R a d io lo g y a n d O n c o lo g y I V o lu m e 5 1 I N u m b e r 4 I P a g e s 3 6 9 -4 7 4 I D e c e m b e r 2 0 1 7 december 2017 vol.51 no.4 Radiol Oncol 2017; 51(4): A. December 2017 Vol. 51 No. 4 Pages 369-474 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Publisher Association of Radiology and Oncology Affiliated with Slovenian Medical Association – Slovenian Association of Radiology, Nuclear Medicine Society, Slovenian Society for Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medical Association – Croatian Society of Radiology Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups of S.I.R.M. Italian Society of Medical Radiology Aims and scope Radiology and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chief Gregor Serša, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Executive Editor Viljem Kovač, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Deputy Editors Andrej Cör, University of Primorska, Faculty of Health Science, Izola, Slovenia Maja Čemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Igor Kocijančič, University Medical Centre Ljubljana, Institute of Radiology, Ljubljana, Slovenia Karmen Stanič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Editorial Board Sotirios Bisdas, National Hospital for Neurology and Neurosurgery, University College London Hospitals, London, UK Karl H. Bohuslavizki, Facharzt für Nuklearmedizin, Hamburg, Germany Serena Bonin, University of Trieste, Department of Medical Sciences, Trieste, Italy Boris Brkljačić, University Hospital “Dubrava”, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Metka Filipič, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Maria Gődény, National Institute of Oncology, Budapest, Hungary Janko Kos, University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Robert Jeraj, University of Wisconsin, Carbone Cancer Center, Madison, Wisconsin, USA Tamara Lah Turnšek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Luka Milas, UT M. D. Anderson Cancer Center, Houston , USA Damir Miletić, Clinical Hospital Centre Rijeka, Department of Radiology, Rijeka, Croatia Häkan Nyström, Skandionkliniken, Uppsala, Sweden Maja Osmak, Ruder Bošković Institute, Department of Molecular Biology, Zagreb, Croatia Dušan Pavčnik, Dotter Interventional Institute, Oregon Health Science Universityte, Oregon, Portland, USA Geoffrey J. Pilkington, University of Portsmouth, School of Pharmacy and Biomedical Sciences, Portsmouth, UK Ervin B. Podgoršak, McGill University, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany , Mirjana Rajer, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Centre Ljubljana, Department of Gastroenterology, Ljubljana, Slovenia Katarina Šurlan Popovič, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Justin Teissié, CNRS, IPBS, Toulouse, France Gillian M.Tozer, University of Sheffield, Academic Unit of Surgical Oncology, Royal Hallamshire Hospital, Sheffield, UK 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 Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psychology, 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 Stojan Plesničar, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Tomaž Benulič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Radiol Oncol 2017; 51(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. Reader for English Vida Kološa Secretary Mira Klemenčič Zvezdana Vukmirović Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanović Layout Matjaž Lužar Printed by Tiskarna Ozimek, Slovenia Published quarterly in 400 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJE Zaloška cesta 2 1000 Ljubljana Slovenia Beneficiary bank account number: SI56 02010-0090006751 IBAN: SI56 0201 0009 0006 751 Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2, 1520 Ljubljana; Slovenia SWIFT: LJBASI2X Subscription fee for institutions EUR 100, individuals EUR 50 The publication of this journal is subsidized by the Slovenian Research Agency. Indexed and abstracted by: This journal is printed on acid- free paper On the web: ISSN 1581-3207 http://www.degruyter.com/view/j/raon http://www.radioloncol.com • Celdes • Chemical Abstracts Service (CAS) • Chemical Abstracts Service (CAS) - SciFinder • CNKI Scholar (China National Knowledge Infrastructure) • CNPIEC • DOAJ • EBSCO - Biomedical Reference Collection • EBSCO - Cinahl • EBSCO - TOC Premier • EBSCO Discovery Service • Elsevier - EMBASE • Elsevier - SCOPUS • Google Scholar • J-Gate • JournalTOCs • Naviga (Softweco) • Primo Central (ExLibris) • ProQuest - Advanced Technologies Database with Aerospace • ProQuest - Health & Medical Complete • ProQuest - Illustrata: Health Sciences • ProQuest - Illustrata: Technology • ProQuest - Medical Library • ProQuest - Nursing & Allied Health Source • ProQuest - Pharma Collection • ProQuest - Public Health • ProQuest - Science Journals • ProQuest - SciTech Journals • ProQuest - Technology Journals • PubMed • PubsHub • ReadCube • SCImago (SJR) • Summon (Serials Solutions/ProQuest) • TDOne (TDNet) • Thomson Reuters - Journal Citation Reports/Science Edition • Thomson Reuters - Science Citation Index Expanded • Ulrich's Periodicals Directory/ulrichsweb • WorldCat (OCLC) Radiol Oncol 2017; 51(4): C. review 369 Carcinogenesis induced by low-dose radiation Igor Piotrowski, Katarzyna Kulcenty, Wiktoria Maria Suchorska, Agnieszka Skrobała, Małgorzata Skórska, Marta Kruszyna-Mochalska, Anna Kowalik, Weronika Jackowiak, Julian Malicki nuclear medicine 378 Clinical significance of fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography in the follow-up of colorectal cancer: searching off approaches increasing specificity for detection of recurrence Semra Ince, Kursat Okuyucu, Oguz Hancerliogulları, Engin Alagoz, Huseyin San, Nuri Arslan radiology 386 Is there a role for contrast-enhanced ultrasound in the detection and biopsy of MRI only visible breast lesions? Aki Nykänen, Otso Arponen, Anna Sutela, Ritva Vanninen, Mazen Sudah 393 Baseline tumor Lipiodol uptake after transarterial chemoembolization for hepatocellular carcinoma: identification of a threshold value predicting tumor recurrence Yusuke Matsui, Masahiro Horikawa, Younes Jahangiri Noudeh, John A. Kaufman, Kenneth J. Kolbeck, Khashayar Farsad 401 The relationship between chondromalacia patella, medial meniscal tear and medial periarticular bursitis in patients with osteoarthritis Mustafa Resorlu, Davut Doner, Ozan Karatag, Canan Akgun Toprak experimental oncology 407 Phytotherapeutics oridonin and ponicidin show additive effects combined with irradiation in pancreatic cancer in vitro Jakob Liermann, Patrick Naumann, Franco Fortunato, Thomas E. Schmid, Klaus-Josef Weber, Jürgen Debus, Stephanie E. Combs 415 Focused transhepatic electroporation mediated by hypersaline infusion through the portal vein in rat model. Preliminary results on differential conductivity Clara Pañella, Quim Castellví, Xavier Moll, Rita Quesada, Alberto Villanueva, Mar Iglesias, Dolores Naranjo, Patricia Sánchez-Velázquez, Anna Andaluz, Luís Grande, Antoni Ivorra, Fernando Burdío contents contents Radiol Oncol 2017; 51(4): D. 422 Minimally invasive electrochemotherapy procedure for treating nasal duct tumors in dogs using a single needle electrode Felipe Maglietti, Matías Tellado, Nahuel Olaiz, Sebastian Michinski, Guillermo Marshall 431 Metformin enhanced in vitro radiosensitivity associates with G2/M cell cycle arrest and elevated adenosine-5′-monophosphate-activated protein kinase levels in glioblastoma Sebastian Adeberg, Denise Bernhardt, Semi B. Harrabi, Nils H. Nicolay, Juliane Hörner-Rieber, Laila König, Michael Repka, Angela Mohr, Amir Abdollahi, Klaus-Josef Weber, Juergen Debus, Stefan Rieken clinical oncology 438 Evaluation of deformable image registration (DIR) methods for dose accumulation in nasopharyngeal cancer patients during radiotherapy Wannapha Nobnop, Imjai Chitapanarux, Hudsaleark Neamin, Somsak Wanwilairat, Vicharn Lorvidhaya, Taweap Sanghangthum 447 Long term results of radiotherapy in vulvar cancer patients in Slovenia between 1997–2004 Helena Barbara Zobec Logar 455 Association between SLC19A1 gene polymorphism and high dose methotrexate toxicity in childhood acute lymphoblastic leukaemia and non Hodgkin malignant lymphoma: introducing a haplotype based approach Barbara Faganel Kotnik, Janez Jazbec, Petra Bohanec Grabar, Cristina Rodriguez-Antona, Vita Dolžan 463 Impact on radiation dose and volume V57 Gy of the brain on recurrence and survival of patients with glioblastoma multiformae Igor Stojkovski, Valentina Krstevska, Snezhana Smichkoska radiophysics 469 Quality assurance procedures based on dosimetric, gamma analysis as a fast reliable tool for commissioning brachytherapy treatment planning systems Grzegorz Zwierzchowski, Grzegorz Bieleda, Janusz Skowronek I slovenian abstracts contents Radiol Oncol 2017; 51(4): 369-377. doi: 10.1515/raon-2017-0044 369 review Carcinogenesis induced by low-dose radiation Igor Piotrowski1, Katarzyna Kulcenty1,2, Wiktoria Maria Suchorska1,2, Agnieszka Skrobała2,3, Małgorzata Skórska3, Marta Kruszyna-Mochalska2,3, Anna Kowalik3, Weronika Jackowiak4, Julian Malicki2,3 1 Radiobiology Laboratory, Department of Medical Physics, Greater Poland Cancer Centre, Poznań, Poland 2 Department of Electroradiology, University of Medical Sciences, Poznań, Poland 3 Department of Medical Physics, Greater Poland Cancer Centre, Poznań, Poland 4 Radiotherapy Ward I, Greater Poland Cancer Centre, Poznań, Poland Radiol Oncol 2017; 51(4): 369-377. Received 4 July 2017 Accepted 25 September 2017 Correspondence to: Dr. Katarzyna Kulcenty, Radiobiology Laboratory, Department of Medical Physics, Greater Poland Cancer Centre, Garbary 15 Street, Poznań, Poland. E-mail: katarzyna.kulcenty@wco.pl Igor Piotrowski and Katarzyna Kulcenty contributed equally to this work. Disclosure: No potential conflicts of interest were disclosed. Background. Although the effects of high dose radiation on human cells and tissues are relatively well defined, there is no consensus regarding the effects of low and very low radiation doses on the organism. Ionizing radiation has been shown to induce gene mutations and chromosome aberrations which are known to be involved in the process of carcinogenesis. The induction of secondary cancers is a challenging long-term side effect in oncologic patients treated with radiation. Medical sources of radiation like intensity modulated radiotherapy used in cancer treatment and computed tomography used in diagnostics, deliver very low doses of radiation to large