can regulate the function of different types of im- mune cells belonging to both innate and adaptive immunity. These include monocytes/macrophages as well as important antigen-presenting cells such as dendritic cells (DCs). MSCs can also suppress the activity of T and B lymphocytes, natural killer (NK) cells, neutrophils, and mast cells. On the oth- er hand, MSCs have been shown to promote the generation of regulatory immune cells such as reg- ulatory T cells (Tregs), tolerogenic DCs, and mye- loid-derived suppressor cells.15 MSCs markedly in- hibit T cell proliferation both in an allogeneic set- ting and upon exposure to various mitogens.16,17 In co-cultures with helper Th1-type T cells, MSCs can greatly reduce the secretion of pro-inflammatory cytokines (e.g., IFN-γ and TNF-α) and increase the proportion of T cells producing anti-inflammato- ry cytokines (e.g., IL -10), such as Tr1 cells.18 They may also inhibit the activation of CD8+ cytotoxic T cells, resulting in a decreased response to allo- antigens and decreased antigen-specific lysis of al- logeneic cells.19,20 In summary, MSCs can suppress immune activation and promote a tissue regenera- tion through mechanisms involving the produc- tion/expression of various cell-bound and soluble factors. An important issue in the clinical translation of MSC-based advanced therapy medicinal prod- ucts (ATMPs) is the known heterogeneity of MSC preparations used in clinical trials. Variations in final product formulations may result from donor variability, tissue source variability, frequent and extensive cell passage variability during manufac- turing, and variable use of fresh or directly thawed IN TE RV EN TI O N D0 D1 D5 D28 M4 • oropharyngeal SCC • cura�ve (chemo)RT • remission ≥2years • RT plan evalua�on • Clinical assessment (CTCAE v5) • salivary flow measurements (uns�mulated, s�mulated) 0  4 weeks • blood tests • MRI • US elastography • scin�graphy [99mTc]TcO4- • biopsy • ques�onnaires • saliva composi�on analysis G NI NEERCS S N OITA NI MAXE To xi ci ty a ss es sm en t ( CT CA E v5 ) Sc in �g ra ph y (c on tr ol ) • toxicity (CTCAE v5) • blood tests) • salivary flow • saliva composi�on • MRI • US elastography • ques�onnaires • toxicity (CTCAE v5) • blood tests • salivary flow • Saliva composi�on • MRI • US elastography • scin�graphy [99mTc]TcO4- • biopsy • ques�onnaires Sc in �g ra ph y [9 9m Tc ]T c- O 4- HM PA O m ar ke d M SC FIGURE 1. Design of the clinical trial. CTCAE v5 = Common Terminology Criteria for Adverse Events version 5.0; MRI = magnetic resonance imaging; MSC = allogeneic mesenchymal stromal stem cells; RT = radiotherapy; SCC = squamous cell carcinoma; US = ultrasound; [99mTc]HMPAO = Technetium 99m-hexamethylpropyleneamine oxime; [99mTc]TcO4- = Technetium 99m-pertechnetate Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy 541 cryopreserved MSCs, the suboptimal functional- ity of which has been reported.21,22 In the present study, we will address these issues by introducing a novel protocol for the preparation of MSCs that allows for equivalent final product formulations for each recruited patient by using only freshly cultured cells with very low passages (p ≤ 2) and high viability, as described below. Advanced therapy medicinal products based on MSCs also represent an interesting and novel ther- apeutic option for patients with radiation-induced salivary gland damage and existing xerostomia because they act through different mechanisms that have both immunomodulatory and regenera- tive effects.23 Since xerostomia significantly limits patients’ quality of life, exploring the potential efficacy of MSC-based therapy is of high priority and with direct clinical relevance.11 Methods and design The study is being conducted at the Institute of Oncology Ljubljana in collaboration with the Slovenian Institute for Transfusion Medicine, the Clinic of Nuclear Medicine at the University Medical Centre Ljubljana, and the Institute of Biochemistry and Molecular Genetics at the Faculty of Medicine, University of Ljubljana. It is a non-randomized, single-center, open-label, phase I exploratory study. The study will include 10 pa- tients (intervention group) and 10 healthy volun- teers (control group) (Figure 1). The aim of the study is to evaluate the safety and preliminary efficacy of treatment of xerosto- mia after irradiation with allogeneic MSC from umbilical cord tissue. Thus, we hypothesize that the treatment of post-radiation xerostomia with allogeneic MSC from umbilical cord tissue is safe and effective. The study protocol was approved by the National Medical Ethics Committee (No. 0120-193/2023/3) and registered in the ClinicalTrial.gov database (NCT06012604) under the title: “Treatment of Post- radiation Xerostomia with Allogeneic Mesenchymal Stromal Stem Cells: A Pilot Study”, registered on 22 August 2023; https://classic.clinicaltrials.gov/ct2/ show/NCT06012604 Objectives The primary objective of the study is to evaluate the safety of administering allogeneic MSCs to the submandibular and parotid glands of patients with radiation-induced salivary gland dysfunction and xerostomia, with a 4-month follow-up after the procedure. Secondary objectives of the study include evalu- ation of the efficacy of the procedure and radiolog- ic, functional, and morphologic changes in glan- dular tissue after application of allogeneic MSC (Table 1). Eligibility criteria Patients who were successfully treated with (chemo)radiotherapy for squamous cell carcinoma of the oropharynx >2 years ago and have grade 2 or 3 xerostomia after radiotherapy according to the Common Terminology Criteria for Adverse Events (CTCAE) v.5.0 scale will participate in the study.24 Inclusion and exclusion criteria will be used to de- termine whether they can be included in the study (Table 2). Criteria for subsequent exclusion of patients from the study are: pregnancy, infection at the graft site, allergy to local anesthetics or citric acid, and withdrawal of consent to participate in the study. If a patient is excluded from the study prior to evaluation of the effect of the intervention under study, she or he will be replaced by another patient, leaving the final number of included patients at 10. Inclusion of patients Candidates for inclusion in the study will be se- lected from patients who are regularly monitored in the follow-up clinics for patients with head and neck cancer at the Institute of Oncology Ljubljana. Patients who meet the basic inclusion criteria will be informed about the study and invited to partici- pate. The study will not be randomized. Pre- and post-treatment assessment After being informed about participation in the study and signing the Informed Consent form, patients will undergo the following tests and complete the following questionnaires during the 4-week period before MSC application, i.e., the procedure (Table 3). The same measurements will be performed and questionnaires completed at both the 4-week and 4-month follow-up, with the exception of core needle biopsy and salivary gland scintigraphy (these will be repeated only at the 4-month follow-up). All procedures in the study are listed in Table 3. Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy542 The study also involves 10 volunteers (control group) who do not participate in the procedure. They will sign the Informed Consent to Participate in the Study and provide saliva samples for the measurement of unstimulated and stimulated salivary flow and for the determination of salivary composition (for comparison with the intervention group). Measurement of unstimulated total salivary flow rate Patients will be instructed to drink at least 2 liters of fluid the day before saliva sample delivery and to abstain from food, drink, and oral hygiene for at least 1 hour before. After a 5-minute break, they will rinse the mouth with a sip (15-20 ml) of cold water from the refrigerator. Unstimulated saliva will be collected for 10 minutes in a pre-weighed disposable plastic container. Saliva samples will be frozen in liquid nitrogen 15 minutes after collec- tion and stored at -80°C for further analysis. Saliva collection will take place between 10:00 and 12:00 in the same room and in the presence of the same physician, before the procedure, 4 weeks and 4 months after the procedure. The flow rate will be calculated assuming that 1 g of saliva is equivalent to 1 ml.25 Measurement of stimulated total salivary flow rate The procedures before and after saliva collection will be the same as those for measuring unstimu- lated saliva. After a 5-min break, patients will chew tasteless kerosene wax for 1 min and rinse the mouth with a sip (15-20 ml) of cold water from the refrigera- tor. Stimulated saliva collection (while chewing the wax) will be performed for 5 minutes in a disposable plastic container, before the procedure, at 4 weeks, and at 4 months after the procedure. Analysis of the composition of unstimulated and stimulated saliva samples Several characteristics or components will be de- termined in the saliva samples: • pH measurement (SevenCompact™ pH meter S210 with the electrode InLab Micro Pro-ISM, Mettler Toledo, Columbus, Ohio); • total protein concentration, spectrophotom- etry at 280 nm (NanoDrop One, ThermoFisher Scientific Inc., Waltham, MA, USA); • α-amylase activity, spectrophotometry at 405 nm (GENESYS™ 150 Vis/UV, ThermoFisher Scientific Inc., Waltham, MA, USA); TABLE 1. Primary and secondary objectives of the study Objective Definition of objective Time of evaluation Primary Evaluation of the safety of administration of allogeneic MSCs Adverse event assessment (CTCAE v.5 criteria): pain at application site, mouth sensation, infection From the start of therapy to the last follow-up (days 1, 5, 28, and 120) Secondary Efficacy of the procedure Measurement of unstimulated/stimulated salivary flow and saliva composition Assessment of subjective degree of xerostomia (questionnaires) Scintigraphic evaluation of grafting, retention, and migration of allogeneic MSCs Assessment of patients’ quality of life During recruitment, day 28 and 120 post-procedure During recruitment, days 28 and 120 after the procedure Immediately after the procedure (day 0) During recruitment, on days 28 and 120 after the procedure Radiological changes of the salivary tissue Magnetic resonance imaging (MRI): volume, signal, and diffusivity changes Ultrasonography (US): consistency (firmness) During recruitment, on days 28 and 120 after procedure Functional changes of the salivary tissue Scintigraphy with ([99mTc]TcO4- (pertechnetate): uptake of radioisotope in parenchyma, ejection fraction During recruitment, on days 28 and 120 after procedure Morphological changes of the salivary tissue Core needle biopsy specimens: composition, inflammatory infiltrate, metaplasia, reactive changes During recruitment, on day 120 after the procedure CTCAE v.5 = Common Terminology Criteria for Adverse Events version 5.0; MSCs = allogeneic mesenchymal stromal stem cells Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy 543 • concentration of mucins (MUC1), ELISA (ThermoFisher Scientific Inc., Waltham, MA, USA); • total esterase activity, spectrophotom- etry at 270 nm (GENESYS™ 150 Vis/UV, ThermoFisher Scientific Inc., Waltham, MA, USA)26; • paraoxonase activity, spectrophotom- etry at 405 nm (GENESYS™ 150 Vis/UV, ThermoFisher Scientific Inc, Waltham, MA, USA).27,28 Contrast-enhanced magnetic resonance imaging (MRI) of the neck MRI scans will be performed before, 4 weeks, and 4 months after MSC application using a 1.5T General Electric Optima 450W scanner. The protocol will include T1 FSE sequences in the axial plane, T2 PROPELLER sequences, diffusion weighted se- quences (b=0, b=400, b=800) with calculation of ADC maps and 3D T1 SPGR sequences. Volumetric analysis of salivary glands using AW server soft- ware, signal changes and diffusivity analysis will be performed. Ultrasonographic examination of the salivary glands Ultrasonography elastography (UE) will be per- formed before, 4 weeks, and 4 months after MSC application to assess the consistency (firm- ness) of the parotid and submandibular glands. Measurements will be taken using the Hitachi Arrieta 850 device immediately prior to ultra- sound-guided biopsy of one of the glands. Scintigraphy with [99mTc]Tc-HMPAO-labeled MSCs. One hour and twenty-four hours (the next day) af- ter MSC application, planar head and whole-body images and SPECT/CT images of the head will be performed to assess the distribution, retention, and migration of MSCs from the application site. Scintigraphy with [99mTc]TcO4- Before and 4 months after MSC transplantation, scintigraphy of the salivary glands will be per- formed with the radiopharmaceutical pertechne- tate ([99mTc]TcO4-). After intravenous administra- tion, the radiopharmaceutical accumulates in the functioning parenchyma of the salivary glands and is subsequently excreted into the oral cavity after stimulation with citric acid. After adminis- tration of the radiopharmaceutical, a 10-minute planar head recording will be made, followed by oral administration and stimulation of the sali- vary glands with citric acid and another 10-minute planar recording. In this study, the uptake of the radiopharmaceutical in the functioning parenchy- ma and the excretory fraction