volumes of healthy tissue, which might contribute to increased cancer rates in long surviving patients and in the general population. Research shows that because of the phenomena characteristic for low dose radiation the risk of cancer induction from expo- sure of healthy tissues to low dose radiation can be greater than the risk calculated from linear no-threshold model. Epidemiological data collected from radiation workers and atomic bomb survivors confirms that exposure to low dose radiation can contribute to increased cancer risk and also that the risk might correlate with the age at exposure. Conclusions. Understanding the molecular mechanisms of response to low dose radiation is crucial for the proper evaluation of risks and benefits that stem from these exposures and should be considered in the radiotherapy treat- ment planning and in determining the allowed occupational exposures. Key words: low-dose radiation; carcinogenesis; DNA; radiation induced bystander effect Introduction Ionizing radiation is widely used in medical pro- cedures, including cancer radiotherapy and the use in diagnosis.1 For over a century the biological effects of medium- and high-dose radiation (over 0.5 Gy) on human health have been investigated. Induction of cancer is one of the most severe long- term effects of radiotherapy. While the relationship between radiation effects and dose is well defined at higher doses, the effects of doses below 0.5 Gy are still unclear. Even though the use of radiother- apy resulted in a significant increase in cancer sur- vivors2, it is important to investigate the potential negative long-term effects of radiation, considering that further advancements in therapy result in in- creased length of patients life after therapy. Table 1 presents the classification of radiation doses ac- cording to Kadhim et al.3 paired with examples of exposures. The damage to DNA is induced by radiation through two mechanisms: the direct and indirect effect.9 Directly induced damage results from the deposition of radiation energy in the DNA mol- ecule creating a break. Damage induced indirectly is a result of an attack of the reactive species, which might be produced from ionization of water, on DNA molecule. These processes induced by radia- Radiol Oncol 2017; 51(4): 369-377. Piotrowski I et al. / Carcinogenesis indiced by low-dose radiation370 tion result in creation of single-strand breaks (SSB) and double-strand breaks (DSB) in DNA mole- cule.10 DSBs are prime lesions induced by ionizing radiation, and are responsible for its deleterious ef- fect. Densely ionizing radiation has the ability to induce more damage per cell, making it possible for two or more lesions to be induced in proximity. These groups of breaks are called clustered DNA lesions, and are harder to repair than single DSBs and SSBs.11 Density of radiation correlates with reparability of DNA lesions, with densely ion- izing radiation being harder to repair. Radiation stimulates DNA damage repair through non-ho- mologous end-joining (NEHJ)12, an error prone pathway that can lead to induction of chromosome aberrations, which in turn may cause genomic in- stability.13 Genomic instability has been connected to induction of most of the human cancers, how- ever currently the exact mechanism of radiation- induced carcinogenesis is not clear. The dose-rate of radiation plays a major role in how much damage an exposed cell receives. High dose-rate irradiation, like radiation created by atomic bomb, results in deployment of energy in very short time to every irradiated cell. However if radiation is protracted over long periods of time, like the dose-rates considered in calculations of oc- cupational and environmental radiation risks, the cell turnover influences how many ‘hits’ a cell will receive. Low-dose rates also influence how cell re- acts regarding the repair of DNA damage, since low-dose rates allow more time for damage to be repaired which makes it more favorable for cells than high-dose rate radiation.14 This review contains a description of biological consequences of low-dose radiation and possible induction of cancer (Figure 1). DNA damage Damage to DNA is one of the most important fac- tors in radiation induced cancer transformation. Even though ionizing radiation induces one DSB per 20 SSBs, research indicates that DSBs are much more impactful effect of irradiation (IR).15 This dif- ference might be caused by the fact that SSBs are repaired by error-free mechanisms and are not sustained, while DSBs induced by radiation are repaired by mechanisms prone to mis-repair and repair failure, making DSBs a main cause of radia- tion induced cell death.16 Recently many authors pointed towards important differences between DNA damage response exerted by high and low doses of radiation.17,18 Research of low dose effect on normal tissues is especially significant consider- ing side effects of radiotherapy treatment. Induction of DSBs is considered to be one of the main mechanisms by which radiation exerts its deleterious effect. One of the earliest events appearing in response to DSB induction is phos- phorylation of histone H2AX by protein kinases: DNA-dependent protein kinase catalytic subu- nit (DNA-PKcs), ataxia telangiectasia and Rad3- related (ATR) and ataxia telangiectasia mutated (ATM).19 Phosphorylated H2AX (γH2AX) foci ap- pears around the DNA break as a part of DSB re- pair mechanism20 and it corresponds directly with the number of DSBs.21 The correlation between the radiation dose and the initial number of γH2AX foci it induces has already been thoroughly de- scribed.21,22 Rothkamm et al.23 have shown that this linear correlation holds true also for the X-ray doses as low as 1 mGy (at dose rate of 6–60 mGy/ min) for nondividing human fibroblasts cultured in vitro. Interestingly, authors came to the conclu- TABLE 1. Dose ranges and sources of radiation exposure Dose range Examples of exposure Very low doses < 0.05 Gy Mammography4, chest X-ray4 Low doses 0.05–0.5 Gy Cardaic CT angiogram4 Medium doses 0.5–5 Gy One fraction dose in standard fractionated radiotherapy5, dose absorbed by workers during Fukushima accident6 High doses 5–15 Gy Intraoperative radiotherapy (as boost)7 Very high doses > 15 Gy Intraoperative radiotherapy8, cumulative dose delivered during fractionated radiotherapy5 targeted effects non-targeted effects ssDNA breaks dsDNA breaks cell cycle arrest HRS RIBE RIGI FIGURE 1. Diagram illustrating targeted and non-targeted effect induced by radiation (all described in the main text). Radiol Oncol 2017; 51(4): 369-377. Piotrowski I et al. / Carcinogenesis indiced by low-dose radiation 371 sion that the damage induced by these very low doses remains unrepaired and evidence suggests that the cells with unrepaired DSBs are eliminated. Similar results were observed recently by Osipov et al. in research conducted on human MSCs isolated from oral mucosa.24 Authors measured kinetics of γH2AX and pATM foci formation after irradiation with X-rays at doses ranging from 20 mGy to 250 mGy and a dose rate of 40 mGy/min. Linear cor- relation between the number of γH2AX foci and radiation dose was observed at all time-points af- ter irradiation. For the intermediate doses (160 and 250 mGy) the highest number of foci was observed at 60 min followed by a significant decrease, which is typical for γH2AX kinetics observed at higher doses. However for the lower doses (20, 40 and 80 mGy) after initial rise in γH2AX foci number no decrease was observed. Considering the fact that pATM foci did not co-localize with γH2AX foci at low doses and the fact that low dose radiation stim- ulates the MSC proliferation25, authors propose that the persistence of γH2AX foci after irradia- tion with low dose X-rays might be a consequence of new DSBs appearing as a result of replication stress rather than resulting from inefficient repair of DSBs. It is important to note that doses used in these experiments are in range of doses used in di- agnostic procedures like CT scan.26 However high linear energy transfer (LET) radiation has also been shown to induce ATM-dependent responses at low doses.27 Fibroblasts isolated from patients with ataxia telangiectasia and healthy persons were ir- radiated with carbon ion beams (LET = 70 keV/µm) or with X-rays at doses between 0.1 to 2 Gy. Results have shown that mutation frequency and cell sur- vival after low dose carbon ion irradiation were dependent on ATM status. Authors also compared the number of γH2AX foci in cells with different activity of ATM irradiated with 0.2 Gy of carbon ion radiation. Results indicate that ATM plays an important role in early DSB recognition and can be responsible for inefficient DSB repair, which is in line with conclusions reached by Osipov et al.24 Authors also observed that doses higher than 0.5 Gy of carbon ion radiation caused an early G2/M arrest dependent on ATM. Some authors suggest that occurrence of this arrest might support DNA DSB repair, while a failure to induce early G2 arrest can be one of the factors responsible for low dose hyper-radiosensitivity (HRS).28 Low-dose hyper radiosensitivity (HRS) HRS is described as an increase in radiosensitivity in cells exposed to low-dose radiation, usually be- low 0.2–0.3 Gy for low LET radiation. When cells are exposed to doses higher than 0.3 Gy an increase in radioresistance is observed and the transition towards it on dose response curve is described as increased radioresistance (IRR).29 Figure 2 presents the dose-response curves with and without the evi- dence of HRS and IRR. Occurrence of HRS and IRR in cells was first described using an in vitro culture of V79 Chinese hamster cells.30 Cells were exposed to X-ray radiation doses ranging between 0.01 and 10 Gy. Using clonogenic assay authors have shown increased effectiveness of X-ray doses be- low 0.2 Gy. Cells exposed to doses higher than 0.4 Gy experienced a rise in radioresistance. Authors hypothesized that only X-ray doses above certain threshold can induce enough damage to activate DNA damage repair mechanisms. The dependence of HRS occurrence on cell cycle phase was also firstly noted in this research. Since then, research of HRS on different cell lines reported that HRS is increased in G2-phase cells.31,32 To prove this con- cept Short et al.31 used two glioma cell lines – T98G exhibiting HRS and U373 that did not exhibit HRS. Using cell counting authors have shown that G2 population of T98G cells irradiated with low dose X-rays demonstrated more pronounced HRS than cells in asynchronous population. Interestingly the G2 population of U373 cells also exhibited HRS in- dicating that in the asynchronous population the response of G1 and S-phase cells dominates. These results show that actively proliferating cells might 0,5 0,6 0,7 0,8 0,9 1 1,1 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 Su rv iv in g Fr a ct io n Dose [Gy] HRS IRR FIGURE 2. Schematic diagram illustrating cell survival curve for cells exhibiting low dose hyper radiosensitivity (HRS). Solid line shows survival curve with evidence of HRS and increased radioresistance (IRR). Dashed line shows survival curve with no evidence of HRS and IRR. Image adapted from.29 Radiol Oncol 2017; 51(4): 369-377. Piotrowski I et al. / Carcinogenesis indiced by low-dose radiation372 be more sensitive to low doses of radiation. It is im- portant to note, that HRS can potentially be used to therapeutic advantage. Using ultra-fractionation with doses in HRS range it is possible to achieve more cytotoxic effects than after administration of same total dose at once.33 Cell cycle arrest In order to repair the damaged DNA, irradiated cells utilize cell cycle arrest to allow sufficient time for the DNA repair. The most important check- point pathways that initiate the radiation-induced arrest inhibit the cell cycle progression from G2 phase to mitosis. Two checkpoints are utilized for the repair of DNA damage induced by radiation. The classic G2/M checkpoint is activated after high doses of radiation and its role is to arrest the cells damaged in S or G1 cell cycle phase.34 The early G2/M checkpoint is activated shortly after expo- sure to radiation and it exclusively protects cells in G2 phase of cell cycle from radiation effects.35 This checkpoint is especially relevant to assessment of low-dose radiation risks because induction of this checkpoint occurs after irradiation with doses up to 10 Gy of low LET radiation35 and a threshold for its activation is observed at radiation doses of 0.3 Gy.36 This threshold dose coincides with radia- tion doses at which a transition between HRS and IRR is observed, suggesting a correlation between the two phenomenon, which was first suggested by Marples et al.36 The activation of early G2/M checkpoint is mediated by radiation-induced, DSB- dependent phosphorylation of ATM.37 It has been hypothesized that ATM acts by an interplay with Chk1 and Chk2.32 Inhibition of these factors in fi- broblast cells results in an enhanced HRS response, however it also causes an increase in IRR. It is im- portant to note that the response varied depending on the HRS status of the cell lines used. While even very low doses of radiation are capable of inducing DSBs, there is evidence sug- gesting a threshold number of 10–20 DSBs for the triggering of ATM dependent G2/M checkpoint.38 This can mean that low doses of radiation inducing few DSBs may not trigger G2/M arrest in irradiated cells, making it possible for cells with unrepaired DSBs to enter mitosis, which in turn might result in loss of genetic material.39 Deckbar et al.40 investi- gated induction of G2 arrest by X-ray radiation on immortalized fibroblasts and on primary human cells. γH2AX foci count confirms, that cells irra- diated with X-ray doses inducing fewer than ~20 DSBs (below 0.6 Gy for immortalized fibroblasts and below 0.2 Gy for primary human cells) did not enter G2 arrest. In cells irradiated with doses above the threshold dose, cells that exited the G2 checkpoint still had ~20 unrepaired DSBs and 1–2 chromosome breaks. These results indicate that a certain threshold of DSBs and chromosome aberra- tions is necessary for both activating and maintain- ing the G2 checkpoint arrest. The threshold dose at which G2 arrest is induced can play a role in low- dose radiosensitivity of a cell. Different cell lines show variability in this threshold dose. Although this threshold usually varies between 0.2 Gy and 0.6 Gy, in some cell lines the cell cycle arrest is in- duced without threshold.41 Currently the mecha- nism behind this difference in threshold dose is not known, however a hypothesis has been proposed stating that cell lines which show early G2 check- point after exposure to very low doses might have higher base level of DSBs, making it much easier to reach the number of DSBs needed for G2 arrest induction.41 Mutation in ATM gene observed in Ataxia telan- giectasia, has been connected with increased chro- mosomal radiosensitivity and increased suscepti- bility to cancer.42 Since both DNA damage repair and G2 checkpoint induction depend on ATM ki- nase, Ataxia telangiectasia (AT) cells are often used to investigate the mechanisms of these processes. Using AT cells researchers were able to show for the first time that G2 checkpoint facilitates not only the repair of DSBs, but also the repair of chromo- somal breaks, after irradiation with 1 Gy ionizing radiation.43 Failure to activate the G2 checkpoint might be one of the reasons for increased cancer rates in patients with Ataxia telangiectasia, since unrepaired damage to DNA might alter cells ge- nome leading to malignant transformation.42 Non-targeted effects Radiation induced genomic instability (RIGI) Radiation induced DNA damage, specifically DSBs, are very important in occurrence of chromo- somal instability, which may involve aneuploidy, deletions, and aberrations, which in turn might contribute to carcinogenesis.44 The non-targeted ef- fects, including bystander effects and genomic in- stability, contribute to the induction of cancer in a less clear way.45 The progeny of exposed cell might exhibit phenotypes such as chromosomal aberra- tions and rearrangements, gene mutations and en- Radiol Oncol 2017; 51(4): 369-377. Piotrowski I et al. / Carcinogenesis indiced by low-dose radiation 373 hanced cell death rate.46 Acquisition of alterations in the genome of progeny of irradiated cells is de- scribed as genomic instability (GI) and it has been accepted as the hallmark of cancer cells and one of the most important factors involved in the de- velopment of some cancers.44 RIGI was firstly ob- served in one-cell mouse embryos irradiated with X-rays and with neutrons.47 The results suggested that chromosomal aberrations might appear de no- vo two or three mitoses after initial exposure and also that radiation of a different quality (different LET) induces aberrations at different frequencies. In the context of RIGI the effect on genome is mediated via the accumulation of genetic changes in the progeny of surviving irradiated cells through many generations.48 The capacity to induce genom- ic instability depends on both dose and quality of radiation, with high LET radiation generally in- ducing chromosome- and chromatid-type aberra- tions more effectively.49 This effect was observed in murine bone marrow cells irradiated with 0.25 Gy, 0.5 Gy and 1 Gy of densely ionizing α-radiation.50 Colonies arising from irradiated cells exhibited high frequency of non-clonally induced chromatid aberrations consistent with them arising de novo and not directly from irradiation. Early studies us- ing human lymphocytes irradiated in vitro show the ability of low dose X-rays to induce chromo- somal aberrations.51 Linearity of chromosomal ab- erration yield was observed above doses of 20 mGy while doses below 20 mGy resulted in yields lower than background. Many researchers have tried to elucidate the mechanisms behind RIGI, especially induced by low dose radiation. Maxwell et al. ob- served that in HMEC cells irradiated with X-ray doses ranging from 10 to 200 cGy frequency of chromosomal aberrations increased with dose.52 In cells irradiated with 50 cGy the frequency of chro- mosome aberrations increased with time after ir- radiation up to 72 h. Interestingly, administration of TGF-β, a growth factor whose activity is induced by radiation, resulted in elimination of cells with chromosome aberrations. The elimination was me- diated by induction of p53-dependent apoptosis in genomically unstable cells. Authors also con- cluded that chromosomal aberrations induced in this experiment are a targeted effect, as induction of chromosomal aberrations above 10 cGy was pro- portional to dose. TGF-β signaling can be induced in nontrans- formed cells irradiated with very low doses of radiation. It has been shown that TGF-β signaling selectively induces reactive oxygen species (ROS) production in transformed cells leading to their apoptosis.53 This signaling pathway could be a part of surveillance network protecting cells from malignant transformation. It is important to note, that radiation induced EMT phenotype can be me- diated by TGF-β, making it a “two-edged sword” in need of further investigation.54 Authors suggest that while TGF-β might play a role in elimination of irradiated, genomically unstable cells, the chron- ic exposition to radiation and TGF-β signaling can induce EMT in remaining cells.53 Dicentric chromosomes are an established marker used to assess IR induced chromosomal aberrations.55 Using the measurement of dicentric frequency in human fibroblasts after irradiation, researchers were able to investigate the role of dose-rate effect in induction of chromosome ab- errations.13] In cells irradiated with a single X-ray dose the frequency of dicentrics correlated expo- nentially with doses higher than 0.2 Gy. Irradiation of cells with a single 1 Gy dose of X-rays resulted in similar rise of frequency of dicentrics as irradia- tion with fractionated 1 Gy dose (fractionation: 10 x 0.1 Gy; 5 x 0.2 Gy; 2 x 0.5 Gy) given with inter- vals of 1 min between fractions. When intervals between fractions were increased above 5 min, the frequency of dicentric chromosomes was signifi- cantly lower, which suggests that the maximum of DNA damage repair, in relation to chromosome di- centrics, is reached after 5 min. These results show that high dose rate irradiation is more harmful to the genome than low dose rate. The measurement of frequency of dicentrics in patients lymphocytes could be utilized in examination of radiosensitiv- ity. Linear dose response of dicentric frequency was observed in human lymphocytes after irradia- tion with X-ray doses above 20 mGy.51 Frequency of dicentrics was also used to evaluate human lym- phocytes response to low LET γ-rays. While for doses above 20 mGy a linear increase in dicentric frequency was observed, authors were not able to detect statistically significant changes in dicentric frequencies below 20 mGy doses, despite