of each gland will be evaluated qualitatively and semiquantitatively. Both parameters, determined before and after MSC transplantation will be compared. Percutaneous core needle biopsy of the salivary gland. A sample will be taken from the parotid gland in the first 5 patients enrolled in the study, while a sample will be taken from the submandibular gland in the last 5 patients. The gland on the side of the head or neck that received a higher dose of radiation during RT will be punctured. The biopsy will be performed in each patient before the proce- dure and 4 months after, both times from the same gland. The biopsy will be performed under local an- esthesia and ultrasound guidance. Before the procedure, blood coagulation parameters will be determined (platelet count, prothrombin time, international normalized ratio). Patients taking anticoagulant medications will be instructed to discontinue them one week prior to the procedure. Tissue samples stained with hematoxylin and eosin will be examined by a pathologist. For both pre- and post-procedure specimens, the patholo- gist will semi-quantitatively evaluate and com- pare the percentage of serous, mucinous, and mixed acini, ducts, adipose tissue, and fibrosis in the specimen. The pathologist will also evalu- ate the presence, amount, and composition of the inflammatory infiltrate, possible metaplasia (e.g., squamous or oncocytosis), and reactive changes such as hyperplasia, atrophy, and signs of regen- eration. Any other pathologic findings will also be described. If necessary and if sufficient tissue will be available, additional special and immunohisto- chemical staining will be performed. Questionnaires for subjective assessment of xerostomia Patients will complete the Visual Analog Scale (VAS) questionnaire and the Xerostomia question- naire, before the procedure, at the follow-up visits Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy544 at 4 weeks and 4 months after the procedure.29,30 The VAS questionnaire consists of 8 questions that assess two basic aspects of salivary secretion: mucosal dryness and functional abilities (swal- lowing, speaking) resulting from mucosal dry- ness. Patients are asked to mark their response on a 100-mm horizontal line for each question.29 The Xerostomia questionnaire also consists of eight questions: the first four questions refer to dryness of the mucous membranes during eating or chew- ing, whereas the last four questions refer to situ- ations in which the person does not eat or chew. Patients rate each symptom on an 11-point Likert scale from 0 to 10, with a higher number indicating more pronounced dryness or greater discomfort due to lack of saliva.30 Quality of life assessment questionnaire Patients will complete the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Head and Neck Module (QLQ-H&N35) before procedure and at follow-up visits at 4 weeks and 4 months after pro- cedure. This questionnaire consists of 35 questions divided into seven domains and 11 individual questions. The questionnaire assesses symptoms and side effects of head and neck cancer treat- ment, social integration, and sexual functioning. Questions are rated using a four-point Likert scale, except for the last five questions, which offer two response options (yes/no).31 Blood tests Venous blood samples will be collected before the procedure and at the follow-up visits 4 weeks and 4 months after the procedure, following the blood collection protocol used at the Institute of Oncology Ljubljana. The values of standard param- eters of complete blood count, differential blood count, liver function tests, as well as electrolyte levels, renal markers, total proteins, albumin and C-reactive protein will be analyzed. Prothrombin time and international normalized ratio will also be determined during the first blood draw. Intervention MSC-based investigational medicinal products (IMPs) will be prepared at the Slovenian Institute for Transfusion Medicine according to validated standard operating procedures approved by the National Medical Ethics Committee (No. 0120- 60/2018/7, 24.4.2018). The umbilical cord tissue will TABLE 2. Inclusion and exclusion criteria Inclusion criteria Exclusion criteria Squamous cell carcinoma of the oropharynx, UICC TNM (8th ed.) stage cT1–2N+ or cT3–4cN0–3 M0, treated with curative intent RT (TD 66–70 Gy, bilateral neck irradiation) with or without concurrent chemotherapy Newly diagnosed malignant tumor anywhere in the body within the last two years Two years or more after treatment with no evidence of locoregional recurrence or systemic metastasis Active smoker Nonsmoker or former smoker (quit smoking ≥ 2 years ago) Use of xerogenic medications (e.g., tricyclic antidepressants, antipsychotics, decongestants, bronchodilators, antihypertensive agents such as beta blockers and diuretics, antihistamines, hypnotic sedatives, opioids, and muscle relaxants) Mean radiation dose > 26 Gy to each of the parotid glands and > 35 Gy to each of the submandibular glands Other salivary gland disorders (e.g., Sjoegren’s syndrome, scleroderma, sialolithiasis, etc.) Grade 2 or 3 xerostomia as assessed by the CTCAE v5.0 scale Patients receiving anticoagulant therapy that cannot be discontinued during the procedure Clinically decreased salivation and hyposalivation (unstimulated total salivary flow of 0.05–0.20 ml/min) Pregnancy or planned pregnancy within the next two years Age between 18–75 years Breastfeeding Both sexes Active, uncontrolled infection Signed “Informed Consent Form” to participate in the study Other medical (including psychiatric) conditions that, in the opinion of the investigators, preclude safe administration of the planned therapy and completion of follow-up visits Known substance abuse or alcoholism CTCAE v.5 = Common Terminology Criteria for Adverse Events version 5.0; RT = radiotherapy: TD = tumor doses; UICC = Union for International Cancer Control Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy 545 be used as the primary biological source. The tis- sue is first seeded in an ex vivo culture. After the first 10-14 days (passage 0), adherent cells begin to proliferate extensively and reach adequate conflu- ence within the next 7 days. They are then har- vested in passage 0 and reseeded for further cell expansion (passage 1). At the end of passage 1, cells from 4 different donors are harvested and pooled into a single cell suspension in equal ratios. They are then redistributed into identical aliquots and cryopreserved as “off-the-shelf units” (EQ-MSC) available for “per patient” orders. For each pa- tient, an identical aliquot of EQ -MSCs is thawed and seeded in cell culture for 3-5 days prior to the procedure (passage 2) to achieve optimal numbers and cell fitness for the final product. The final drug formulation will be prepared as a suspen- sion of 50×106 MSCs/ml in a physiological solution with an addition of 0.5% human albumin. It will be filled in syringes of 0.5 ml or 1.0 ml with a 23G 0.6x25 mm hypodermic needle. In the intervention group, each patient will receive an ultrasound-guided injection of 50×106 MSCs into each parotid gland and an injection of 25×106 MSCs into each submandibular gland, with- out anesthesia. To ensure even distribution of the MSC suspension, each parotid gland will receive injections in two areas: the tail and the body of the gland. Approximately 5% of the volume of the pre- pared MSC injectate will be removed under aseptic conditions. The cells will be labeled with hexam- ethylpropyleneamine oxime ([99mTc]Tc-HMPAO) and resuspended with the remaining injection material for administration to the patient. Course of the study After the procedure (day 0), patients (intervention group) will be examined at day 1, day 5, after 4 weeks, and after 4 months. An assessment of toxic- ity will be performed at each visit. At 4 weeks and 4 months after the intervention, the same meas- urements and questionnaires will be performed as when patients were enrolled in the study, except for core needle biopsy and scintigraphy, which will be repeated only at 4 months (Figure 1). No follow-up is scheduled for the volunteers (control group) after enrollment in the study and the initial examinations (day 0). The study will continue until completion of fol- low-up for the last enrolled patient or for a maxi- mum of two years. Tentatively, the study is expect- ed to run from October 2023 to September 2025. Termination guidelines The study will be terminated early if serious ad- verse events are noted during the intervention or follow-up. An allergic reaction, injection site infection, or other local or systemic event as- sessed as possibly/probably/surely related to the intervention and graded according to CTCAE v5.0 grade 3 or higher (serious adverse reac- tion, SAR) will be considered a discontinuation criterion. If a SAR is registered, the principal investigator will notify the study coordinator within 24 hours and later the National Center for Pharmacovigilance of the Agency for Medicinal Products and Medical Devices of the Republic of Slovenia and the Ethics Committee of the Institute of Oncology Ljubljana. Confidentiality On all documents collected for data analysis, patients will be identified by code only. The log of subject identification data will be maintained by the principal investigator. The name or other information that might reveal the identity of the patient will not be included in any document that leaves the research center and will not be used for clinical research data analysis. As a research participant, the patient has the right at any time to obtain information about the personal data collected and processed by the research provid- er, to request their correction or deletion, and to complain to the supervisory authorities, i.e. the Information Commissioner of the Republic of Slovenia and the Data Protection Commissioner of the Institute of Oncology Ljubljana. Data Monitoring Committee As a body independent of studies, sponsors and competing interests, the Ethics Committee of the Institute of Oncology Ljubljana is responsible for reviewing and monitoring all ongoing stud- ies conducted at the clinic. Each year during the study, the principal investigator prepares an an- nual report on the progress of the study, regis- tered adverse effects and SAEs, which is evalu- ated by the aforementioned committee. Availability of data and materials Electronic study records will be stored on the hospital server and will be accessible via pass- word-protected network computers. Data will be Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy546 retained for up to 10 years, after which they will be deleted. Statistical methods Since this is an exploratory study, no formal sam- ple size calculation was performed. Data will be managed in databases with Excel spreadsheets and analyzed with statistical analy- sis software such as SPSS, GraphPad Prism, or similar tools. Changes in absolute values and percent deviations from baseline salivary flow rate values before and after the procedure will be analyzed and compared at different time points within the intervention group; baseline values will also be compared with those of the control group. Numeric variables will be compared with paired t tests or nonparametric tests if the assumptions for parametric tests are not met. The scores of the individual items of the VAS questionnaire and the Xerostomia questionnaire will be summed, and the total score is linearly transformed into a scale from 0 to 100.29,30 Scoring and interpretation of the EORTC QLQ -H&N35 questionnaire will be performed according to EORTC guidelines.31 Comparisons of MRI, ultrasound, scintigraphy, and analysis of core-needle biopsies taken before and after procedure will be descriptive in nature. Discussion The aim of this phase I study is to evaluate the safety and preliminary efficacy of treatment of xerostomia after irradiation of patients with oro- pharyngeal carcinoma with allogeneic MSCs derived from umbilical cord tissue. These cells are characterized by a high rate of stemness and marked immunomodulatory activity. Compared with autologous MSCs or allogeneic MSCs from adipose or bone marrow tissue, their collection and processing are less invasive and simpler.32 If TABLE 3. Procedures of the study Procedures Inclusion Intervention Follow-up examinations W4→0 D0 D1 D5 D28 D120 Patient screening with clinical and objective evaluation of xerostomia1 X Complete blood count, biochemistry X X X Coagulation profile X Measurement of unstimulated and stimulated salivary flow rate X X X Determination of salivary composition X X X Magnetic resonance imaging X X X US elastography X X X Scintigraphy with [99mTc]Tc-HMPAO labeled MSCs X Scintigraphy with ([99mTc]TcO4-) X X Core needle biopsy of the gland2 X X Visual Analog Scale questionnaire X X X Xerostomia Questionnaire X X X EORTC QLQ-H&N35 X X X Toxicity assessment, CTCAE v.5 X X X X X CTCAE v.5 = Common Terminology Criteria for Adverse Events version 5.0; D = day; EORTC QLQ-H&N35 = European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module; MSCs = allogeneic mesenchymal stromal stem cells; US = ultrasound; W = week; [99mTc]HMPAO = Technetium 99m-hexamethylpropyleneamine oxime; [99mTc]TcO4- = Technetium 99m-pertechnetate 1 Inclusion criteria, see Table 2. 