counting over 5000 metaphases.56 This means that dicentric frequency count might not be a precise enough method for assessment of response to very low doses. Aneuploidy is a phenotype very often observed in tumors and it arises from incorrect chromosome segregation during mitosis.57 Delayed appearance of aneuploid cells induced by low-dose radiation has been observed in vitro in human fibroblasts.58 Fibroblasts were irradiated with X-ray doses of 20, 50 or 100 cGy and cultured for five passages. As an end point authors picked assessment of mi- Radiol Oncol 2017; 51(4): 369-377. Piotrowski I et al. / Carcinogenesis indiced by low-dose radiation374 cronuclei (MN) formation with special probes for analysis of structural and numerical aberrations59, with chromosomes 1 and 4 picked for aneuploidy analysis. Micronuclei are generated in the progeny of irradiated cells as a result of de novo generation of chromosome aberrations.49 After X-ray irradia- tion with doses of 50 and 100 cGy frequencies of centromere-positive and negative MNs increased significantly. Frequency of aneuploidy increased with dose in a dose-dependent manner and after irradiation with 50 and 100 cGy the increase in ane- uploidy with time was observed. The data show that after irradiation with low doses, chromosomal aberrations appear de novo up to five population doublings, which as authors suggest, might be connected with variability in the number of cen- trosomes.60 One of the radiation induced phenotypes used to investigate genetic effects of low-dose radiation is loss of heterozygosity (LOH). LOH is convention- ally associated with cancer as a mechanism inacti- vating tumor suppressor genes, however it is also found in regions of genome responsible for cancer induction.61 For research on induction of mutations in vitro, TK6 cells are often used, as this cell line contains heterozygosity at the thymidyne kinase (TK) locus. In one of the experiments irradiation of TK6 cells with 10 cGy X-rays caused a signifi- cant increase in hemizygous LOH.61 Hemizygous type of LOH is a result of homologous end join- ing, a repair pathway observed after irradiation. Umebayashi et al. analyzed induction of LOH in TK6 cells after irradiation with low-dose, low dose- rate γ-irradiation.62 Even irradiation with the dose as low as 30 mGy increased the frequency of early mutations in TK gene almost 2-fold. Authors claim that detection of LOH is an efficient system for estimation of genetic effects induced by low-dose radiation. Radiation induced bystander effect Another non-targeted effect playing a role in car- cinogenesis is radiation induced bystander effect (RIBE). This effect together with other non-target- ed effects, is often described as most relevant to low-dose radiation63 and is mediated through two mechanisms: secretion of soluble factors by irradi- ated cells64 and also by signaling through cell-to- cell junctions.65 One of the early works in which this effect was described used Chinese hamster ovary (CHO) cells irradiated with low doses of α-radiation.66 The authors chose very low radia- tion dose (0.31 mGy), so that less than 1% of cells were traversed through by the radiation. After ir- radiation close to 30% cells exhibited presence of sister chromatid exchanges which suggests, that the genetic damage was induced even in the cells whose nuclei was not traversed through by radia- tion. Authors speculated that this effect might have been mediated through the production of ROS by the cells which were irradiated directly. The oc- currence of RIBE was also confirmed for very low doses of X-ray radiation in human fibroblasts.67 After irradiation with doses ranging from 1.2 to 200 mGy the induction of DSBs, measured by ATM phosphorylation, followed a supralinear relation- ship. While cells were treated with an inhibitor of gap junction intercellular communication, the number of DSBs induced by radiation was smaller than in untreated cells. The largest differences be- tween treated and untreated cells were observed at doses between 1.2 to 5 mGy, meaning that RIBE has the most influence on DSB induction at doses up to 10 mGy. RIBE has also been shown to induce mutations in bystander cells and the mechanism of these mutations is different than in the cells irra- diated directly.68 In bystander CHO cells, the fre- quency of deletions was higher after using 10 cGy α-radiation and after using 0.5 cGy α-radiation the frequency of point mutations was higher. Very low doses of α-radiation also have the ability to induce ROS production in irradiated cells69 and ROS has been shown to induce point mutations70 making it the likely mechanism for induction of mutations in bystander cells. However it is not clear whether the effect is mediated through ROS produced by irra- diated cells and transferred to unirradiated cells, or the irradiated cells produce factors inducing ROS production in uniradiated cells. The role of factors secreted by irradiated cells has also been confirmed for low-dose induced RIBE. Seymour and colleagues71 irradiated human keratinocytes with doses ranging from 0.01 to 0.5 Gy of γ-radiation. Then the medium from irradi- ated cells was collected and transferred to unir- radiated cell culture. The results of clonogenic as- says measuring clonogenic death of cells show that RIBE mediated by the secreted substances is most predominant in doses below 0.5 Gy. RIBE has also been confirmed to occur in vivo and cause chro- mosomal instabilities. To first demonstrate this effect in vivo, Watson et al.72 transplanted a mix- ture of irradiated (0.5 Gy, neutrons) and unirradi- ated bone marrow cells into unirradiated recipient. After transplantation some of the progeny of the unirradiated donor cells showed chromosomal aberrations induced through the bystander effect. Radiol Oncol 2017; 51(4): 369-377. Piotrowski I et al. / Carcinogenesis indiced by low-dose radiation 375 Evidence has also been presented for oncogenic ef- fect of RIBE in in vivo mouse model, however the effect was observed only after irradiation with 3 Gy of X-rays.73 Currently no research was able to con- firm with confidence the oncogenic effect of low- dose RIBE, and even the exact mechanism of RIBE in vivo is not clear.74 Presented results suggest that bystander effect plays an important role in induc- tion of mutations after low dose irradiation, mak- ing it a potential oncogenic factor. Conclusions Radiation to which humans are exposed comprises mainly of low-dose and low-dose rate radiation from both natural and man-made sources. In re- cent years the biological effects of low-dose radia- tion became a point of interest due to the increase in popularity of radiation therapy and diagnostic radiology. Even though many studies point to- ward a link between carcinogenesis and exposure to radiation, the exact mechanism is still not clear. Induction of genomic instability is suspected to play a major role in malignant transformation after high-dose irradiation, and it might be responsible for carcinogenesis after exposure to lower doses. Latest research suggests that phenomena charac- teristic for low-dose exposures like HRS and RIBE might be the factors contributing to induction of genomic instability after exposure. Better under- standing of these processes is crucial for the proper estimation of low-dose exposure risks for radia- tion workers, patients and people exposed to high background radiation. Acknowledgement This work was supported by the National Science Centre [grant number: 2015/19/B/NZ7/03811]. References 1. Ron E. Ionizing radiation and cancer risk: evidence from epidemiology. Pediatr Radiol 2002; 32: 232-7. doi: 42-410.1007/s00247-002-0672-0 2. 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Received 19 July 2017 Accepted 10 September 2017 Correspondence to: Semra Ince M.D., University of Health Sciences, Gulhane Training and Research Hospital, Department of Nuclear Medicine, 06010 Etlik-Ankara, Turkey. Phone: +90 312 304 48 28; E-mail: drsemra@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Nearly 40% of colorectal cancer (CRC) recurs within 2 years after resection of primary tumor. Imaging with fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) is the most recent modality and often applied for the evaluation of metastatic spread during the follow-up period. Our goal was to study the diagnostic importance of 18F-FDG-PET/CT data of maximum standardized uptake value (SUVmax), total lesion glycolysis (TLG) and the difference of SUVmax on dual-time imaging in CRC. Patients and methods. We examined the SUVmax value of lesions on control or restaging 18F-FDG-PET/CT of 53 CRC patients. All lesions with increased SUVmax values were confirmed by colonoscopy or histopathology. We compared PET/CT results with conventional imaging modalities (CT, MRI) and tumor markers (carbohydrate antigen 19-9 [Ca 19- 9], carcinoembryonic antigen [CEA]). Results. Mean SUVmax was 6.9 ± 5.6 in benign group, 12.7 ± 6.1 in malignant group. Mean TLG values of malignant group and benign group were 401 and 148, respectively. 18F-FDG-PET/CT was truely positive in 48% of patients with nor- mal Ca 19-9 or CEA levels and truely negative in 10% of cases with elevated Ca 19-9 or CEA. CT or MRI detected suspi- cious malignancy in 32% of the patients and 18F-FDG-PET/CT was truely negative in 35% of these cases. We found the most important and striking statistical difference of TLG value between the groups with benign and recurrent disease. Conclusions. Although SUVmax is a strong metabolic parameter (p = 0.008), TLG seems to be the best predictor in recurrence of CRC (p = 0.001); both are increasing the specificity of 18F-FDG-PET/CT. Key words: metabolic tumor markers; recurrent colorectal cancer; 18F-fluorodeoxyglucose positron emission tomog- raphy; computed tomography Introduction Colorectal cancer (CRC) ranks in the third line amongst the most common cancers all over the world. Roundly 40% of patients recur within 2 years after resection of primary tumor by surgery.1 In the follow-up, most guidelines recommend thoracoabdominal CT usually at 12th, 36th months after surgery or any time in case of clinical doubt as well as routine serial carcinoembryonic antigen Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer 379 (CEA) and carbohydrate antigen 19-9 (Ca 19-9) assays.2 Imaging has the main role for the evalu- ation of metastatic spread during the follow-up. Molecular imaging with 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography (18F-FDG-PET/CT) is the most recent modality for this purpose.3 18F-FDG- PET/CT has been used for baseline staging, assess- ment of treatment response and restaging of CRC as in many other cancers and is concerned to be more sensitive and specific imaging method than routine tools in cases of dubious recurrence and/ or metastasis.2,3 CEA is expressed by a lot of epithelial tumors and its serum levels may increase in non-malig- nant conditions such as inflammatory bowel dis- eases.4 Approximately 70% CRC patients exhibit an elevated CEA level at the time of diagnosis and this fact made it a routine monitoring marker for the disease recurrence.5,6 Nevertheless, recent studies of meta-analyses revealed controversies about its utility for the detection of recurrence with a sensi- tivity of 64% and a specificity of 90% which might be considered poor as a biomarker on its own goal.7 Ca 19-9 assays have also a poor performance. It has been reported that Ca 19-9 was positive only in 20- 40% of metastatic CRCs.8 18F-FDG-PET has the ability to detect recurrent CRC (as in many other cancers), through patho- logically increased tissue metabolism, which pre- cedes the appearance of morphological chang- es.3,9,10 18F-FDG-PET, however has some intrinsic limitations and its use in the monitoring of CRC is vexed.11,12 Latest data offer no indication except the cases with inconclusive CT with suspicion of dis- tant metastasis or in the existence of negative CT and serial CEA rises.13 Some current interventions on 18F-FDG-PET/CT such as dual-time or voxel- based dual-time parametric imaging and use of metabolic tumor parameters have been suggested to improve its diagnostic accuracy in several can- cers.14 Previously, quantitative analyses based on volume-of-interest FDG uptake were introduced. Maximum standardized uptake value (SUVmax) is the vanguard of them. Determination of a cutoff lev- el of SUVmax which differentiates between benign conditions and recurrence of CRC would certainly be helpful. The goal of this paper is to appraise clinical significance of 18F-fluorodeoxyglucose up- take on FDG-PET/CT in the aftermath of primary curative surgery and/or chemoradiotherapy with respect to recurrence in patients with CRC. We also aimed to research the diagnostic power of 18F-FDG- PET in recurrent CRC over total lesion glycolysis (TLG), the difference of SUVmax on dual-time imaging, calculation of a cutoff point of SUVmax discriminating metastasis/recurrence from benign conditions on restaging 18F-FDG-PET/CT. Patients and methods This retrospective cohort study was carried out be- tween 2011 and 2016. It was conducted at nuclear medicine department of a tertiary health care hos- pital. Inclusion criteria were: histopathologically proven CRC by surgical specimen after primary curative surgery, pathologic FDG uptake on con- trol (evaluation of treatment response) 18F-FDG- PET/CT or restaging 18F-FDG-PET/CT performed for the existence of suspicious recurrence or metas- tasis by routine conventional screening methods in the follow-up, confirmation of all these abnormal uptakes by colonoscopy or histopathologic exami- nation. All cases were treated by surgery and/or chemoradiotherapy. The files of the patients were retrieved from the archive and looked over retro- spectively. We evaluated the lesions on 18F-FDG-PET/CT in 53 patients. Indications for 18F-FDG-PET/CT were suspicion of recurrence/metastasis (27 patients) and treatment response monitoring (26 patients). Elevated CEA and/or Ca 19-9 levels raised the sus- picion of recurrence in 10 cases, conventional im- aging (CT or MRI) in 17 cases. All foci of FDG up- take were confirmed by colonoscopic findings and/ or histopathologically. Normal range of Ca 19-9 is 0-35 U/mL, CEA < 2.5 ng/ml for nonsmokers and <5 ng/ml for smokers. 18F-FDG-PET/CT imaging protocol 370-555 MBq of 18F-FDG, calculated according to body weight, was administered to patients by in- travenous injection. They fasted for 6 hours prior to the examination and their blood glucose level needed to be below 150 mg/dl before the injec- tion. Image acquisition was performed 1 hour after the injection with an integrated PET/CT scanner (Discovery 690-GE Healthcare, WI, USA). A low- dose unenhanced CT was performed. CT data were obtained with the automated dose modulation technique of 120 kVp (maximal 100 mA), collimat- ed by 64×0.625 mm, measured field of view (FOV) of 50 cm, noise index of 20% and reconstructed to images of 0.625 mm transverse pixel size and 3.75 mm slice thickness. PET emission data were ob- tained from the middle of thigh up to vertex of the Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer380 skull while the patient was in supine position with the arms rised over head. Acquired PET data were in 3D mode with scanning time of 2 min per bed position and an axial FOV of 153 mm. A standard- ized way (random, scatter and attenuation) and it- erative reconstruction (matrix size 256×256, Fourier rebinning, VUE Point FX [3D] with 3 iterations, 18 subsets) were used for correction of emission data. Dual-time 18F-FDG-PET/CT was performed in 28 patients. 105 ± 10 minutes post-injection after the completion of standard protocol, delayed imaging for the whole abdomen with the CT scan and repo- sitioning was performed. Visual and quantitative interpretation 18F-FDG-PET/CT images were interpreted visually by two nuclear medicine specialists aware of pa- tient history. Focally or heterogeneously increased FDG uptake, diffuse or heterogeneously increased FDG uptake and/or soft tissue mass on CT compo- nent, hipodense or nodular lesion on CT (with or without FDG uptake), diffuse uptake accompanied by wall tickening, consolidation or ambiguous lesions on CT (with or without uptake) were ac- cepted pathologic. SUVmax was calculated for all patients. Other quantitative parameters of average standardized uptake value (SUVmean), metabolic tumor volume (MTV) and TLG were calculated in 20 cases on 18F-FDG-PET/CT. We calculated TLG values by multiplying MTV and SUV mean. The corresponding CT scans of lesions were used as a guideline to demarcate them if their bounda- ries were difficult to define for the calculation of SUVmax. Quantitative PET/CT parameters were calcu- lated from a routine protocol used on a sophisti- cated workstation (Volumetrix for PET-CT and AW volume share 4.5, GE Healthcare, Waukesha, WI, USA). Standard methods computed SUVmax and SUVmean, corrected for body weight, from the voxel having the most intense activity in three-di- mensional tumor region on transaxial whole-body images of attenuation-corrected PET/CT images. MTV (cm3) was measured with a half-automatic PET analysis computer program, having an au- tomatic isocontour threshold method based on a notion of being greater than 42% of the SUV max value within the tumor. Statistical analysis The whole data were analysed by IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp. Number and percentage values were used for the description of categorical data. Mean, median, standard de- viation (SD), minimum and maximum values de- scribed continuous data. Intergroup (benign condi- tions versus malignant group) comparisons were carried out by Mann-Whitney U test for SUVmax, TLG and the difference between SUVmax values of dual-time imaging. Wilcoxon test was used for ingroup comparison between SUVmax values of early and delayed imaging for benign and malig- nant group. The variables having a value of p < 0.05 were accepted as statistically meaningful. ROC curve analysis was plotted to see the diagnostic value of SUVmax on recurrent disease. Informed consent was supposed as a retrospective study which permitted to use records, documents and data of patients applied on our clinic for the test. The study was ratified by our Institutional Review Ethics Committee (approval number 80/2016). This study conforms to the Declaration of Helsinki. Results Mean age was 58.6 ± 10.9 years (30–89). 24 of the patients were male (45%), 29 of them were female (55%). Primary tumor sites were rectum (n = 23), sigmoid colon (n = 5) and other colonic segments (n = 25). The most common localization of FDG uptake was rectosigmoid region (43.3%). Locations of pathologic FDG uptake were demonstrated in Table 1. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 18F-FDG-PET/CT in the detection of recurrence TABLE 1. Locations of pathologic FDG uptake Sites of abnormal FDG uptake n Anostomosis line (area) 7 Rectum 9 Rectosigmoid region 11 Liver 9 Caecum 1 Kidney 1 Abdominal mass 4 Presacral mass 5 Sigmoid region 3 Descending colon 2 Lung 1 Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer 381 and/or metastasis were 100%, 51.7%, 63.1% and 100%, respectively. 18F-FDG-PET/CT results and fi- nal histopathologic diagnosis were represented in Table 2. 18F-FDG-PET/CT was truely positive in 48% of patients with normal Ca 19-9 and/or CEA levels and truely negative in 10% of cases with elevated Ca 19-9 and/or CEA levels according to histopatho- logical confirmation or colonoscopy findings. In the follow-up, conventional imaging tests (CT or MRI) detected suspicious malignancy in 32% of the patients (17/53) and further examination with 18F-FDG-PET/CT was truely negative in 35% of these cases (6/17) according to histopathology. 18F-FDG-PET/CT findings in histopathologically proven recurrence according to tumor markers (Ca 19-9 and/or CEA) and conventional imaging mo- dalities (CT-MRI) were described in Table 3,4. Mean SUVmax was 6.9 ± 5.6 (1–22) in benign group and 12.7 ± 6.1 (3.6–24) in malignant group. There is a statistically significant difference (p = 0.008) between them according to SUVmax. A box- plot graph illustrates the distribution of SUVmax in benign conditions versus disease recurrence (Figure 1). ROC curve of SUVmax was plotted for the differentiation between benign conditions and malignant group (Area Under Curve: 0.755) (Figure 2). Neverthless, sensitivity and specificity couldn’t be calculated due to undersampling and inconvenient SUVmax data not suggesting a de- terminant cutoff value. There is also a statistically significant difference between early and delayed SUVmax values of both groups separately in them (p = 0.013 for benign group, p = 0.012 for malig- nant group). However, we don’t see a significant difference between them according to early and delayed SUVmax values (p = 0.238). Mean TLG of malignant group was 401 ± 226. Mean TLG of be- nign group was 148 ± 126 (median value: 44). The most important and striking statistical difference between them was found for TLG (p = 0.001). Discussion Recurrent disease is seen in 30–50% of patients with CRC after curative resection.6 The recurrence rate TABLE 2. 