2 From the parotid gland: the first 5 patients included in the study; from the submandibular gland: the last 5 patients included. The gland on the side that received a higher dose of radiation will be punctured. Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy 547 our hypothesis is confirmed, our results will make an important contribution to the optimization of MSC treatment of xerostomia after radiation and will be used in the design of the next clinical trials. Treatment of xerostomia after radiation with MSCs is one of the newest therapeutic approaches. Due to the complex pathophysiology of radiation- induced salivary gland hypofunction, which also involves immune mechanisms, MSCs represent an interesting therapeutic agent because they have both immunoregulatory and regenerative ef- fects.33 The mechanism of action of MSCs is there- fore multifaceted and well suited for the treatment of diseases with a complex etiologic background. Although MSCs are naturally present in affected tissues, their numbers are significantly reduced after irradiation. The first reports on the treatment of xerostomia with MSCs in animal models appeared about ten years ago. In irradiated mouse models, systemic therapy with adipose-derived MSCs was shown to improve salivary flow.34 Following therapy, there was an increase in mucin secretion and amylase production.35 In addition, MSC therapy affected the architecture of the gland by increasing the number of functional acini and improving the mi- crovascular structure. Compared with the untreat- ed group, MSC-treated mice had a lower number of apoptotic cells, confirming the known anti-ap- optotic and mitogenic effects of MSCs.35,36 There is less clinical evidence for the effica- cy of xerostomia therapy with MSCs compared with some other indications, e.g., orthopedics or hematology. However, significant progress has been made in recent years. In 2018, Grønhøj et al. were the first to publish the results of a phase I/ II clinical trial evaluating the safety and efficacy of therapy for xerostomia after RT with autologous MSCs.37 They performed a randomized, placebo- controlled trial in 30 patients with squamous cell carcinoma of the oropharynx who had been cured with (chemo)radiotherapy two or more years ago. MSCs were applied to both submandibular glands under ultrasound guidance. The amount of cells (drug dose) was calculated using available preclin- ical data, taking into account gland volume in hu- mans. The maximum dose of MSCs was approxi- mately 4.5×107 MSCs per gland (2.86×105 – 2.86×106 MSCs/cm3 of gland). No side effects were observed during the study and up to two years after treat- ment. Compared to the placebo group, the treated group showed a statistically significant increase in unstimulated salivary flow both one month (33% increase, p=0.048) and four months after treat- ment (50% increase, p=0.003). Four months after MSC application, treated patients reported signifi- cant improvement in xerostomia symptoms (VAS questionnaire) compared to the placebo group. In a subsequent report with a median follow-up of 3.6 years after treatment, the authors reported no serious adverse events associated with the inter- vention, but maintained a positive clinical effect of MSC treatment.38 In addition, Lynggaard et al. published encour- aging results on the treatment of radiation-in- duced xerostomia with allogeneic adipose-derived MSCs.39 The use of allogeneic MSCs offers numer- ous logistical advantages: surgical intervention is no longer required to obtain the starting material, and the quality of the final preparations can be bet- ter controlled.40 In this study, 25×106 MSCs were in- serted into each submandibular gland and 50×106 MSCs were inserted into each parotid gland in ten patients; the follow-up period was four months. During this period, an increase in mean unstimu- lated salivary flow was measured from an initial 0.13 ml/min to 0.18 ml/min. Stimulated salivary flow also increased after therapy from 0.66 ml/ min to 0.75 ml/min. Patients’ subjective perception of an improvement in xerostomia was confirmed by questionnaire results, and no serious adverse events were reported during the study period.39 Recently, a dose-escalating phase I study pro- tocol (3 + 3 design) was published for the treat- ment of radiation-induced xerostomia in patients with head and neck cancer with interferon-gam- ma-stimulated autologous bone marrow stromal cells.41 The results of this study are not yet avail- able. However, in a pilot study, the same group has already used MSC prepared in this manner in six patients who received a single injection of 10x106 MSC into a single mandibular salivary gland and found that the therapy was well tolerated and showed a trend toward improvement in salivary volume and quality of life.42 To our knowledge, there has been no research using allogeneic MSCs from umbilical cord tissue. We believe that this may have several advantages. First, the collection of umbilical cord tissue is non- invasive for both the patient and the donor. The relative ease of obtaining umbilical cord tissue al- lows for greater flexibility in cryobanking “off the shelf” MSC products. This is also accelerated by the biological advantages of MSCs from umbilical cord tissue compared with, for example, bone mar- row or adipose tissue, namely their high prolifera- tion rate, smaller cell size, and lower rate of senes- cence during cell culture, which allows for higher Radiol Oncol 2023; 57(4): 538-549. Strojan P et al. / Post-radiation xerostomia therapy548 cell yield during manufacturing.32,43 Last but not least, umbilical cord tissue-derived MSCs have been reported to have excellent immunomodula- tory potential, very low immunogenicity, and doc- umented regenerative properties that may outper- form tissue-derived MSCs in certain aspects.44,45 Because this is an academic clinical trial focusing on a very limited number of patients, we chose a single dose consistent with previous report of safe use of allogeneic adipose-derived MSCs for the treatment of xerostomia.39 We believe that this is also a safe dose, both because of the small cell size of MSCs from umbilical cord tissue and because of their low immunogenicity profile.46 Because this is the first attempt to evaluate the use of MSCs from umbilical cord tissue for the treatment of xerostomia, we made every effort to design our study to be as similar as possible to the designs of previously published studies. For ex- ample, we used comparable study inclusion crite- ria (oropharyngeal cancer, at least 2 years after RT, and no evidence of recurrence) and exclusion cri- teria, examinations (unstimulated and stimulated salivary flow measurements with salivary compo- sition analysis; biopsy, MRI, and US of the salivary glands; questionnaires on xerostomia and quality of life), and time points for assessing treatment ef- fect (4 weeks and 4 months after procedure).39,41,47 The nuclear medicine studies in our protocol aim to provide additional definition of the effect of the administered therapy. This should enable us to make a more credible comparison of our results with those of other authors, which will undoubt- edly help to evaluate the potential of our approach compared with previously studied methods for the treatment of xerostomia after irradiation with MSCs. Conclusions The treatment of xerostomia after irradiation with MSCs represents a promising new therapeutic method, which is expected to trigger the regenera- tion of the glandular tissue and improve its func- tion, with a positive impact on patients’ quality of life. Moreover, a crucial aspect is the fact that no se- rious adverse effects related to MSC therapy have been observed up to 3.6 years (median) after this type of treatment in the clinical trials performed so far.32 We expect that the results of our study will contribute significantly to a deeper understand- ing of the effects of treatment with allogeneic MSC from umbilical cord tissue and optimize this ther- apy. The research results will not only be valuable from a scientific or academic point of view but will also have practical and clinical significance. Acknowledgement This work was supported by the Slovenian Research Agency (ARRS), grant number P3-0307. References 1. 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Potency of umbilical cord blood and Wharton’s jelly-derived mesenchymal stem cells for scarless wound healing. Sci Rep 2016; 6: 18844. doi: 10.1038/srep18844 47. Grønhøj C, Jensen DH, Glovinski PV, Jensen SB, Bardow A, Oliveri RS, et al. First-in-man mesenchymal stem cells for radiation-induced xerostomia (MESRIX): study protocol for a randomized controlled trial. Trials 2017; 18: 108. doi: 10.1186/s13063-017-1856-0 Radiol Oncol 2023; 57(4): 550. doi: 10.2478/raon-2023-0049 550 erratum Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses Angelika Vizintin1, Stefan Markovic2, Janez Scancar2, Jerneja Kladnik3, Iztok Turel3, Damijan Miklavcic1 1 Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia 2 Department of Environmental Sciences, Jožef Stefan Institute, Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 326-335. Received 7 June 2022 Accepted 19 June 2022 Correspondence to: Prof. Damijan Miklavčič, Ph.D., Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, SI-1000 Ljubljana, Slovenia. E-mail: Damijan.Miklavcic@fe.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.2478/raon-2022-0028 In Figure 3A, three horizontal bars representing the standard deviation were incorrectly drawn. The corrected figure is shown below. FIGURE 3. Cell survival as a function of the number of cisplatin molecules per cell for (A) CHO cells and (B) B16F1 cells in nonelectroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/ cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation. Survival data were combined from the previous8 (for non-electroporated CHO cells and CHO cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse). 0.1 1 10 100 0 1 2 3 4 5 6 7 8 Su rv iv al /% Cisplatin molecules per cell non-EP 25×400 ns 1×200 ns 8×100 µs × 107A B Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. I Radiol Oncol 2023; 57(4): 411-418. doi: 10.2478/raon-2023-0057 Slikanje mikrovaskularnih sprememb pri neoftalmoloških onkoloških kliničnih aplikacijah z angiografijo z optično koherentno tomografijo. Pregled literature Hren R, Serša G, Simončič U, Milanič M Izhodišča. Angiografija z optično koherentno tomografijo (angl. optical coherence tomography angi- ography - OCTA) je nova metoda medicinskega slikanja, ki omogoča neinvazivno vizualizacijo in analizo vaskulature tumorja. OCTA je bila še posebej uporabna v klinični oftalmološki onkologiji, medtem ko smo v tem članku ovrednotili OCTA pri ocenjevanju mikrovaskularnih sprememb v klinični neoftalmološki on- kologiji s sistematičnim pregledom literature. Metode. Vključitveni kriterij za iskanje literature v elektronskih bazah podatkov PubMed, Web of Science in Scopus je bila uporaba OCTA v neoftalmološki klinični onkologiji. Rezultati. Kriteriju za vključitev je ustrezalo enajst člankov. Anatomske lokacije novotvorb v izbranih člankih so bile prebavila (2 članka), glava in vrat (1 članek) ter koža (8 člankov). Zaključki. Medtem ko je OCTA pokazala velik napredek v oftalmologiji, se v neoftalmološki klinični onkologiji sooča z več omejitvami, pri čemer pomanjkanje standardiziranih protokolov in smernic za interpretacijo meritev predstavlja največji izziv. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. II Radiol Oncol 2023; 57(4): 419-429. doi: 10.2478/raon-2023-0059 Lokoregionalna terapija v kombinaciji s sistemsko terapijo (LRT+ST) za inoperabilen in metastatski intrahepatični holangiokarcinom. Sistematični pregled in metaanaliza Zhang M, Qi W, Qiu X, Yu C, Qiu W, Wang S, Qiu Z Izhodišča. Rezultati sistemskega zdravljenja inoperabilnega in metastatskega intrahepatičnega holan- giokarcinoma (angl. intrahepatic cholangiocarcinoma, iCCA) so slabi. Namen te raziskave je nadalje oceniti učinkovitost in varnost lokoregionalne terapije v kombinaciji s sistemsko terapijo (LRT+ST) v pri- merjavi s samo sistemsko terapijo pri inoperabilnem in metastatskem iCCA s sistematičnim pregledom literature in metaanalizo. Bolniki in metode. Do 3. novembra 2022 smo opravili obsežno iskanje v PubMed, Web of Science, EMBASE in Cochrane Library. Primarni cilj je bil ugotoviti celokupno preživetje (angl. overall survival, OS), sekundarni cilji pa so bili preživetje brez napredovanja bolezni (angl. progression-free survival, PFS), objek- tivna stopnja odgovora (angl. objective response rate, ORR) in neželeni dogodki (angl. adverse events, AE). Rezultati. V raziskavo smo vključili deset retrospektivnih kohortnih raziskav s 3791 inoperabilnimi ali metastatskimi bolniki z iCCA, vključno s 1120, ki smo jih zdravili z ablacijo, arterijsko usmerjeno tera- pijo (angl. arterially directed therapy, ADT) ali zunanjim obsevanje (angl. external beam radiation therapy, EBRT), v kombinaciji s sistemsko terapijo. Metaanaliza je pokazala, da je imela skupina LRT+ST boljši OS (HR = 0,51; 95 % CI = 0,41–0,64; p vrednost < 0,001), PFS (HR = 0,40, 95 % CI = 0,22–0,71, p vrednost = 0,002) in ORR (OR = 1,68; 95 % CI = 1,17–2,42; vrednost p = 0,005). Analiza podskupin je pokazala, da lahko sistemska terapija v kombinaciji z ADT (HR = 0,42, 95 % IZ = 0,31–0,56, p vrednost < 0,001) in EBRT (HR = 0,67, 95 % CI = 0,63–0,72, p vrednost < 0,001) izboljšata OS. Nevtropenija, trombo- citopenija, anemija, anoreksija in bruhanje niso pokazale pomembnih razlik med skupinami (vrednost p > 0,05). Zaključki. V primerjavi s samo sistemsko terapijo je LRT+ST izboljšala rezultate preživetja za inoperabilne in metastatske bolnike z iCCA brez povečanja resnih neželenih učinkov, kar je lahko podlaga za smer- nice. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. III Radiol Oncol 2023; 57(4): 430-435. doi: 10.2478/raon-2023-0045 Retrofaringealni kalcinirajoči tendinitis v Službi urgentne nevrologije, poročilo o treh primerih in pregled literature Filipović T, Avsenik J Izhodišča. Retrofaringealni kalcinirajoči tendinitis je razmeroma benigno stanje kalcinacije tetive mišice longus colli neznanega izvora, ki povzroča hude akutne bolečine v vratu. Vendar pa ga pogosto ne prepoznamo, kar vodi do prepozne diagnoze in nepotrebnega zdravljenja. Bolniki in metode. V PubMed in Google Scholar smo pregledali publikacije zadnjih 20 let, ki so po- ročale o vsaj enem bolniku z retrofaringealnim kalcinirajočim tendinitisom. Literaturo smo analizirali po protokolu PRISMA-S. Predstavimo tudi tri bolnike z retrofaringealnim kalcinirajočim tendinitisom, ki smo jih pregledali v Službi urgentne nevrologije Univerzitetnega kliničnega centra Ljubljana, Slovenija, od 1. janu- arja 2020 do 1. junija 2022. Opisujemo njihove klinične znake, diferencialno diagnozo in proces odločanja ter na kratko klinični potek. Pri prikazu primerov smo upoštevali protokol CARE. Rezultati. Analizirali smo skupno 112 naslovov objav z 231 bolniki. Najpogostejši simptomi in znaki so bili: bolečina v vratu, okorelost vratu in odinofagija, kot je bilo tudi v naših predstavljenih primerih. Zaključki. Retrofaringealni kalcinirajoči tendinitis je dramatično, vendar samoomejujoče stanje, ki ga pogosto ne prepoznamo pravočasno. Potrebna je čim večja ozaveščenost nevrologov o tem stanju. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. IV Radiol Oncol 2023; 57(4): 436-445. doi: 10.2478/raon-2023-0055 Kvantitativni SSTR-PET/CT za napovedovanje odziva na zdravljenje in rezultatov preživetja pri bolnikih z nevroendokrinimi tumorji trebušne slinavke, ki prejemajo capecitabin in temozolomid Ingenerf M, Karim H, Auernhammer C, Zacherl M, Wenter V, Winkelmann M, Ricke J, Berger F, Schmid-Tannwald C Izhodišča. Namen raziskave je bil ovrednotiti napovedno in spremljajočo vlogo somatostatinskih re- ceptorjev (SSTR) s pozitronsko emisijsko tomografijo–računalniško tomografijo (PET/CT) ter kliničnih para- metrov pri bolnikih z nevroendokrinim tumorjem trebušne slinavke in metastazami v jetrih, ki so prejemali capecitabin in temozolomid (CAPTEM). Bolniki in metode. V retrospektivno raziskavo smo vključili 22 bolnikov z nevroendokrinim tumorjem trebušne slinavke z metastazami v jetrih, ki so prejemali CAPTEM in pri katerih je bila pred in po terapiji narejena preiskava 68Ga-DOTATATE/-TOC PET/CT. Ocenili smo slikovne parametre (vključno s standardi- zirano vrednostjo privzema [SUV] ciljnih lezij v primerjavi z vranico in jetri) ter klinične parametre (kromo- granin A [CgA], Ki-67). Izid zdravljenja smo ocenjevali kot odziv na zdravljenje glede na kriterije RECIST 1.1, preživetje brez napredovanja bolezni (angl. progression free survival, PFS) in celokupno preživetje (angl. overall survival, OS). Rezultati. Mediana PFS (mPFS) je bila 7 mesecev. Osebe, ki so se odzivale na terapijo, so imele bistve- no daljši mPFS v primerjavi z osebami, ki se niso odzivale (10 v primerjavi s 4 meseci; p = 0,022). Mediana OS (mOS) je bila 33 mesecev (mOS pri odzivnih 80 mesecev, pri neodzivnih 24 mesecev; p = 0,182). Izhodiščno slikanje je pokazalo višjo SUV pri bolnikih, ki so se odzivali, vključno z absolutno SUV, razmerji med tumorjem in vranico (angl. tumor-to-spleen, T/S) in tumorjem z jetri (angl. tumor-to-liver, T/L) (p < 0,02). Vsi parametri SUV so se med spremljanjem spremenili le pri odzivnikih. Univariatna Coxova regre- sijska analiza je ugotovila izhodiščno razmerje med maksimalnim kopičenjem v tumorju in povprečnim v vranici (Tmax/Smean) ter odstotno spremembo velikosti pankreatičnega nevroendokrinega tumorja (pNET) kot pomembne dejavnike, ki so povezani s PFS. Izhodiščno razmerje Tmax/Smean < 1,5 je bilo v korelaciji s krajšim mPFS (10 v primerjavi s 4 meseci; p < 0,05). Napovedni dejavniki za OS so vključevali starost, odstotek spremembe CgA in razmerja T/S pri univariatni Coxovi regresiji. Zaključki. SSTR-PET/CT je lahko koristen za napovedovanje rezultatov odziva in preživetja pri bolnikih z nevroendokrinim tumorjem trebušne slinavke ki prejemajo CAPTEM. Višje izhodiščne vrednosti SUV, zlasti razmerja Tmax/Smean jetrnih metastaz, so povezane z boljšim odzivom in podaljšanim PFS. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. V Radiol Oncol 2023; 57(4): 446-454. doi: 10.2478/raon-2023-0051 Ocena lezij dojk z uporabo slikanja s prenosom amidnih protonov in z dinamično kontrastno magnetno resonanco Zhuang L, Lian C, Wang Z, Zhang X, Wu Z, Huang R Izhodišča. Dosedanje preiskave so pokazale, da bi lahko slikanje s prenosom amidnih protonov (angl. amide proton transfer-weighted imaging, APTWI) uporabili za razlikovanje med benignimi in malignimi tumorji. Tehnologijo APTWI v zadnjih letih vse pogosteje uporabljamo za raziskave tumorjev dojk. Vendar do sedaj niso objavili raziskav, ki bi primerjale vrednost APTWI in dinamičnega kontrastnega slikanja (angl. dynamic contrast-enhanced, DCE) z magnetno resonanco pri razlikovanju med benignimi in malignimi lezijami. Zato smo v pričujoči raziskavi primerjali uporabo APTWI in DCE. Bolniki in metode. APTWI smo naredili pri 40 bolnicah (42 lezij), ki smo jih vključili v prospektivno razi- skavo. Lezije so bile glede na rezultate histoloških preiskav razdeljene v dve skupini, maligne lezije dojk (n = 28) in benigne lezije dojk (n = 14). Izmerjene značilnosti slik (vrednost prenosa amidnih protonov [angl. amide proton transfer, APT], vrednost difuzijskega kvocienta [angl. apparent diffusion coefficient, ADC] in tip krivulje časa intenzivnosti [angl. time-of-intensity-curve, TIC]) smo primerjali med obema skupinama, krivulja ROC pa uporabili za količinsko opredelitev diagnostične učinkovitosti na podlagi teh dejavni- kov. Korelacija med vrednostmi APT in ravnmi izražanja estrogenskega receptorja, progesteronskega receptorja, humanega epidermalnega rastnega faktorja 2 (HER-2) in Ki-67 ter histološkimi stopnjami smo preverili s Spearmanovim korelacijskim koeficientom. Rezultati. Izmerjene vrednosti APT in ADC so se glede na intraklasne korelacijske koeficiente (0,954 in 0,825) med opazovalci močno ujemale. V primerjavi z benignimi lezijami so imele maligne lezije bistveno višje vrednosti APT (3,18 ± 1,07 in 2,01 ± 0,51, p < 0,001). Na podlagi APTWI; DCE; DWI in ADC + APTWI; ADC + DCE; DCE + APTWI so bile vrednosti območja pod krivuljo 0,915; 0,815; 0,878; 0,921; 0,916 oziroma 0,936. Zaključki. APTWI je obetavna metoda za razlikovanje med benignimi in malignimi lezijami dojk in lahko v prihodnosti postane odličen nadomestek za preiskavo DCE. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. VI Radiol Oncol 2023; 57(4): 455-464. doi: 10.2478/raon-2023-0048 Meritve difuzijskega koeficienta vzorcev infiltracije kostnega mozga pri diseminiranem plazmocitomu za oceno tumorskega bremena. Raziskava uporabnosti Xiong X, Ma Y, Dai Y, Hu C, Zhang Y Izhodišča. Namen raziskave je bil raziskati in primerjati tumorsko breme pri različnih infiltracijah kostnega mozga ter oceniti uporabnost vrednosti difuzijskega koeficienta (angl. apparent diffusion coefficient, ADC) za prepoznavo diseminiranega plazmocitoma. Bolniki in metode. Pri 93 bolnikih z na novo diagnosticiranim diseminiranim plazmocitomom in pri 23 kontrolnih bolnikih smo od januarja 2019 do novembra 2020 naredili navadno magnetnoresonnčno slikanje (MRI) in difuzno obteženo slikanje (angl. diffusion-weighted MRI, DWI). Pet vzorcev infiltracije ko- stnega mozga smo opredelili glede na navadno MRI. Analizirali in primerjali smo laboratorijske podatke in vrednosti ADC pri različnih vzorcih. Analizo ROC smo uporabili za določitev najboljše diagnostične mejne vrednosti ADC za prepoznavo teh vzorcev in razlikovanje normalnega vzorca od kontrol. Dodatno smo ocenili korelacijo med vrednostmi ADC difuznega vzorca in razmerjem plazemskih celic. Rezultati. Vrednosti hemoglobina, beta-2 mikroglobulina (β2-MG), plazemskih celic, proteina M, odstotkov stadijev, fluorescence in situ hibridizacije z visokim tveganjem in vrednosti ADC so pokazale pomembno razliko med vzorci. Povprečje ADC pri določeni vrednosti (368,5 × 10−6 mm2/s) je dalo največjo specifičnost (95,5 %) in občutljivost (92,0 %) pri diagnosticiranju diseminiranega plazmocitoma. Specifična vrednost (335,5 × 10−6mm 2/s) je dala največjo specifičnost (84,7 %) in občutljivost (88,0 %) pri razlikovanju vizualno normalnega vzorca od kontrol. Med razmerjem plazemskih celic in ADC difuznih vzorcev infiltracije je bila zmerna pozitivna korelacija (r = 0,648; P < 0,001). Zaključki. Vzorci infiltracije kostnega mozga pri bolnikih z diseminiranim plazmocitomom lahko kažejo na tumorsko breme, vrednosti ADC pa lahko objektivno razlikujejo te vzorce. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. VII Radiol Oncol 2023; 57(4): 465-472. doi: 10.2478/raon-2023-0054 Ocena kratkoročnega učinka zdravljenja tendinoze s trombocitno plazmo ob uporabi teksturne analize ultrazvočnih slik Pintarić K, Salapura S, Snoj Ž, Vovk A, Božič Mijovski M, Vidmar J Izhodišča. Orodja za računalniško podprto diagnostiko postajajo vse bolj uporabne metode za spre- mljanje subtilnih sprememb tkiva. Namen pričujoče pilotne raziskave je bil ugotoviti kratkoročni odgovor na zdravljenje s trombocitno plazmo (angl. platelet rich plasma) tendinoze tetive supraspinatusa in sku- pne tetive iztegovalke podlakti z metodo teksturne analize ultrazvočne (UZ) slike in s kliničnimi vprašalniki. Bolniki in metode. Trinajst bolnikov (7 moških and 6 žensk, starosti 36–60 let, povprečno 51,2 ± 5,2 le- ta) smo spremljali po UZ vodenem zdravljenju s trombocitno plazmo tendinoze dveh tetiv (9 bolnikov z lateralnim epikondilitisom in 4 s tendinozo tetive supraspinatusa). Klinično oceno tendinoze smo izvedli z validiranimi kliničnimi vprašalniki in z UZ oceno tetiv pred zdravljenjem s trombocitno plazmo in 3 mesece po njem. Tkivni odgovor v tetivah smo merili z uporabo metode teksturne analize UZ slik (angl. gray level run length matrix method GLRLM). Rezultati. Pri vseh bolnikih so se simptomi tendinoze po zdravljenju s trombocitno plazmo izboljšali glede na klinične vprašalnike. Skoraj vsi opazovani kazalniki GLRLM so se statistično izboljšali 3 mesece po zdra- vljenju. Kazalnik GLRLM-dolg zagon z visokim poudarkom sivine (angl. long run high gray level emphasis, LRLGLE) je imel najboljšo zmerno pozitivno in statistično značilno korelacijo po aplikaciji trombocitne plazme (r = 0,4373, p = 0,0255), sledil mu je GLRLM-z nizkim poudarkom sivine (angl. low gray level run emphasis, LGLRE) (r = 0,3877, p = 0,05). Zaključki. Teksturna analiza UZ slik tendinoze se je pokazala kot uporabna kvantitativna metoda za oceno remodelacije tetiv po minimalno invazivnem zdravljenju s trombocitno plazmo. Kazalnike GLRLM bi lahko potencialno uporabljali kot slikovne biološke označevalce za spremljanje prostorsko in časovno omejenega odziva tkiva po zdravljenju s trombocitno plazmo, vendar so zato potrebne večje raziskave s podobnimi protokoli. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. VIII Radiol Oncol 2023; 57(4): 473-486. doi: 10.2478/raon-2023-0061 Povezava genetskih dejavnikov s serumsko koncentracijo kalretinina pri azbestnih boleznih Zupanc C, Franko A, Štrbac D, Kovač V, Vita Dolžan, Goričar K Izhodišča. Izpostavljenost azbestu je povezana z različnimi azbestnimi boleznimi, tudi z malignim mezo- teliomom. Diagnozo malignim mezoteliomom potrdijo z imunohistokemično analizo več označevalcev, eden izmed njih je kalretinin. Pri malignem mezoteliomu so opazili tudi povišano izražanje kalretinina v krvi. Namen pričujoče raziskave je bil določiti, ali so polimorfizmi CALB2 ali polimorfizmi v genih, ki lahko regulirajo izražanje kalretinina, povezani s serumsko koncentracijo kalretinina ali dovzetnostjo za maligni mezoteliom. Subjekti in metode. V raziskavo smo vključili 288 bolnikov z malignim mezoteliomom in 616 preiskovan- cev brez te bolezni, ki so bili poklicno izpostavljeni azbestu (153 z azbestozo, 380 s plevralnimi plaki in 83 brez azbestnih bolezni). Pri vseh preiskovancih smo s kompetitivnim alelno specifično polimerazno veri- žno reakcijo (angl. polymerase chain reaction, PCR) določili sedem polimorfizmov v genih CALB2, E2F2, MIR335, NRF1 in SEPTIN7. Serumsko koncentracijo kalretinina smo določili z ELISO pri 545 preiskovancih. V statistični analizi smo uporabili neparametrične teste, logistično regresijo in analizo krivulj ROC (angl. receiver operating characteristic). Rezultati. Nosilci vsaj enega polimorfnega alela CALB2 rs889704 so imeli nižjo koncentracijo kalretinina (P = 0.036). V skupini preiskovancev brez malignega mezotelioma so imeli nosilci dveh polimorfnih alelov MIR335 rs3807348 višjo koncentracijo kalretinina (P = 0.027), nosilci vsaj enega polimorfnega alela NRF1 rs13241028 pa nižjo koncentracijo kalretinina (P = 0.034). Nosilci dveh polimorfnih alelov E2F2 rs2075995 so imeli večjo verjetnost za razvoj malignega mezotelioma (razmerje obetov [RO] = 0.64, 95 % interval zaupanja [IZ] = 0.43-0.96; P = 0.032), vendar povezava ni bila več statistično značilna po prilagoditvi za starost (P = 0.093). Serumske koncentracije kalretinina, ki so najbolje razlikovale med bolniki z malignim mezoteliomom in ostalimi preiskovanci, so bile različne glede na genotip CALB2, NRF1, E2F2 in MIR335. Zaključki. Rezultati raziskave kažejo, da bi genetska variabilnost lahko vplivala na serumsko koncen- tracijo kalretinina. Te ugotovitve bi lahko pripomogle k boljšemu razumevanju regulacije kalretinina in potencialno k hitrejši diagnozi malignega mezotelioma. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. IX Radiol Oncol 2023; 57(4): 487-492. doi: 10.2478/raon-2023-0030 Povezanost mesta preloma translokacije t(14;18) s kliničnimi značilnostmi pri folikularnem limfomu Panjan M, Boltežar L, Novaković S, Koković I, Jezeršek Novaković B Izhodišča. Za folikularni limfom je značilna translokacija t(14;18)(q32;q21), ki povzroča prekomerno izra- žanje antiapoptotičnega proteina BCL2. Na kromosomu 18 je prelom na različnih mestih, vendar spre- membe ne vplivajo na BCL2. Najpogosteje se prelom pojavi v regiji glavne prelomne točke (angl. major breakpoint region, MBR), redkeje pa v regiji manjšega grozda (angl. minor cluster region, mcr) ter med MBR in mcr v regijah 3'MBR, regiji vmesnega grozda (angl. intermediate cluster region, icr) in regiji 5'mcr. Namen raziskave je bil analizirati povezanost mesta preloma translokacije t(14;18)(q32;q21) s kliničnimi značilnostmi folikularnega limfoma. Bolniki in metode. Vključili smo bolnike z diagnozo folikularnega limfoma, ki so prejeli vsaj en krog sistemskega zdravljenja in so imeli pred prvim zdravljenjem z verižno reakcijo s polimerazo (PCR) dokaza- no translokacijo t(14;18)(q32;q21) na mestih MBR, mcr ali 3`MBR. Bolnike z različnimi mesti preloma smo primerjali glede na spol, starost, gradus, stadij, simptome B, mednarodni napovedni indeks folikularnega limfoma (angl. follicular lymphoma international prognostic index, FLIPI), prisotnost večje limfomske mase, čas brez napredovanja bolezni in celokupno preživetje. Rezultati. Med 84 bolniki s folikularnim limfom jih je imelo 63 prelom v regiji MBR, 17 v mcr in 4 v regiji 3`MBR. Skupina MBR je imela statistično značilno nižji stadij kot skupina mcr. Imela je tudi daljši čas brez napredovanja bolezni in daljše celokupno preživetje, več primerov nižjega gradusa, nižjo starost in FLIPI ter manj simptomov B, vendar te razlike niso bile statistično značilne. Zaključki. Nakazuje se, da je pri folikularnem limfomu mesto preloma MBR povezano z ugodnejšimi kliničnimi značilnostmi kot mcr. Da bi to ugotovitev podprli, bi bila potrebna večja klinična raziskava. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. X Radiol Oncol 2023; 57(4): 493-506. doi: 10.2478/raon-2023-0046 Napovedni pomen celic tumorskega-imunskega mikrookolja v ascitesu bolnic s seroznim rakom jajčnika visokega gradusa Miceska S, Škof E, Buček S, Grašič Kuhar C, Gašljević G, Smrkolj Š, Kloboves Prevodnik V Izhodišča. Serozni rak jajčnika visokega gradusa običajno diagnosticiramo v napredovalnem stadiju bolezni, ko se pojavi ascites. V raziskavi smo želeli določiti delež imunskih celic v ascitesu bolnic s seroznim rakom jajčnika visokega gradusa pred začetkom zdravljenja ter oceniti njihov vpliv na preživetje. Bolniki in metode. V raziskavo smo vključili 47 bolnic s primarnim seroznim rakom jajčnika visokega gradusa, ki so imele ascites. S pretočno citometrijo smo določili delež limfocitov T (CD3+) in njihovih pod- skupin (CD4+, CD8+, regulatornih limfocitov T [Treg] in naravnih celic ubijalk T [NKT]), limfocitov B, naravnih celic ubijalk (NK) (CD56++CD16- in CD56+CD16+), makrofagov in dendritičnih celic (DC). Na limfocitih T in njihovih podskupinah smo določili izražanje CD103 ter na vseh imunskih celicah izražanje PD-1 in PD-L1. Skupine z majhnim in velikim deležem imunskih celic smo določili na podlagi srednje vrednosti. Izračunali smo tudi korelacijo med deležem imunskih celic in preživetjem brez napredovanja bolezni ter celoku- pnim preživetjem. Rezultati. V vzorcih ascitesa so prevladovali limfociti T (srednja vrednost 51 %), medtem ko je bil delež ostalih imunskih celic veliko manjši (srednja vrednost ≤ 10 %). CD103 je bil izražen predvsem na CD8+ lim- focitih T, PD-1 pa na vseh CD3+ limfocitih T (srednja vrednost 20 %); manjše izražanje obeh označevalcev smo opazili na ostalih imunskih celicah (srednja vrednost ≤ 10 %). PD-L1 na imunskih celicah ni bil izražen. Velik delež CD103+CD3+ limfocitov T, PD-1+Treg celic, CD56++CD16- NK celic in DC je bil povezan z daljšim preživetjem brez napredovanja bolezni in celokupnim preživetjem, medtem ko je bil velik delež CD8+ limfocitov T, makrofagov in PD-1+CD56++CD16- NK celic ter majhen delež CD4+ limfocitov T povezan le z boljšim celokupnim preživetjem. DC so se med vsemi imunskimi celicami pokazale kot edini neodvisni napovedni dejavnik preživetja. Zaključki. Rezultati raziskave nakazujejo, da bi na podlagi podatkov o imunskih celicah v ascitesu lahko sklepali na napoved poteka bolezni pri bolnicah s seroznim rakom jajčnika visokega gradusa, vendar bodo za potrditev naših rezultatov potrebne dodatne raziskave. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. XI Radiol Oncol 2023; 57(4): 507-515. doi: 10.2478/raon-2023-0056 Obvladovanje sprememb tumorskega volumna med predoperativnim obsevanjem mehkotkivnih sarkomov okončin. Nova strategija adaptivne radioterapije Geneau De Lamarliere M, Lusque A, Attal Khalifa J, Esteyrie V, Chevreau C, Valentin T, Gangloff D, Meresse T, Courtot L, Rochaix P, Boulet B, Graulieres E, Ducassou A Izhodišča. Z uporabo adaptivne radioterapije (ART) smo želeli določiti objektivna klinična merila, ki iden- tificirajo bolnike z mehkotkivnimi sarkomi okončin (angl. extremity soft tissue sarcoma, ESTS), ki potrebujejo prilagoditev njihovega predoperativnega načrta obsevanja (RT). Bolniki in metode. Vključili smo 17 bolnikov z ESTS spodnjih okončin, zdravljenih med letoma 2019 in 2021 s predoperativno radioterapijo (RT) ob uporabi helikoidno intenzivno modulirane tomoterapije (IMRT) pred kirurško resekcijo. Zbrali smo klinične podatke ter podatke o tumorskih parametrih in zdra- vljenju. Ponastavitve smo zagotovili z dnevnim slikanjem z megavoltno računalniško tomografijo (angl. megavoltage computed tomography, MVCT). S tehnologijo PreciseART smo retrospektivno ročno dolo- čili vsaj en MVCT za vsakega pacienta na teden ter zabeležili volumne in dozimetrične parametre. Več kot 5-odstotna sprememba med dozimetrično pokritostjo tarčnega volumna in planiranega tarčnega volumna (PTV) od začetnega načrtovanja CT skeniranja do vsaj enega MVCT je bila opredeljena kot klinično pomembna. Rezultati. Vseh 17 bolnikov je med zdravljenjem imelo znatne spremembe tumorskega volumna; 7 tumorjev je zraslo (41 %) in 10 se jih je zmanjšalo (59 %). Trije bolniki (18 %), vsi nediferencirani pleomorfni sarkomi s povečanimi spremembami volumna, so imeli znatno zmanjšanje pokritosti tumorja z odmerkom sevenja. Sedem bolnikov je potrebovalo prilagoditev načrta, kot je bilo določeno s praktičnimi merili, ki jih uporabljamo v naši klinični praksii. Med temi bolniki je le pri enem na koncu prišlo do pomembne spre- membe v pokritosti PTV. Trije bolniki so imeli zmanjšanje pokritosti PTV. Med njimi 2 nista prejela prilagodi- tve načrta po naših kriterijih. Nobeden od bolnikov z zmanjšanim tumorskim volumnom ni imel zmanjšane pokritosti tarčnega volumna. Zdi se, da je spremljanje variacij volumna z ocenjevanjem makroskopskega tumorskega volumna (angl. gross tumor volume, GTV) na MVCT poleg aksialnih in sagitalnih linearnih dimenzij tumorja najučinkovitejše za odkrivanje zmanjšanja pokritosti PTV med zdravljenjem. Zaključki. Spremembe volumna ESTS so očitne med predoperativno RT, vendar so pomembne dozi- metrične spremembe redke. V prvih dveh tednih zdravljenja je treba posebno pozornost nameniti ne- diferenciranim pleomorfnim sarkomom stopnje 2-3. Ker v rutinski klinični praksi ni namenske programske opreme, lahko spremljanje sprememb volumna tumorja z ocenjevanjem GTV predstavlja uporabno strategijo za prepoznavanje bolnikov, pri katerih je treba ponovno načrtovati zdravljenje. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. XII Radiol Oncol 2023; 57(4): 516-523. doi: 10.2478/raon-2023-0050 Dozimetrična primerjava postoperativne intersticijske brahiterapije z visoko hitrostjo doze in sodobnih oblik teleradioterapije pri tumorjih jezika in ustnega dna glede doze, ki jo prejmejo kritični organi Ferenczi Ö, Major T, Fröhlich G, Béla D, Tódor S, Polgár C, Akiyama H, Bukovszky B, Takácsi-Nagy Z Izhodišča. Namen raziskave je bil dozimetrično primerjati intersticijsko brahiterapijo z visoko hitrostjo doze (angl. high-dose-rate brachytherapy, HDR BT) in sodobne oblike teleradioterapije, kot sta volu- metrično modulirana ločna terapija (angl. volumetric modulated arc therapy, VMAT) in stereotaktična radioterapija s kibernetskim nožem (angl. cyberknife, CK), pri tumorjih jezika in ustnega dna. Primerjali smo dozo, ki jo prejmejo kritični organi. Bolniki in metode. Med marcem 2013 in avgustom 2022 smo postoperativno obsevali 20 bolnikov (11 moških in 9 žensk) s tumorjem jezika (n = 14) in dna ustne votline (n = 6), stadija T1–3N0M0. Indikacije za obsevanje so bili bližina ali pozitivnost kirurškega roba in/ali limfovaskularna in/ali perinevralna invazija. Zdravili smo jih z intersticijsko brahiterapijo z visoko hitrostjo doze in uporabili fleksibilne plastične katetre ter aplicirali skupno dozo 15 x 3 Gy. Poleg načrtov za brahiterapijo smo pri vsakem bolniku naredili tudi načrt za VMAT in stereotaktično CK. Načrtovali smo enak režim frakcioniranja in predpisane odmerke. Kar zadeva zdrave, kritične organe, smo primerjali doze, ki so jih prejeli čeljust ter slinavke na ispsilateralni in kontralateralni strani. Rezultati. Povprečni volumen planirnega tarčnega volumna (angl. planning target volume, PTV) je bil 12,5 cm3; 26,5 cm3 in 17,5 cm3 pri tehnikah BT, VMAT in CK, ob različnih protokolih glede varnostnega robu. Odmerki na čeljust so bili najugodnejši pri tehniki BT, kar zadeva slinavke (parotid in submandibular- nih) pa je bila doza najnižja pri tehniki CK. Najvišje doze na kritične organe smo ugotovili pri tehniki VMAT. Povprečne vrednosti D2cm3 in D0.1cm3 za kritične organe pri tehnikah BT, VMAT in CK so bile naslednje: 47,4 % in 73,9 %; 92,2 % in 101,8 %; 68,4 % in 92,3 % za čeljust; 4,8 % in 6,7 %; 7,3 % in 13,8 %; 2,3 % in 5,1 % za ipsilateralno parotidno žlezo; 3,5 % in 4,9 %; 6,8 % in 10,9 %; 1,5 % in 3,3 % za kontralateralno parotidno žlezo ter 7,3 % in 9,4 %; 9,0 % in 14,3 %; 3,6 % in 5,6 % za kontralateralno submandibularno žlezo. Zaključki. Rezultati raziskave potrjujejo, da je BT, kljub temu da gre za invazivno tehniko, v dozimetrič- nem pogledu pri zdravljenju tumorjev ustne votline nedvomno ugodna in da je tehnika, na katero velja pomisliti pri uporabi radioterapije, ne le kot prvega zdravljenja, temveč tudi po operaciji. Uporaba CK v področju glave in vratu potrebuje nadaljnje raziskave. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. XIII Radiol Oncol 2023; 57(4): 524-529. doi: 10.2478/raon-2023-0060 Fazni kot, izmerjen z metodo bioelektrične impedančne analize, kot napovedni kazalnik kirurških izidov pri bolnikih z rakom prebavil Gulin J, Ipavic E, Mlakar Mastnak D, Brecelj E, Edhemović I, Rotovnik Kozjek N Izhodišča. Podhranjenost in slaba zmogljivost pri bolnikih z rakom črevesja, pri katerih je predviden kirurški poseg, povečata tveganje za negativne izide zdravljenja v pooperativnem obdobju. Fazni kot, izmerjen pri meritvi sestave telesa z metodo bioelektrične impedančne analize, predstavlja kazalec pre- snovnega in funkcionalnega stanja posameznika ter je lahko pomemben napovedni kazalnik kliničnih izidov kirurškega zdravljenja bolnikov z rakom črevesja. Namen raziskave je bila meritev vrednosti faznih kotov na skupini bolnikov z rakom črevesa v predoperativnem obdobju in ocena njegove napovedne vrednosti na pooperativne zaplete ter čas hospitalizacije. Zanimalo nas je, ali nižje vrednosti faznih kotov napovedujejo več pooperativnih zapletov in podaljšano hospitalizacijo. Želeli smo tudi določiti vrednost faznega kota, ki bi lahko napovedala večje tveganje negativnih izidov pri zgoraj omenjeni skupini bol- nikov. Bolniki in metode. Pri 70 bolnikih, ki so imeli elektivno operacijo raka prebavil, smo pred operativnim posegom opravili bioelektrično impedančno analizo, na osnovi katere smo dobili vrednosti faznega kota. V prvem mesecu smo iz bolnišnične dokumentacije beležili pooperativne zaplete teh bolnikov in jih klasificirali po sistemu za oceno kirurških zapletov Clavien Dindo. Zabeležili smo čas hospitalizacije. Podatke smo statistično obdelali s pomočjo programa SPSS. Zasnova raziskave je bila prospektivna. Rezultati. Ugotovili smo statistično značilno razliko (p = 0,036, test Kruskal-Wallis) v povprečni vrednosti faznih kotov med skupino bolnikov, ki je imela pooperativne zaplete (fazni kot 5,09°), in skupino, ki jih ni imela (5,64°). Poleg tega smo zaznali težnjo padanja vrednosti faznega kota z naraščanjem časa hospi- talizacije (Pearsonov koeficient korelacije R = -0,40; p = 0,001). Z metodo krivulje ROC smo izračunali mej- no vrednost faznega kota (5,5°), ki napovedovala večje tveganje za pooperativne zaplete (p = 0,037). Zaključki. Nižje vrednosti faznega kota pred operacijo so bile povezane z več zapleti v obdobju enega meseca po operaciji in z daljšim časom hospitalizacije. Ugotovili smo, da bi vrednost faznega kota 5,5° lahko služila kot mejna vrednost, ki napove večje tveganje za pooperativne zaplete. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. XIV Radiol Oncol 2023; 57(4): 530-537. doi: 10.2478/raon-2023-0053 Povezava med največjo srčno razdaljo in spremembami prsnega premera ter znižanjem prepone pri bolnicah z levostranskim rakom dojke med globokim in zadržanim vdihom Wu HG, Zhang GW, Liu JF, Yang JG, Su XH Izhodišča. Pri radioterapiji bolnic z levostranskim rakom dojke je pomembna zaščita srca. Odlično si lahko pomagamo s tehniko globokega in zadržanega vdiha. Tudi ob uporabi te tehnike vpliva giba- nje prsnega koša in diafragme na obseg, s katerim srce vstopi v polje sevanja. Namen raziskave je bil analizirati povezavo med največjo razdaljo, s katero srce vstopi v obsevalno polje, imenovano največja razdalja srca (angl. maximum heart distance, MHD), ter spremembami premera prsnega koša in zniža- njem (relaksacijo) prepone pri bolnicah z levostranskim rakom dojke med globokim in zadržanim vdihom. Bolniki in metode. V retrospektivno raziskavo smo vključili 98 bolnic z levostranskim rakom dojke. Izvedli so simulacijo globokega in zadržanega vdiha s pomočjo sistema Sentinel, uporabili dva niza slik CT pri prostem dihanju ter globokem in zadržanem vdihu. Izmerili smo položaj prepone, anteriorno-posteriorni prsni premer (angl. anteroposterior thoracic diameter, ATD), prečni prsni premer (angl. transverse tho- racic diameter, TTD), proženje s koincidenčnim časovnim oknom (angl. gating window level, GWL) in MHD ter izračunali spremembe (Δ) vsakega parametra pri globokem in zadržanem vdihu v primerjavi s prostim dihanjem. Za analizo korelacije med spremembo največje razdalje srca in spremembami drugih parametrov smo uporabili Pearsonov ali Spearmanov test. Rezultati. Pri vseh bolnikih z globokim in zadržanim vdihom je bila povprečna sprememba ΔMHD -8,3 mm, povprečna vrednost ΔATD in ΔTTD 11,0 in 8,6 mm, srednja vrednost znižanja levega dela prepone (angl. left diaphragmatic descent, LDD) in desnega dela prepone (angl. right diaphragmatic descent, RDD) 35,0 mm, srednja vrednost GWL pa 11,1 mm. Korelacijski koeficienti med zmanjšanjem MHD (ΔMHD) in LDD, RDD ter ΔTTD so bili -0,430 (p = 0,000), -0,592 (p = 0,000) oziroma 0,208 (p = 0,040), niso pa bili pomembno povezani z ΔATD ali GWL. Zaključki. Zmanjšanje MHD je bilo zmerno povezano z relaksacijo prepone ob simulaciji globokega in zadržanega vdiha s pomočjo sistema Sentinel pri bolnicah z levostranskim rakom dojke. Povezava s spremembami prsnega premera ali GWL pa je bila šibka ali je sploh ni bilo. Abdominalno dihanje lahko bolj zniža prepono in je lahko zato bolj koristno za srce, ki na ta način ostane dlje od tangencialnega obsevalnega polja. Slovenian abstracts Radiol Oncol 2023; 57(4): I-XV. XV Radiol Oncol 2023; 57(4): 538-549. doi: 10.2478/raon-2023-0052 Zdravljenje kserostomije po obsevanju pri bolnikih z rakom glave in vratu z alogenskimi mezenhimskimi stromalnimi matičnimi celicami. Študijski protokol za klinično preskušanje I. faze Strojan P, Plavc G, Kokalj M, Mitrović G, Blatnik O, Lezaić L, Sočan A, Bavec A, Tesić N, Hartman K, Švajger U Izhodišča. Kserostomija je pogost stranski učinek radioterapije pri bolnikih s tumorji glave in vratu, ki ne- gativno vpliva na kakovost življenja. Za kserostomijo ni znanega učinkovitega standardnega zdravljenja. Predstavljamo protokol študije namenjen oceni varnosti in predhodne učinkovitosti alogenskih mezen- himskih stromalnih matičnih celic, pridobljenih iz tkiva popkovnice. Bolniki in metode. V nerandomizirano, odprto klinično preskušanje I. faze bomo vključili deset bolnikov z rakom orofarinksa in kserostomijo po obsevanju, ki bodo brez znakov ponovitve bolezni 2 ali več let po (kemo)radioterapiji (intervencijska skupina) in 10 zdravih prostovoljcev (kontrolna skupina). Mezenhimske stromalne matične celice iz tkiva popkovnice bomo pod ultrazvočnim nadzorom aplicirali v obe paro- tidni žlezi in obe submandibularni žlezi bolnikov. Toksičnost postopka bomo ocenjevali v skladu s skupni- mi terminološkimi merili za neželene dogodke (angl. common terminology criteria for adverse events, CTCAE), 5. verzija, na dneve 0, 1, 5, 28 in 120. Učinkovitost bomo ocenjevali z merjenjem pretoka sline in analizo njene sestave, s scintigrafsko oceno presaditve, ohranjenosti in migracije mezenhimskih stromal- nih matičnih celic ter z vprašalniki za oceno subjektivne kserostomije in kakovosti življenja. Poleg tega bo- mo pred posegom ter 4 tedne in 4 mesece po posegu ugotavljali radiološke, funkcionalne in morfološke značilnosti tkiva slinavk. Pri preiskovancih iz kontrolne skupine bomo ocenjevali le pretok in sestavo sline. Diskusija. Uporaba alogenskih mezenhimskih stromalnih matičnih celic iz tkiva popkovnice predstavlja inovativen pristop v zdravljenju kserostomije po obsevanju. Zaradi neinvazivnega postopka zbiranja, prilagodljivosti kriobanke in bioloških prednosti ima lahko terapija kserostomije z uporabo alogenskih mezenhimskih stromalnih matičnih celic iz tkiva popkovine prednost pred drugimi podobnimi terapijami. abemaciklib vsak dan dvakrat na dan DAJTE JI VEČ KOT UPANJE Od 24.3.2023 na Pozitivni listi zdravil P100* tudi za adjuvantno zdravljenje HR+, HER2- zgodnjega raka dojk2 SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA IME ZDRAVILA: Verzenios 50 mg/100 mg/150 mg filmsko obložene tablete KAKOVOSTNA IN KOLIČINSKA SESTAVA: Ena filmsko obložena tableta vsebuje 50 mg/100 mg/150 mg 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) pozitivnim, na receptorje humanega epidermalnega rastnega faktorja 2 (HER2) negativnim zgodnjim rakom dojk s pozitivnimi bezgavkami, pri katerih obstaja veliko tveganje za ponovitev. Pri ženskah v pred- ali perimenopavzi je treba endokrino zdravljenje z zaviralcem aromataze kombinirati z agonistom gonadoliberina (LHRH – luteinizing hormone–releasing hormone). Napredovali ali metastatski rak dojk: Zdravilo Verzenios je indicirano za zdravljenje žensk z lokalno napredovalim ali metastatskim, na hormonske receptorje (HR) pozitivnim in na receptorje humanega epidermalnega rastnega faktorja 2 (HER2) 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 zdravljenje rakavih bolezni. 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 predvidenem času; dodatnega odmerka ne sme vzeti. Obvladovanje nekaterih neželenih učinkov lahko zahteva prekinitev in/ali zmanjšanje odmerka. Zdravljenje z abemaciklibom prekinite v primeru povišanja vrednosti AST in/ali ALT >3 x ZMN SKUPAJ s celokupnim bilirubinom > 2,0 x ZMN v odsotnosti holestaze ter pri bolnicah z intersticijsko pljučno boleznijo (ILD)/pnevmonitis stopnje 3 ali 4. Sočasni uporabi močnih zaviralcev CYP3A4 se je treba izogibati. Če se uporabi močnih zaviralcev CYP3A4 ni mogoče izogniti, je treba odmerek abemacikliba znižati na 100 mg dvakrat na dan. Pri bolnicah, pri katerih je bil odmerek znižan na 100 mg abemacikliba dvakrat na dan in pri katerih se sočasnemu dajanju močnega zaviralca CYP3A4 ni mogoče izogniti, je treba odmerek abemacikliba dodatno znižati na 50 mg dvakrat na dan. Pri bolnicah, pri katerih je bil odmerek znižan na 50 mg abemacikliba dvakrat na dan in pri katerih se sočasnemu dajanju močnega zaviralca CYP3A4 ni mogoče izogniti, je mogoče z odmerkom abemacikliba nadaljevati ob natančnem spremljanju znakov toksičnosti. 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. Zdravila se ne sme jemati z grenivko ali grenivkinim sokom. Bolnice naj odmerke vzamejo vsak dan ob približno istem času. Tableto je treba pogoltniti celo (bolnice tablet 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 loperamid, 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 motnjami, kot so intoleranca za galaktozo, popolno pomanjkanje laktaze ali malapsorpcija glukoze/galaktoze, tega zdravila ne smejo jemati. Bolnice je treba spremljati glede pljučnih simptomov, ki kažejo na ILD/pnevmonitis, in jih ustrezno zdraviti. 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 abema-cikliba in zaviralcev CYP3A4 lahko poveča plazemsko koncentracijo abemacikliba. Uporabi močnih zaviralcev CYP3A4 sočasno z abe-maciklibom se je treba izogibati. Če je močne zaviralce CYP3A4 treba dajati sočasno, je treba odmerek abemacikliba zmanjšati, nato pa bolnico skrbno spremljati glede toksičnosti. Pri bolnicah, zdravljenih z zmernimi ali šibkimi zaviralci CYP3A4, ni potrebno prilagajanje odmerka, vendar jih je treba skrbno spremljati za znake toksičnosti. Sočasni uporabi močnih induktorjev CYP3A4 (vključno, vendar ne omejeno na: karbamazepin, fenitoin, rifampicin in šentjanževko) se je treba izogibati zaradi tveganja za zmanjšano učinkovitost abema-cikliba. 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, pove-čana 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 Nederland B.V., Papendorp-seweg 83, 3528BJ, Utrecht, Nizozemska. Datum prve odobritve dovoljenja za promet: 27. september 2018. Datum zadnjega podaljšanja: 23. junij 2023 Datum zadnje revizije besedila: 23.6.2023. Režim izdaje: Rp/Spec - Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Reference: 1. Povzetek glavnih značilnosti zdravila Verzenios, zadnja odobrena verzija. Pomembno: Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Pred predpisovan-jem zdravila Verzenios si preberite zadnji veljavni Povzetek glavnih značilnosti zdravil. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila http:// www.ema.europa.eu Eli Lilly farmacevtska družba, d.o.o., Dunajska cesta 167, 1000 Ljubljana, telefon 01 / 580 00 10, faks 01 / 569 17 05 PP-AL-SI-0228, 17.8.2023, Samo za strokovno javnost. 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 Referenca: 1. Keytruda EU SmPC SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila! • 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; za adjuvantno zdravljenje odraslih z nedrobnoceličnim pljučnim rakom, ki imajo visoko tveganje za ponovitev bolezni po popolni kirurški odstranitvi in kemoterapiji na osnovi platine; 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 HER2- 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; v kombinaciji s trastuzumabom,  uoropirimidinom in kemoterapijo, ki vključuje platino, je indicirano za prvo linijo zdravljenja lokalno napredovalega neoperabilnega ali metastatskega HER2-pozitivnega adenokarcinoma želodca ali gastroezofagealnega prehoda pri odraslih, 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, NSCLC 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 je znašala 37 % za vse stopnje in 9 % od 3. do 5. stopnje, pri metastatski bolezni pa 25 % za vse stopnje in 6 % 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 3.473 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 (54 %), navzea (54 %), utrujenost (37 %), diareja (36 %), nevtropenija (34 %), zaprtost (34 %), alopecija (32 %), bruhanje (29 %) in zmanjšanje apetita (28 %). Pojavnost neželenih učinkov 3. do 5. stopnje je pri bolnikih z NSCLC pri kombiniranem zdravljenju s pembrolizumabom znašala 67 % in pri zdravljenju samo s kemoterapijo 66 %, pri bolnikih s HNSCC pri kombiniranem zdravljenju s pembrolizumabom 85 % in pri zdravljenju s kemoterapijo v kombinaciji s cetuksimabom 84 %, pri bolnikih z rakom požiralnika pri kombiniranem zdravljenju s pembrolizumabom 86 % in pri zdravljenju samo s kemoterapijo 83 %, pri bolnikih s TNBC pri kombiniranem zdravljenju s pembrolizumabom 80 % in pri zdravljenju samo s kemoterapijo 77 %, pri bolnicah z rakom materničnega vratu pri kombiniranem zdravljenju s pembrolizumabom 82 % in pri zdravljenju s kemoterapijo z ali brez bevacizumaba 75 %, in pri bolnikih z rakom želodca pri kombiniranem zdravljenju s pembrolizumabom (kemoterapija in trastuzumab) 71 % in pri kemoterapiji v kombinaciji s trastuzumabom 65 %. 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, 10/2023; SI-KEY-00561 Samo za strokovno javnost. H - Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. (pembrolizumab, MSD) KLJUČ ZA VEČ PRILOŽNOSTI PRI ZDRAVLJENJU VAŠIH BOLNIKOV KEYTRUDA je odobrena za zdravljenje 23 indikacij rakavih obolenj1 Čas, ki ga ona preživi na intraveneski aplikaciji, je čas, ki ga ne moreta preživeti skupaj. Zdravilo PHESGO je fiksna kombinacija pertuzumaba in trastuzumaba v eni viali, namenjena takojšnji podkožni aplikaciji v 5 – 8 minutah. Bolniki s HER2 + rakom dojk lahko preživijo manj časa na kliniki zaradi aplikacije zdravila in s tem več časa s svojimi bližnjimi.1 85 % bolnikov je v raziskavi Phrancesca izbralo zdravljenje z zdravilom PHESGO v primerjavi z intravenskim zdravljenjem.2 Zadnji veljavni Povzetki glavnih značilnosti zdravil so dosegljivi v Registru zdravil Skupnosti na povezavi Reference: 1. Povzetek glavnih značilnosti zdravila Phesgo https://ec.europa.eu/health/documents/community-register/2020/20201221150167/anx_150167_sl.pdf (dostopano maja 2021). 