18F-FDG-PET/CT results, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) according to final histopathologic diagnosis Histopathologic diagnosis 18F-FDG-PET/CT Results Positive Negative Sensitivity Specificity PPV NPV Total (n) Malignant TP = 24 FN = 0 24 Benign FP = 14 TN = 15 29 Total (n) 38 15 100% 51.7% 63.1% 100% 53 FN = False negative; FP = False positive; TN = True negative; TP = True positive TABLE 3. FDG-PET/CT findings according to serum Ca 19-9 or CEA levels for histopathologically proven recurrence 18F-FDG-PET/CT results Ca 19-9 or CEA levels True positive True negative Total (n) Elevated 9 1 10 Normal 13 14 27 Total (n) 22 15 37 TABLE 4. Overlap between 18F-FDG-PET/CT findings and conventional imaging modalities (CT or MRI) in histopathologically proven recurrence 18F-FDG-PET/CT results CT/MRI True positive True negative Total (n) Malign 11 6 17FIGURE 1. Box-plot graph illustrating the distribution of SUVmax through benign conditions and disease recurrence. Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer382 in our study was 45% and it is accordant with lit- erature. The main goal of follow-up surveillance is to reveal recurrences as soon as possible at an early stage for an immediate cure.5 Most of the relapsed cases are not operable at the time of diagnosis and 1/3 of the patients with isolated locoregional or dis- tant metastases survive 5 years.15 Several studies have proven that 18F-FDG-PET/ CT is very sensitive, but not that much specific for detection of recurrence in CRC and affects patient management.3 Sobhani et al.16 included 130 patients with recurrent CRC in their study and compared 18F-FDG-PET/CT and conventional follow-up. They found no difference in recurrence rate, but recurrences were detected earlier by 18F-FDG-PET/ CT. Scott et al.17 showed that 18F-FDG-PET/CT de- tected 45 additional lesions in a multicenter pro- spective trial of 93 patients. Lu et al.18 studied sen- sitivity and specificity of diagnostic CT in a meta- analysis and found 51% and 90%, respectively. The sensitivity and specificity of conventional imaging tests (CT or MRI) and 18F-FDG-PET/CT were 71%, 87% and 100%, 52%, respectively in our study. In Figure 3, example of a true positive recurrent le- sion suspected on MRI and confirmed by 18F-FDG- PET/CT is shown. Maas et al.19, in a meta-analysis of 14 studies, investigated which imaging modality has the highest accuracy in CRC recurrence/metas- tasis suspected clinically or because of the elevated tumor marker levels. They reached the conclusion that 18F-FDG-PET/CT is absolutely more accurate (areas under curves were 0.94 for PET, 0.94 for PET/CT, 0.83 for CT). We found true positivity on 18F-FDG-PET/CT in 48% of patients with normal Ca 19-9 and/or CEA levels, although there was true negativity in 10% of cases with elevated Ca 19-9 FIGURE 2. ROC curve drawn to indicate the detection and diagnostic accuracy of SUVmax in recurrence/metastasis. FIGURE 3. A female patient aged 51 years with rectum cancer was operated and treated by chemoradiotherapy. MRI findings revealed suspected metastasis with serum CEA and Ca 19-9 in normal range during the follow-up. Her axial PET (A), CT (B), fusion (C) and coronal PET (D), CT (E), fusion (F) images on 18F-FDG-PET/CT showed a focal uptake in sigmoid colon with a SUVmax of 13.7 and TLG of 272 accompanied by wall thickening causing a mass lesion on CT component (arrows). Histopathology established the diagnosis as recurrence. A B C D E F Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer 383 and/or CEA levels. All our findings are in line with data from literature. Although functional imaging with 18F-FDG PET is a useful technique for evaluating treatment re- sponse, it has some limitations. 18F-FDG is taken up at a relatively higher rate in cancer cells and ac- cumulates during glucose metabolism. However, cancer cells are not the only cells that are metaboli- cally hyperactive. Inflammation, infection and oth- er non-neoplastic conditions such as hyperplastic colorectal polyps may have increased FDG accu- mulation.20,21 Depending upon this, PET scans have a high sensitivity but a low specificity for CRC21 as it was also the case in our study. Compared to PET alone, combination of PET and CT, having the advantage of detecting metabolic abnormality with anatomic localization, is superior in localizing le- sions and differentiating between physiologic and malignant uptake of FDG. The benign pathologies diagnosed in our study were granulation tissue, fibrin and inflammation, fibrosis, pyelonephritis, ulceration of colonic mucosa, fibrosis and inflam- mation, polip, as well as changes secondary to ra- diotherapy or operation. Some manipula- tive registration or in- tervention methods on 18F-FDG-PET/CT imag- ing have been suggested to increase the specificity. Recently, Prieto et al.22 suggested voxel-based du- al-time 18F-FDG-PET im- ages for brain tumors and demonstrated that para- metric images provided enhanced tumor identifi- cation. Voxel-based analy- sis is different from VOI- based one in that it is an attempt enabling calcu- lation of real increase in delayed scan of the same voxel. Choi et al.12 inves- tigated voxel-based dual- time 18F-FDG-PET para- metric imaging in the evaluation of residual tu- mor for rectal cancer and found promising results. Dual-time 18F-FDG-PET has also been reported to improve diagnostic accu- racy for several cancers.14 This is based on the different pattern of FDG up- take in malignant tumors displaying a slow incre- ment, whereas benign lesions show an earlier peak and then a decline.23 Lots of benign conditions and physiologic FDG uptakes displaying focal or diffuse FDG accumulations in gastrointestinal tract can be seen in patients with CRC during the follow-up and may be confused with true patho- logic lesions. When such a pattern is observed on 18F-FDG-PET/CT, invasive interventions (colonos- copy, biopsy) are recommended. So it is essential to distinguish them. An example of false positivity is shown in Figure 4. Miyake et al.24 showed that delayed scans in- creased correct differentiation of physiologic FDG uptakes causing false positivities from foci of path- ologic uptake in CRC. Yoon et al.25 reported that dual-time 18F-FDG-PET/CT had better accuracy in diagnosing tumor response and recurrence. We researched the difference of SUVmax on early and delayed images of dual-time 18F-FDG-PET between A B C D E FIGURE 4. A female patient aged 73 years with sigmoid colon cancer was operated and treated by chemoradiotherapy. Her serum CEA level was 3.2 ng/ml, and Ca 19-9 was 7.3 U/ml. In the evaluation of treatment response; axial PET (A), CT (B), fusion (C) and coronal PET (D), fusion (E) images on 18F-FDG-PET/CT exhibited a diffuse uptake in sigmoid colon with a SUVmax of 10.1 and TLG of 154 accompanied by wall thickening on CT component (arrows). This uptake raised the suspicion of a probable recurrence, but histopathology confirmed it as benign. Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer384 benign and malignant group. We didn’t find a meaningful statistics and discriminative power for this parameter. We investigated the value of SUVmax and TLG too. Most of the studies including SUVmax and TLG evaluation in CRC are related to prognosis es- timation. Marcus et al.26 declared SUVmax and TLG to be higher in patients having bad prognosis. Gade et al.2 found a lower mean SUVmax of 8.6, Marcus et al.26 7.3 in recurrent CRC when compared to ours (12.7). Shamim et al.27 found a significant increase according to mean SUVmax in recurrence (11.8 for recurrence versus 3.7 for benign conditions) in a study of 32 patients with CRC. They were 12.7 for recurrence against 6.9 for benign group in our study and there was a significant difference (p = 0.008). Giacomobono et al.28 assessed SUVmax in CRC patients with increased CEA levels and found a worse overall survival for SUVmax values great- er than 5.7. Inflammatory pathologies, fibrosis or edema following irradiation and/or surgery can also be FDG-avid and their SUVmax values can be as high as malignant ones due to the degree of cel- lular metabolism reflected by 18F-FDG uptake.11,12 Therefore, big overlaps in SUVmax values of both benign conditions and recurrent/metastatic disease may be seen just like in our study. However, we found SUVmax was very helpful for the differen- tiation of recurrent disease from benign conditions. As our results trended towards improved diag- nostic accuracy, a determinative cutoff value for SUVmax was not obtained. Undersampling and inconvenient SUVmax data didn’t let an estimated cutoff value on ROC curve for related sensitivity and specificity calculation. Quantitative PET parameters have been used in prognosis estimation and evaluation of treatment response for several cancers. There is not a specific study in literature which investigated metabolic tumor parameters for the differentiation of benign conditions from recurrence. As far as we know, our study is the first one in literature. Higher metabolic activity and glucose consumption of tumor cells are measured by these parameters. SUVmax is the first one and represents the highest FDG uptake within the tumor. More lately volume-based metabolic parameters emerged out. TLG is a volume-based metabolic tumor parameter having widespread use and increasing recognition in many cancers as a predictor. Arslan et al.29 reported it has a role in prediction of survival for patients with small cell lung cancer. Caglar et al.30 investigated metabolic tumor markers in recurrent CRC and found mod- erate correlation. Marcus et al.26 found mean TLG 280, Caglar et al.30 55 in recurrent CRC. It was 401 in our study. There was significant difference be- tween benign conditions and recurrence according to mean TLG and TLG was the most striking factor for this purpose in the study (p = 0.001). Again, we didn’t intentionally calculate a cutoff value which could be misleading due to insufficient sampling. But first impressions imply determinative cutoff values for SUVmax and TLG increasing the speci- ficity may be obtained from studies with larger patient numbers. Higher SUVmax and TLG values in suspected cases would favour in recurrence/me- tastasis and support the necessary invasive inter- ventions. Absence of an estimated cutoff