2. O´Shaughnessy J, Sousa S, Cruz J, et al., ESMO Breast 2020 (Abstract 80O): https://cslide.ctimeetingtech.com/global_storage/media/content/breast2020/ANNONC_31_S2_Breast_2020_Final_LBA1_content_embargo.pdf (dostopano maja 2021). HER2 – receptor za epidermalni rastni dejavnik tipa 2 DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o., Stegne 13G, Ljubljana Datum priprave informacije: oktober 2021 M-SI-00000301 (v 2.0) Ime zdravila: Phesgo 600 mg/600 mg in 1200 mg/600 mg raztopina za injiciranje. Kakovostna in količinska sestava: Ena viala z 10 ml raztopine vsebuje 600 mg pertuzumaba in 600 mg trastuzumaba. En ml raztopine vsebuje 60 mg pertuzumaba in 60 mg trastuzumaba (Phesgo 600 mg/600 mg). Ena viala s 15 ml raztopine vsebuje 1200 mg pertuzumaba in 600 mg trastuzumaba. En ml raztopine vsebuje 80 mg pertuzumaba in 40 mg trastuzumaba (Phesgo 1200 mg/600 mg). Terapevtske indikacije: Zgodnji rak dojk: Zdravilo Phesgo je v kombinaciji s kemoterapijo indicirano za neoadjuvantno zdravljenje odraslih bolnikov s HER2-pozitivnim, lokalno napredovalim, vnetnim ali zgodnjim rakom dojk z visokim tveganjem za ponovitev in adjuvantno zdravljenje odraslih bolnikov s HER2-pozitivnim zgodnjim rakom dojk z visokim tveganjem za ponovitev. Razsejani rak dojk: Zdravilo Phesgo je v kombinaciji z docetakselom indicirano za zdravljenje odraslih bolnikov s HER2-pozitivnim, razsejanim ali lokalno ponovljenim neoperabilnim rakom dojk, ki pred tem še niso prejeli anti-HER2 terapije ali kemoterapije za razsejano bolezen. Odmerjanje in način uporabe: Zdravilo Phesgo je lahko uvedeno le pod nadzorom zdravnika, ki ima izkušnje z uporabo zdravil proti raku. Zdravilo Phesgo mora dajati zdravstveni delavec, ki je usposobljen za obvladovanje anafilaksije, in v okolju, kjer je takoj na voljo celotna oprema za oživljanje. Bolniki, ki trenutno prejemajo pertuzumab in trastuzumab intravensko, lahko preidejo na zdravilo Phesgo. Odmerjanje: Bolniki, zdravljeni z zdravilom Phesgo, morajo imeti HER2-pozitivni tumor, imunohistokemijsko opredeljen kot 3+ in/ali razmerje pri ISH > 2,0. Bolniki, ki prejemajo taksan, morajo zdravilo Phesgo dobiti pred taksanom. Kadar se zdravilo Phesgo uporablja sočasno z docetakselom, je priporočeni začetni odmerek docetaksela 75 mg/m2, nato pa se ga poveča na 100 mg/m2 glede na izbrano shemo in prenašanje začetnega odmerka. Druga možnost je odmerek docetaksela 100 mg/m2 po 3-tedenskem razporedu že od začetka, ponovno glede na izbrano shemo. Če se uporablja shemo, ki temelji na karboplatinu, je priporočeni odmerek docetaksela ves čas 75 mg/m2. Kadar se zdravilo Phesgo uporablja sočasno s paklitakselom v adjuvantnem zdravljenju, je priporočeni odmerek paklitaksela 80 mg/m2 enkrat na teden v 12-tedenskih ciklih. Bolniki, ki prejemajo shemo na osnovi antraciklina, morajo zdravilo Phesgo dobiti po koncu celotne sheme na osnovi antraciklina. Razsejani rak dojk: Zdravilo Phesgo je treba uporabljati v kombinaciji z docetakselom. Zdravljenje z zdravilom Phesgo se lahko nadaljuje do napredovanja bolezni ali pojava neobvladljivih toksičnih učinkov, tudi če se zdravljenje z docetakselom ukine. Zgodnji rak dojk: Pri neoadjuvantnem zdravljenju, ki predstavlja del celostnega zdravljenja zgodnjega raka dojk, je treba zdravilo Phesgo dajati 3 do 6 ciklov v kombinaciji. V okviru adjuvantnega zdravljenja je treba zdravilo Phesgo uporabljati v skupnem trajanju eno leto, ki predstavlja del celostnega zdravljenja zgodnjega raka dojk in ne glede na čas operacije. Zdravljenje mora vključevati standardno kemoterapijo na osnovi antraciklina in/ali taksana. Zdravilo Phesgo naj se začne uporabljati 1. dan prvega cikla, ki vsebuje taksan, in ga je treba uporabljati še naprej, tudi če se kemoterapija ukine. Zamujeni ali izpuščeni odmerki: Če je čas med dvema zaporednima injiciranjema: krajši od 6 tednov: vzdrževalni odmerek zdravila Phesgo 600 mg/600 mg je treba dati čimprej. Nato nadaljujte s 3-tedenskim režimom. 6 tednov ali več: ponovno je treba dati polnilni odmerek zdravila Phesgo 1200 mg/600 mg, sledi pa mu vzdrževalni odmerek zdravila Phesgo 600 mg/600 mg na vsake 3 tedne. Prilagoditev odmerka: Za zdravilo Phesgo ni priporočljivo zmanjševanje odmerka. Po presoji zdravnika bo morda potrebna prekinitev zdravljenja z zdravilom Phesgo. Prehod z intravenskega pertuzumaba in trastuzumaba na zdravilo Phesgo: Bolnikom, ki so zadnji odmerek intravenskega pertuzumaba in trastuzumaba prejeli pred manj kot 6 tedni, je treba dati vzdrževalni odmerek zdravila Phesgo 600 mg pertuzumaba/600 mg trastuzumaba in ta odmerek uporabljati tudi za nadaljnje aplikacije na vsake 3 tedne. Bolnikom, ki so zadnji odmerek intravenskega pertuzumaba in trastuzumaba prejeli pred 6 tedni ali več, je treba dati polnilni odmerek zdravila Phesgo 1200 mg pertuzumaba/600 mg trastuzumaba, ki mu sledi vzdrževalni odmerek 600 mg pertuzumaba/600 mg trastuzumaba na vsake 3 tedne. Način dajanja: Zdravilo Phesgo moramo dajati le kot subkutano injekcijo. Zdravilo Phesgo ni namenjeno intravenskemu dajanju. Mesto injiciranja je treba izmenjevati le med levim in desnim stegnom. Polnilni odmerek je treba dati v 8 minutah, vzdrževalni odmerek pa v 5 minutah. Zaradi z injiciranjem povezanih reakcij je priporočljiv čas opazovanja 30 minut po danem polnilnem odmerku in 15 minut po zaključku vzdrževalnega odmerka zdravila Phesgo. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Disfunkcija levega prekata (vključno s kongestivnim srčnim popuščanjem): Med uporabo zdravil, ki zavirajo aktivnost HER2, so poročali o zmanjšanju LVEF. Večino primerov simptomatskega srčnega popuščanja v okviru adjuvantnega zdravljenja so zabeležili pri bolnikih, zdravljenih s kemoterapijo na osnovi antraciklina. Bolniki, predhodno zdravljeni z antraciklini ali obsevanjem v predelu prsnega koša, imajo lahko večje tveganje za zmanjšanje LVEF glede na študije z intravenskim pertuzumabom v kombinaciji s trastuzumabom in kemoterapijo. Pred uvedbo zdravila Phesgo je treba oceniti vrednost LVEF in jo nato med zdravljenjem tudi redno spremljati ter zagotoviti, da LVEF ostaja znotraj normalnih vrednosti. Če se LVEF poslabša in se ob naslednjem merjenju ne izboljša ali se še dodatno poslabša, je treba resno razmisliti o prenehanju zdravljenja z zdravilom Phesgo, razen če koristi za posameznega bolnika odtehtajo tveganja. Pred uporabo zdravila Phesgo skupaj z antraciklinom je treba skrbno razmisliti o kardiološkem tveganju in ga pretehtati glede na zdravstvene potrebe posameznega bolnika. Z upoštevajnjem farmakološkega delovanja zdravil, usmerjenih proti HER2, in antraciklinov je med sočasno uporabo zdravila Phesgo in antraciklinov mogoče pričakovati večje tveganje za kardiotoksičnost kot med zaporedno uporabo. Z injiciranjem povezane reakcije/z infundiranjem povezane reakcije: Uporabo zdravila Phesgo so spremljale z injiciranjem povezane reakcije. Opredeljene so bile kot katera koli sistemska reakcija s simptomi, kot so zvišana telesna temperatura, mrzlica, glavobol, najverjetneje zaradi sproščanja citokinov, ki se je pojavilo v 24 urah po dajanju zdravila Phesgo. Priporoča se skrbno opazovanje bolnika med dajanjem polnilnega odmerka in še 30 minut po njem ter med dajanjem vzdrževalnega odmerka zdravila Phesgo in še 15 minut po njem. Če se pojavi pomembna z injiciranjem povezana reakcija, injiciranje upočasnimo ali prekinemo ter nudimo ustrezno zdravljenje. Oceniti je treba stanje bolnika in ga skrbno spremljati, dokler znaki in simptomi popolnoma ne izzvenijo. Pri bolnikih s hudo reakcijo je treba razmisliti o dokončni prekinitvi zdravljenja. Klinična ocena mora temeljiti na tem, kako huda je bila prejšnja reakcija, in na odzivu na zdravljenje neželenega učinka. Preobčutljivostne reakcije/anafilaksija: Bolnike je treba skrbno opazovati glede preobčutljivostnih reakcij. Pri pertuzumabu v kombinaciji s trastuzumabom in kemoterapijo so opazili hude reakcije preobčutljivosti, vključno z anafilaksijo in dogodki s smrtnim izidom. Zdravilo Phesgo je treba dokončno ukiniti v primeru preobčutljivostne reakcije 4. stopnje po merilih NCI- CTCAE, bronhospazma ali akutnega respiratornega distresnega sindroma. Febrilna nevtropenija: Pri bolnikih, ki se zdravijo z zdravilom Phesgo v kombinaciji s taksanom, obstaja večje tveganje za nastanek febrilne nevtropenije. Pri bolnikih, ki se zdravijo z intravenskim pertuzumabom v kombinaciji s trastuzumabom in docetakselom, obstaja večje tveganje za nastanek febrilne nevtropenije v primerjavi z bolniki, ki se zdravijo s placebom, trastuzumabom in docetakselom, še posebej med prvimi 3 cikli zdravljenja. Driska: Zdravilo Phesgo lahko izzove hudo drisko. Driska je najpogostejša med sočasnim prejemanjem terapije s taksanom. Starejši bolniki (> 65 let) imajo večje tveganje za drisko v primerjavi z mlajšimi bolniki (< 65 let). Zlasti pri starejših bolnikih in v primeru hude ali dolgotrajne driske je treba razmisliti o zgodnjem zdravljenju z loperamidom in nadomeščanju tekočin ter elektrolitov. Razmisliti je treba o prekinitvi zdravljenja z zdravilom Phesgo, če ne dosežemo izboljšanja bolnikovega stanja. Pljučni dogodki: Pri uporabi trastuzumaba so v obdobju po prihodu zdravila na trg poročali o hudih pljučnih dogodkih. Ti dogodki so bili občasno smrtni. Poleg tega so poročali o primerih intersticijske bolezni pljuč, vključno s pljučnimi infiltrati, sindromom akutne respiratorne stiske, pljučnico, pnevmonitisom, plevralnim izlivom, dihalno stisko, akutnim pljučnim edemom in respiratorno insuficienco. Dejavniki tveganja, povezani z intersticijsko boleznijo pljuč, vključujejo predhodno ali sočasno zdravljenje z drugimi antineoplastičnimi terapijami, za katere je znano, da so z njo povezane, kot so taksani, gemcitabin, vinorelbin in radioterapija. Ti dogodki se lahko pojavijo kot del z infuzijo povezane reakcije ali imajo zapoznel nastop. Bolniki z dispnejo v mirovanju zaradi zapletov napredovale maligne bolezni in sočasnih bolezni imajo lahko večje tveganje za pljučne dogodke. Zato teh bolnikov ne smemo zdraviti z zdravilom Phesgo. Pri pnevmonitisu je potrebna previdnost, zlasti pri bolnikih, ki se sočasno zdravijo s taksani. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Formalnih študij medsebojnega delovanja niso izvedli. Neželeni učinki: Najpogostejši neželeni učinki zdravila (≥ 30%), o katerih so poročali pri bolnikih, zdravljenih z zdravilom Phesgo ali intravenskim pertuzumabom v kombinaciji s trastuzumabom in kemoterapijo, so bili alopecija, driska, navzea, anemija, astenija in artralgija. Najpogostejši resni neželeni dogodki (≥ 1 %), o katerih so poročali pri bolnikih, zdravljenih z zdravilom Phesgo ali intravenskim pertuzumabom v kombinaciji s trastuzumabom, so bili febrilna nevtropenija, srčno popuščanje, zvišana telesna temperatura, nevtropenija, nevtropenična sepsa, zmanjšanje števila nevtrofilcev in pljučnica. 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: 2.0/21 Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Kako poročati o neželenih učinkih, si poglejte skrajšani povzetek glavnih značilnosti zdravila pod ‘‘Poročanje o domnevnih neželenih učinkih‘‘. Če bolnica med zdravljenjem z zdravilom Phesgo ali v 7 mesecih po prejemu zadnjega odmerka zdravila Phesgo zanosi, vas prosimo, da nosečnost takoj poročate podjetju Roche farmacevtska družba d.o.o. (na e-naslov: slovenia.drugsafety@roche.com ali po telefonu na številko 01 3602 606). Prosili vas bomo za dodatne informacije med izpostavljenostjo zdravilu Phesgo v času nosečnosti in v prvem letu otrokovega življenja. S tem bomo v družbi Roche bolje razumeli varnost zdravila Phesgo in zagotovili ustrezne informacije zdravstvenim oblastem, zdravstvenim delavcem in bolnikom. Za dodatne informacije glejte Povzetek glavnih značilnosti zdravila Phesgo. Samo za strokovno javnost. 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*: V kombinaciji z bevacizumabom za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (KRR), ki so prejeli dva predhodna režima zdravljenja raka, vključno s kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF – Vascular Endothelial Growth Factor) in/ali zaviralci receptorjev za epidermalni rastni dejavnik (EGFR – Epidermal Growth Factor Receptor). V monoterapiji za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom, ki so bili predhodno že zdravljeni ali niso primerni za zdravl- jenja, ki so na voljo. Ta vključujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci VEGF in zaviralci EGFR. 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 28dnevnega 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 pri posameznem bolniku: dovoljena so zmanjšanja odmerka na najmanjši odmerek 20 mg/m2 dvakrat dnevno (oz. 15 mg/m2/odmerek dvakrat dnevno pri bolnikih s hudo ledvično okvaro). Potem ko je bil odmerek zmanjšan, povečanje ni dovoljeno. Kadar se zdravilo Lonsurf uporablja v kombinaciji z bevacizumabom za zdravljenje metastatskega KRR, je odmerek bevacizumaba 5 mg/kg telesne mase enkrat na 2 ted- na. KONTRAINDIKACIJE*: Preobčutljivost na učinkovini ali katero koli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI*: Supresija kostnega mozga: Pred uvedbo zdravljenja in po potrebi za spremljanje toksičnos- ti 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 priporo- čljiva pri bolnikih s končno stopnjo ledvične bolezni. Bolnike z ledvično okvaro je potrebno med zdravljenjem skrbno spremljati; bolnike z zmerno ali hudo ledvično okvaro je treba zaradi hematološke toksičnosti bolj pogos- to 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*. Bolnikom, ki želijo spočeti otroka, je treba svetovati, da se odločijo za svetovanje o reprodukciji ter shranjevanje jajčnih celic oz. sperme z zamrzovan- jem pred začetkom zdravljenja z zdravilom Lonsurf. 