value on ROC curve for related sensitivity, specificity calculations due to undersampling and inconvenient SUVmax data was a limitation in the study. The number of subjects were small. Evaluation with larger popu- lations is required for definitive results. Study design was also a limitation. Ideally, prospective studies are needed. The other limitation was that TLG calculations and dual-time imaging could not be performed for all the patients. Conclusions FDG uptake on PET/CT imaging is quite sensitive for both benign and malignant lesions in patients with CRC. 18F-FDG-PET/CT appears to be very ben- eficial in revealing especially true-negative lesions suspected of recurrence or metastasis and may pre- vent unnecessary treatments. Although SUVmax is a strong metabolic parameter (p = 0.008), TLG seems to be the best predictor in recurrence of colo- rectal cancers (p = 0.001). Both are increasing the specificity of 18F-FDG-PET/CT. References 1. Hammond K, Margolin DA. The role of postoperative surveillance in colo- rectal cancer. Clin Colon Rectal Surg 2007; 20: 249-54. doi: 10.1055/s-2007- 984869 2. Gade M, Kubik M, Fisker RV, Thorlacius-Ussing O, Petersen LJ. Diagnostic value of (18)F-FDG PET/CT as first choice in the detection of recurrent colo- rectal cancer due to rising CEA. Cancer Imaging 2015; 15: 11. doi: 10.1186/ s40644-015-0048-y 3. Laurens ST, Oyen WJ. Impact of fluorodeoxyglucose PET/Computed Tomography on the management of patients with colorectal cancer. PET Clin 2015; 10: 345-60. doi: 10.1016/j.cpet.2015.03.007 4. Chen CH, Hsieh MC, Lai CC, Yeh CY, Chen JS, Hsieh PS, et al. Lead time of carcinoembryonic antigen elevation in the postoperative follow-up of colo- rectal cancer did not affect the survival rate after recurrence. Int J Colorectal Dis 2010; 25: 567-71. doi: 10.1007/s00384-010-0889-6 Radiol Oncol 2017; 51(4): 378-385. Ince S et al. / FDG PET/CT in recurrent colorectal cancer 385 5. Chiaravalloti A, Fiorentini A, Palombo E, Rinino D, Lacanfora A, Danieli R, et al. Evaluation of recurrent disease in the re-staging of colorectal cancer by 18F-FDG PET/CT: Use of CEA and CA 19-9 in patient selection. Oncol Lett 2016; 12: 4209-13. doi: 10.3892/ol.2016.5143 6. 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Clin Oncol (R Coll Radiol) 2012; 24: 232-49. doi: 10.1016/j.clon.2011.11.008 14. Basu S, Alavi A. Partial volume correction of standardized uptake values and the dual time point in FDG-PET imaging: should these be routinely employed in assessing patients with cancer? Eur J Nucl Med Mol Imaging 2007; 34: 1527-9. doi: 10.1007/s00259-007-0467-5 15. Bowne WB, Lee B, Wong WD, Ben-Porat L, Shia J, Cohen AM, et al. Operative salvage for locoregional recurrent colon cancer after curative resection: An analysis of 100 cases. Dis Colon Rectum 2005; 48: 897-909. doi: 10.1007/ s10350-004-0881-8 16. Sobhani I, Tiret E, Lebtahi R, Aparicio T, Itti E, Montravers F, et al. Early detec- tion of recurrence by 18FDG-PET in the follow-up of patients with colorectal cancer. Br J Cancer 2008; 98: 875-80. doi: 10.1038/sj.bjc.6604263 17. Scott AM, Gunawardana DH, Kelley B, Stuckey JG, Byrne AJ, Ramshaw JE, et al. 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Dual-time-point 18F-FDG PET/CT in patients with colorectal cancer: clinical value of early delayed scanning. Ann Nucl Med 2012; 26: 492-500. doi: 10.1007/s12149-012-0599-y 25. Yoon HJ, Kim SK, Kim TS, et al. New application of dual time point 18F-FDG PET/CT in the evaluation of neoadjuvant chemoradiation response of lo- cally advanced rectal cancer. Clin Nucl Med 2013; 38: 7-12. doi: 10.1097/ RLU.0b013e3182639a58 26. Marcus C, Wray R, Taghipour M, Marashdeh W, Ahn SJ, Mena E, et al. Journal club: Value of quantitative FDG PET/CT volumetric biomarkers in recurrent colorectal cancer patient survival. AJR Am J Roentgenol 2016; 207: 257-65. doi: 10.2214/AJR.15.15806 27. Shamim SA, Kumar R, Halanaik D, Shandal V, Reddy RM, Bal CS, et al. Role of FDG-PET/CT in detection of recurrent disease in colorectal cancer. Nucl Med Commun 2010; 31: 590-6. doi: 10.1097/MNM.0b013e328338a120 28. Giacomobono S, Gallicchio R, Capacchione D, Nardelli A, Gattozzi D, Lettini G, et al. F-18 FDG PET/CT in the assessment of patients with unexplained CEA rise after surgical curative resection for colorectal cancer. Int J Colorectal Dis 2013; 28: 1699-705. doi: 10.1007/s00384-013-1747-0. 29. Arslan N, Tuncel M, Kuzhan O, Alagoz E, Budakoglu B, Ozet A, et al. Evaluation of outcome prediction and disease extension by quantitative 2-deoxy- 2-[18F] fluoro-D-glucose with positron emission tomography in patients with small cell lung cancer. Ann Nucl Med 2011; 25: 406-13. doi: 10.1007/ s12149-011-0478-y 30. Caglar M, Yener C, Karabulut E. Value of CT, FDG PET-CT and serum tumor markers in staging recurrent colorectal cancer. Int J Comput Assist Radiol Surg 2015; 10: 993-1002. doi: 10.1007/s11548-014-1115-8 Radiol Oncol 2017; 51(4): 386-392. doi: 10.1515/raon-2017-0049 386 research article Is there a role for contrast-enhanced ultrasound in the detection and biopsy of MRI only visible breast lesions? Aki Nykänen1,2, Otso Arponen1, Anna Sutela1, Ritva Vanninen1,2,3, Mazen Sudah1 1 Kuopio University Hospital, Diagnostic Imaging Center, Department of Clinical Radiology, Kuopio, Finland 2 University of Eastern Finland, Institute of Clinical Medicine, School of Medicine, Kuopio, Finland 3 University of Eastern Finland, Cancer Center of Eastern Finland, Kuopio, Finland Radiol Oncol 2017; 51(4): 386-392. Received 17 August 2017 Accepted 2 October 2017 Correspondence to: Aki Nykänen, BM, Department of Clinical Radiology, Kuopio University Hospital, Puijonlaaksontie 2, P.O. Box 100, FI-70029 Kuopio, Finland. Phone: +358 40 532 0129; E-mail: akinyk@uef.fi Disclosure: No potential conflicts of interest were disclosed. Background. This study aimed to evaluate the feasibility of contrast-enhanced ultrasound (CEUS) and CEUS-guided interventions in the diagnostics of MRI visible targeted US occult breast lesions. Patients and methods. This retrospective study examined 10 females with 10 occult, MRI only detected breast lesions between July 2014 and April 2017. Targeted second look US followed by CEUS with 2.4 ml of SonoVue® were performed for all of the lesions. After positive CEUS localization the same dose was repeated for confirmation and CEUS-guided interventions were performed. Results. MRI revealed 8 mass lesions with a mean size of 9 mm (range 5–16 mm) and 2 non-mass enhancing lesions of 10 and 20 mm in largest diameters. Targeted US revealed no morphological correlate for the lesions. Five out of 10 lesions (50%) were visible on CEUS. CEUS-guided core biopsy was performed on 4 lesions and 1 was marked with a clip for later surgical removal. Histopathological analysis confirmed 4 of them to be malignant. Three out of 5 nonvisible lesions on CEUS underwent MRI-guided interventions, 1 lesion was scheduled for follow-up as it was non-amenable for MRI biopsy, and 1 lesion was biopsied under US-guidance. Three of these nonvisible lesions on CEUS were confirmed to be malignant. Conclusions. Based on our preliminary results, CEUS is a feasible tool for detecting many MRI only visible breast le- sions, resulting in a more cost effective and less time-consuming practice. It is a more convenient alternative than MRI guided biopsy and has the potential to be included in the diagnostic algorithm which evaluates MRI only visible breast lesions. Key words: additional lesions; MRI; MRI-guided interventions; contrast-enhanced ultrasound; CEUS; CEUS-guided in- terventions Introduction Breast magnetic resonance imaging (MRI) has emerged as the most sensitive imaging modality in the detection and evaluation of breast lesions. One of the major indications for breast MRI is the preoperative local staging of breast cancer and de- spite controversies about its benefits, the number of preoperatively imaged women has increased.1,2 MRI detects additional lesions in 16% (range 11%– 24%) of patients in the ipsilateral breast and 9.3% (95% confidence interval, 5.8%–14.7%) in the con- tralateral breast, depending on the patient popu- lation and the definition criteria.3-5 MRI-detected primarily mammographically and sonographically (US) occult lesions have been shown to rarely dis- play features that are suggestive of malignancy.6 Therefore, it is important to characterize and man- Radiol Oncol 2017; 51(4): 386-392. Nykänen A et al. / Contrast-enhanced ultrasound and MRI only visible breast lesions 387 age these breast lesions correctly, because if these additional lesions are malignant, this can alter the surgical treatment protocol.2 Additional MRI-detected lesions are character- ized and evaluated using the BI-RADS® lexicon. Lesions categorized as suspicious for malignancy (e.g. BI-RADS® 4 or 5) need to undergo a second look targeted ultrasound7, and when visible, their nature should be verified histologically with core biopsy (CB) under US guidance whenever possible. A recent meta-analysis of 17 articles that included benign and malignant lesions revealed that the sec- ond look ultrasound had a pooled detection rate of 57.5% (range 22.6–82.1%) in the MRI suspicious additional lesions.7 The second look ultrasound is able to observe mass lesions and malignant le- sions better than non-mass-enhancing (NME) or benign lesions.8,9 The additional lesions that have no morphological correlate on targeted ultrasound should then undergo further MRI-guided vacuum assisted biopsy (VAB). Nevertheless, MRI-guided biopsy is a time consuming, expensive and occa- sionally, challenging procedure. Furthermore, not all MRI only detected lesions are accessible for MRI biopsy.10 Therefore, other, more practical, methods to aid in the evaluation of additional MRI-detected lesions are needed. Contrast-enhanced ultrasound (CEUS) is a non- invasive non-nephrotoxic technique that does not subject the patient to ionizing radiation. The meth- od utilizes microbubble contrast agents, blood flow and tissue perfusion specific imaging.11 Tissue perfusion is correlated to microvascular density (MVD)12,13 and angiogenic factor expression14 are analogous in CEUS and contrast-enhanced MRI (CE-MRI).11 MRI contrast agents additionally ex- travasate to the extracellular matrix due to the increased permeability of the microvessels.15,16 However, the microbubbles in CEUS contrast agent do not extravasate; thus it is the contrast agent ac- cumulating in the intravascular, not in the extracel- lular space, that is detected