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, stomatitis. Pogosti: okužba spodnjih dihal, okužba, febrilna nevtropenija, limfopenija, hipoalbuminemija, disgevzi- ja, omotica, glavobol, hipertenzija, dispneja, bolečina v trebuhu, zaprtje, razjede v ustih, bolezni ustne votline, hiperbilirubinemija, izpuščaj, artralgija, mialgija, alopecija, pruritus, suha koža, proteinurija, pireksija, edem, vnetje sluznice, splošno slabo počutje, zvišanje jetrnih encimov, zvišanje alkalne fosfataze v krvi, zmanjšanje telesne mase. Občasni: okužba žolčevoda, gripa, okužba sečil, gingivitis, herpes zoster, okužba s kandido, bakterijska okužba, nevtropenična sepsa, okužba zgornjih dihal, konjunktivitis, bolečina zaradi raka, pancitopenija, monocitopenija, eritropenija, levkocitoza, monocitoza, dehidracija, hiperglikemija, hiperkaliemija, hipoka- liemija, hipofosfatemija, hiponatriemija, hipokalciemija, anksioznost, nespečnost, periferna nevropatija, nevrotoksičnost, parestezija, letargija, vrtoglavica, angina pektoris, aritmija, palpitacije, hipotenzija, vročinski oblivi, pljučna embolija, disfonija, epistaksa, izcedek iz nosu, kašelj, krvavitev v prebavilih, ileus, kolitis, gastritis, moteno praznjenje želodca, abdominalna distenzija, analno vnetje, dispepsija, gastroezofagealna refluksna bolezen, glositis, bolezen zob, siljenje na bruhanje, flatulenca, hepatotoksičnost, sindrom palmarne-plantarne eritrodisestezije, urtikarija, akne, hiperhidroza, bolezni nohtov, bolečina v kosteh, mišična oslabelost, mišični krči, bole- čina v okončinah, ledvična odpoved, motnje mikcije, hematurija, motnje menstruacije, poslabšanje splošnega zdravstvenega stanja, bolečina, občutek spremembe telesne temperature, neugodje v okončinah, zvišanje kreatinina v krvi, povečanje mednarodnega umerjenega razmerja (INR), zvišanje sečnine v krvi, zvišanje laktatne dehidrogenaze v krvi, zvišanje C-reaktivnega proteina, zmanjšan hematokrit. Redki: infekcijski enteritis, tinea pedis, septični šok, granulocitopenija, putika, hipernatriemija, pekoč občutek, disestezija, hiperestezija, hipoestezija, sinkopa, katarakta, suho oko, zamegljen vid, diplopija, zmanjšana ostrina vida, neugodje v ušesu, em- bolija, orofaringealna bolečina, plevralni izliv, ascites, akutni pankreatitis, subileus, slab zadah, bukalni polip, hemoragični enterokolitis, krvavitev dlesni, ezofagitis, parodontalna bolezen, proktalgija, refluksni gastritis, razširitev žolčnih vodov, mehur, eritem, preobčutljivostne reakcije na svetlobo, luščenje kože, otekanje sklepov, neinfektivni cistitis, levkociturija, kseroza, podaljšanje aktiviranega parcialnega tromboplastinskega časa, podaljšanje intervala QT na elektrokardiogramu, znižanje celokupnih proteinov. Post-marketinške izkušnje: intersticijska bolezen pljuč. PREVELIKO ODMERJANJE*: Neželeni učinki, o katerih so poročali v povezavi s pre- velikim odmerjanjem, so bili v skladu z uveljavljenim varnostnim profilom. Glavni pričakovani zaplet prevelikega odmerjanja je supresija kostnega mozga. FARMAKODINAMIČNE LASTNOSTI*: Farmakoterapevtska skupi- na: 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 - Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. 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: julij 2023. *Pred predpisovanjem preberite celoten povzetek glavnih značilnosti zdravila. Celoten povzetek glavnih značilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., Podmilščakova ulica 24, 1000 Ljubljana, www.servier.si. VEČ KOT 10-MESEČNO CELOKUPNO PREŽIVETJE Lonsurf® v kombinaciji z bevacizumabom je pokazal edinstvene rezultate pri zdravljenju mCRC v 3. liniji, saj je bila prvič dosežena mediana celokupnega preživetja (mOS) 10,8 meseca, s skoraj polovico živih bolnikov po enem letu in v dobri kondiciji za nadaljnje zdravljenje.1 Širimo obzorja v 3. liniji zdravljenja metastatskega kolorektalnega raka (mCRC) trifluridin/tipiracil Usmerjen v prihodnost Literatura: 1. Prager GW et al. N Engl J Med 2023;388:1657-67. Družba Servier ima licenco družbe Taiho za zdravilo Lonsurf®. Pri globalnem razvoju zdravila sodelujeta obe družbi in ga tržita na svojih določenih področjih. LON AD1 C1 2023-24. Samo za strokovno javnost. Datum priprave informacije: avgust 2023. 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It is their responsibility to check the proofs carefully and return a list of essential corrections to the editorial office within three days of receipt. Only grammatical corrections are acceptable at that time. Open access Papers are published electronically as open access on https://content.sciendo.com/raon, also papers accepted for publication as E-ahead of print. instructions palbociklib Ustvarjen za mRD HR+/HER2- = pozitiven na estrogenske receptorje in negativen na receptorje humanega epidermalnega rastnega faktorja 2; mRD = metastatski rak dojke Zdravilo IBRANCE že več kot 8 let pomaga do trenutkov, kot so ti.2-5 Zdravilo IBRANCE, ki ga podpirajo obširne izkušnje iz klinične in vsakodnevne dejanske prakse ter dokazana klinična učinkovitost in dobro prenašanje,1,4,6-35 vašim bolnicam pomaga, da najbolje izkoristijo vsak dan.4,5,36,37 DRAGOCENI TRENUTKI BOLNIKA SO MERILO VAŠEGA USPEHA. OMOGOČA JIH ZDRAVILO IBRANCE. ZA ODRASLE BOLNIKE S HR+/HER2- mRD1 PP-IBR-SVN-0018 Datum priprave: avgust 2023. Samo za strokovno javnost. Pfizer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J. F. Kennedy, L-1855 Pfizer, podružnica Ljubljana, Letališka cesta 29a, Ljubljana BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA IBRANCE 75 mg, 100 mg, 125 mg filmsko obložene tablete Sestava in oblika zdravila: Ena filmsko obložena tableta vsebuje 75 mg, 100 mg ali 125 mg palbocikliba. Indikacije: Zdravljenje lokalno napredovalega ali metastatskega na hormonske receptorje (HR – Hormone Receptors) pozitivnega in na receptorje humanega epidermalnega rastnega faktorja 2 (HER2 – Human Epidermal growth factor Receptor 2) negativnega raka dojk: v kombinaciji z zaviralcem aromataze ali v kombinaciji s fulvestrantom pri ženskah, ki so prejele predhodno endokrino zdravljenje. Pri ženskah v pred- in perimenopavzi je treba endokrino zdravljenje kombinirati z agonistom gonadoliberina. Odmerjanje in način uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. Priporočeni odmerek je 125 mg enkrat na dan 21 zaporednih dni, sledi 7 dni brez zdravljenja (shema 3/1), celotni cikel traja 28 dni. Zdravljenje je treba nadaljevati, dokler ima bolnik od zdravljenja klinično korist ali dokler se ne pojavi nesprejemljiva toksičnost. Pri sočasnem dajanju s palbociklibom je treba zaviralec aromataze dajati v skladu s shemo odmerjanja, ki je navedena v Povzetku glavnih značilnosti zdravila (PGZZ). Pri sočasnem dajanju s palbociklibom je priporočeni odmerek fulvestranta 500 mg intramuskularno 1., 15. in 29. dan ter nato enkrat na mesec, glejte PGZZ za fulvestrant. Prilagajanja odmerkov: Za prilagajanja odmerkov zaradi hematološke toksičnosti glejte preglednico 2, zaradi nehematološke toksičnosti pa preglednico 3 v PGZZju. Pri bolnikih s hudo intersticijsko boleznijo pljuč (ILD)/pnevmonitisom je treba zdravljenje trajno prekiniti. Posebne skupine bolnikov: Starejši: Prilagajanje odmerka ni potrebno. Okvara jeter ali ledvic: Pri bolnikih z blago ali zmerno okvaro jeter ali blago, zmerno ali hudo okvaro ledvic prilagajanje odmerka ni potrebno. Pri bolnikih s hudo okvaro jeter je priporočeni odmerek 75 mg enkrat na dan po shemi 3/1. Pediatrična populacija: Varnost in učinkovitost pri otrocih in mladostnikih, starih < 18 let, nista bili dokazani. Način uporabe: Peroralna uporaba. Tablete se lahko jemlje s hrano ali brez nje. Ne smemo jemati z grenivko ali grenivkinim sokom. Tablete zdravila je treba pogoltniti cele. Kontraindikacije: Preobčutljivost na učinkovino ali katerokoli pomožno snov. Uporaba pripravkov s šentjanževko. Posebna opozorila in previdnostni ukrepi: Ženske v pred- in perimenopavzi: Kadar zdravilo uporabljamo v kombinaciji z zavi- ralcem aromataze je obvezna ovarijska ablacija ali supresija z agonistom gonadoliberina. Hematološke bolezni: Pri nevtropeniji stopnje 3 ali 4 je priporočljiva prekinitev odmerjanja, zmanjšanje odmerka ali odložitev začetka ciklov zdravljenja, bolnike pa je treba ustrezno spremljati. ILD/pnevmonitis: Pri bolnikih se lahko pojavita huda, življenjsko ogrožajoča ali smrtna ILD in/ali pnevmonitis, kadar zdravilo jemljejo v kombinaciji z endokrinim zdravljenjem. Bolnike je treba spremljati glede pljučnih simptomov, ki kažejo na ILD/pnevmonitis (npr. hipoksija, kašelj, dispneja), in pri pojavu novih ali poslabšanju respiratornih simptomov oz. sumu na ILD/pnevmonitis zdravljenje prekiniti. Okužbe: Zdravilo lahko poveča nagnjenost k okužbam, zato je bolnike treba spremljati glede znakov in simptomov okužbe ter jih ustrezno zdraviti. Venska trombembolija: Pri bolnikih, zdravljenih s tem zdravilom, so poročali o venskih trombemboličnih dogodkih, zato je bolnike treba spremljati glede znakov in simptomov globoke venske tromboze in pljučne embolije ter jih ustrezno zdraviti. Okvara jeter ali ledvic: Pri bolnikih z zmerno ali hudo okvaro jeter ali ledvic je treba zdravilo uporabljati previdno in skrbno spremljati znake toksičnosti. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Učinki drugih zdravil na farmakokinetiko palbocikliba: Zaviralci CYP3A: Sočasni uporabi močnih zaviralcev CYP3A, med drugim klaritromicina, indinavirja, itrakonazola, ketokonazola, lopinavirja/ritonavirja, nefazodona, nelfinavirja, posakonazola, sakvinavirja, telaprevirja, telitromicina, vorikonazola in grenivke ali grenivkinega soka, se je treba izogibati. Induktorji CYP3A: Sočasni uporabi močnih induktorjev CYP3A, med drugim kar- bamazepina, enzalutamida, fenitoina, rifampicina in šentjanževke, se je treba izogibati. Učinek zdravil za zmanjševanje kisline: Klinično pomembnega učinka na izpostavljenost palbociklibu ni pričakovati. Učinki palbocikliba na farmakokinetiko drugih zdravil: Pri sočasni uporabi bo morda treba zmanjšati odmerek občutljivih substratov CYP3A z ozkim terapevtskim indeksom (npr. alfentanil, ciklosporin, dihidroergotamin, ergotamin, everolimus, fentanil, pimozid, kinidin, sirolimus in takrolimus), saj IBRANCE lahko poveča izpostavljenost tem zdravilom. Študije in vitro s prenašalci: Palbociklib lahko zavira prenos, posredovan s P-gp v prebavilih in beljakovino odpornosti pri raku dojk (BCRP). Uporaba palbocikliba z zdravili, ki so substrati P-gp (npr. digoksin, dabigatran, kolhicin) ali BCRP (npr. pravastatin, rosuvastatin, sulfasalazin) lahko poveča njihov terapevtski učinek in neželene učinke. Palbociklib lahko zavira privzemni prenašalec organskih kationov OCT1. Plodnost, nosečnost in dojenje: Med zdravljenjem in vsaj 3 tedne (ženske) oziroma 14 tednov (moški) po koncu zdravljenja je treba uporabljati ustrezne kontracepcijske metode. Zdravila ne uporabljajte pri nosečnicah in ženskah v rodni dobi, ki ne uporabljajo kontracepcije. Bolnice, ki prejemajo palbociklib, ne smejo dojiti. Zdravljenje s palbociklibom lahko ogrozi plodnost pri moških. Pred začetkom zdravljenja naj moški zato razmislijo o hrambi sperme. Vpliv na sposobnost vožnje in upravljanja s stroji: Ima blag vpliv na sposobnost vožnje in upravljanja strojev. Potrebna je previdnost. Neželeni učinki: Zelo pogosti: okužbe, nevtropenija, levkopenija, anemija, trombocitopenija, pomanjkanje teka, stomatitis, navzea, diareja, bruhanje, izpuščaj, alopecija, suha koža, utrujenost, astenija, pireksija, povečane vrednosti ALT/AST. Način in režim izdaje: Rp/Spec - Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Imetnik dovoljenja za promet: Pfizer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. Datum zadnje revizije besedila: 26.05.2023 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. Literatura: 1. Povzetek glavnih značilnosti zdravila Ibrance, 26.5.2023. 2. Beaver JA, et al. Clin Cancer Res. 2015;21(21):4760-4766. 3. George MA, et al. Front Oncol. 2021;11:693104. 4. Rugo H, et al. Breast Cancer Res Treat. 2019;174(3):719-729. 5. Rugo HS, et al. Ann Oncol. 2018;29:888-894. 6. Cristofanilli M, et al. Clin Cancer Res. 2022;28(16):3433-3442. 7. Xu B, et al. Eur J Cancer. 2022;175:236-245. 8. Finn RS, et al. N Engl J Med. 2016;375(20):1925- 1936. 9. Gelmon K, et al. Breast. 2021;59:321-326. 10. Rugo HS, et al. Eur J Cancer. 2018;101:123-133. 11. Turner NC, et al. 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ESMO 2022; Poster 251P. 29. Karuturi MS, et al. ESMO BC 2022; Poster 190P. 30. Blum J, et al. SABCS 2021; Abstract P1-18-29. 31. Finn R, et al. ASCO 2022; Oral presentation. 32. Finn R, et al. Oncologist. 2021;26:e749-e755. 33. Diéras V, et al. Oncologist. 2019;24(12):1514-1525. 34. Verma S, et al. Oncologist. 2016;21(10):1165-1175. 35. Harbeck N, et al. Ann Oncol. 2016;27(6):1047-1054. 36. Rocque G, et al. ESMO 2022; Poster 266P. 37. Karuturi M, et al. SABCS 2021; Poster P1-18-25. R a d io lo g y a n d O n c o lo g y I V o lu m e 5 7 I N u m b e r 4 I P a g e s 4 1 1 -5 5 0 I D e c e m b e r 2 0 2 3 december 2023 vol.57 no.4