in the angiogenetic le- sions.17 CEUS can be administered intravenously to differentiate benign and malignant breast lesions18, to assess the disease extent19 and to monitor the tu- mour response to neoadjuvant chemotherapy.20,21 The utility of CEUS to detect MRI only visible lesions has not been previously reported, yet it is an attractive additional tool to be incorporated into the routine clinical practise, because when the tar- geted US fails, the patients are already in the US investigation position while rescheduling for MRI- guided biopsy will inevitably delay treatment deci- sions. In our institution, CEUS is a routine practice for various indications; in breast diagnostics it is occasionally applied to characterize breast lesions or to identify the sentinel axillary lymph nodes in appropriate situations.22 The aim of this retrospec- tive study is to report the feasibility of CEUS and CEUS-guided interventions in the detection of US occult MRI visible breast lesions. Patients and methods Study design and patients Patients with malignant or suspicious breast find- ings are referred to our tertiary hospital (catch- ment area 260,000 inhabitants) for consultation and further management. All patients undergo a full mammography as well as a clinical and bilat- eral breast US evaluation upon referral. Patients are referred to breast MRI according to national guidelines which are in concordance with those issued by the European Society of Breast Cancer Specialists working group (EUSOMA).23 Annually, approximately 200 breast MRI examinations are performed for various indications. For the purpos- es of the present study patients with MRI-detected additional lesions that were investigated with CEUS were retrospectively retrieved from the local picture archiving and communication system. The local chair of the hospital district waived the need for written informed consent from the patients. Between July 2014 and April 2017, a total of 10 patients fulfilling the following inclusion criteria were evaluated: 1) bilateral mammograms and breast US performed by a breast radiologist; 2) breast MRI according to a structured protocol; 3) an occult, MRI only detected lesion found on MRI that might alter the surgical plan; the lesion had not been evident initially in mammography, US or clinical examination, 4) no morphological cor- relate of the lesion found during the targeted US performed by a breast radiologist. Breast MRI protocol and image analysis MRI examinations in 8 patients were performed at our institution in the prone position with a seven element, phased-array coil dedicated to breast imaging (Philips Achieva 3.0 T TX, Philips N.V., Eindhoven, The Netherlands). The structural breast MRI protocol consisted of five sequences as seen in Table 1. Breast radiologists (with > 20 years of experi- ence in breast radiology) primarily evaluated the morphological and kinetic features of MRI findings Radiol Oncol 2017; 51(4): 386-392. Nykänen A et al. / Contrast-enhanced ultrasound and MRI only visible breast lesions388 together with mammograms and US examinations guided by the BI-RADS® lexicon. Two patients were referred to our university hospital from two central hospitals for MRI-guided interventions be- cause of MRI only visible lesions. These patients were scanned with 1.5 T scanners yet with a simi- lar structured MRI protocol. MRI-guided interven- tions were performed with automated 10 gauge (G) vacuum assisted biopsy device (EnCore Enspire Breast Biopsy System, C.R. Bard Inc., Tempe, AZ, USA). Second look targeted US examination Patients were meticulously scanned with both grey scale and Doppler US. For MRI lesions detected in the lateral part of the breast, patients were scanned in the opposite lateral decubitus position and con- sequently in the ipsilateral oblique position for medial lesions in an attempt to simulate the MRI position of the breast. If no morphologically con- cordant lesions were detected in targeted US, then a CEUS examination was considered. CEUS procedures The purpose and nature of the CEUS procedure was discussed with the patient and all patients pro- vided verbal consent. Nonlinear harmonic imaging using an Esaote MyLabClassC ultrasound scanner (Esaote S.p.A., Genova, Italy) equipped with a 7–13 MHz linear array transducer was performed at baseline with a low mechanical index (MI) of 0.08 in three patients. The remaining examinations were performed using Logiq E9 class US scanner (GE, Wauwatosa, Wisconsin, USA) with a low MI of 0.11. A microbubble contrast agent (SonoVue®, Bracco S.p.A., Milan, Italy) was used for localizing occult lesions. The contrast agent was adminis- tered according to the guidelines of the manufac- turer: microbubble dispersion was prepared before use by injecting through the septum the contents of the vial a total of 5 ml of sodium chloride 9 mg/ ml (0.9%) solution for injection. The vial was then shaken vigorously for a few seconds until the ly- ophilisate was completely dissolved. A standard dose of 2.4 ml of dispersion was drawn into a sy- ringe and administered intravenously via an 18-G cannula placed in a cubital vein. Injections were flushed with 5 ml of sodium chloride 9 mg/ml (0.9%) solution. After contrast agent administration, the sus- pected area was scanned to detect enhancements morphologically concordant with the MRI find- ing. If the lesion was identified, then the skin area was ink-marked and sterilized. The marked area was confirmed with another SonoVue injection and thereafter, without moving the US probe, the breast area was anesthetized and an incision was made to collect the biopsy. Histopathological CB samples were obtained using 14-G core needle tar- geted at the area of interest. After the CB, the tar- geted area was marked with a coil. Both gray scale targeted US and CEUS breast examination were performed in all cases by or under the direct su- pervision of a breast radiologist with over 20 years of experience in multimodality breast imaging. All interventions were performed by the same senior breast radiologist. Histopathological analysis CB specimens were placed into 10% formalin and embedded in paraffin after fixation. The samples were cut into 5 μm slices at four different levels and stained with haematoxylin and eosin. Biopsy samples were evaluated by two pathologists, first at the time of diagnosis, and then at a multidisci- plinary meeting. Diagnosis was confirmed from final surgical specimens when indicated. Both non- invasive and invasive carcinomas were considered malignant. Results MRI detected 8 mass lesions with a mean size of 9 mm (range 5–16 mm) and 2 non-mass enhance- ment of 10 and 20 mm in largest diameters. Five of these lesions were CEUS positive, of which 4 un- derwent CEUS-guided CB (Figure 1) and one was primarily marked with a clip for later surgical re- moval. Of these 5 lesions, 4 proved to be malignant. TABLE 1. Breast MRI protocol Sequence TR/TE(ms) in-plane resolution mm Slice thickness (mm) Scanning time T1-FFE 4.57/2.3 0.48 × 0.48 0.7 6 min 11 s T2-TSE 5000/120 0.6 × 0.6 2 3 min 20 s STIR 5000 /60 1 × 1 2 5 min 40 s T1 dynamic* 4.67/ 2.31 0.96 × 0.96 1 58.5 s DWI# 7168 /95 1.15 × 1.15 4 min 8 s #DWI = Diffusion weighted echo planar imaging with five respective b factors (0, 200, 400, 600 and 800 s/mm2); *eTHRIVE spectrally adiabatic inversion recovery (SPAIR) fat suppression; pre-contrast and six phases after the gadoterate meglumine (0.2 ml/kg, 3 ml/s) injection followed by a saline chaser; FFE = fast field echo; STIR = Short tau inversion recovery; TSE = turbo spin echo Radiol Oncol 2017; 51(4): 386-392. Nykänen A et al. / Contrast-enhanced ultrasound and MRI only visible breast lesions 389 medical records on final treatment decisions are not available. Discussion MRI-detected primarily occult additional lesions are small, have few specific suspicious features, and are therefore challenging to characterize.6 When imaged by US, these lesions are usually sub- tle and appear as nonspecific findings.24 Searching for such lesions can therefore be challenging and time consuming even in the hands of experienced breast radiologists. Our findings indicate that CEUS and CEUS-guided interventions are feasi- ble in the evaluation of half of the MRI only visible lesions and represent a practicable alternative for MRI-guided biopsy in these situations. In general, CEUS is known to be a safe, rapid, cost-effective and less time-consuming procedure for both the clinician and the patient.25 In our study, half of the CEUS studies were, however, negative, meaning that there were some additional contrast agent costs. Nevertheless, these costs are less than the savings made when MRI-guidance is avoided. In our institution, the basic list price of CEUS and CEUS-guided biopsy and coil place- ment is 313 € compared to 1167 € for MRI-guided biopsy. Furthermore, patients can be evaluated and managed immediately in positive cases, thus eliminating the need for rescheduling for MRI- guided intervention. FIGURE 1. 53 year old female patient with invasive carcinoma of the right breast. MRI detected an occult, BI-RADS 5, oval, 20x16 mm irregular mass lesion in the lower medial quadrant of the left breast (not shown). Targeted US (A) was negative. Contrast-enhanced ultrasound (B) revealed a 21 mm enhancement. Core biopsies were obtained from the enhancement area (C, ARROW) showing the core biopsy needle’s position). Both core biopsy and final histology showed high grade invasive carcinoma. FIGURE 2. 60 year old female patient with invasive carcinoma of no specific type (NST) of the left breast (not shown). MRI detected an occult BI-RADS 4 oval, 7x5 mm mass lesion in the upper medial quadrant of the right breast (A-C) thin slice multiplanar reconstruction in axial, coronal and sagittal orientations). Targeted US was negative (D). Contrast-enhanced ultrasound (E, ARROW) revealed a 5 mm round enhancement. Low grade ductal carcinoma in situ (DCIS) was diagnosed in the core biopsy of the enhanced lesion. The final histology was both DCIS (6x4 mm) and low grade invasive carcinoma NST (6x5 mm) in close vicinity. In one of these patients, CEUS showed a round 5 mm lesion which was evaluated to be low grade ductal carcinoma in situ (DCIS) at CB. At final his- tology, a low grade invasive carcinoma was found in the continuation of the DCIS. It is presumed that the CEUS showed only the DCIS component which was also carefully targeted (Figure 2). Of the 5 CEUS negative patients, 2 underwent MRI-guided vacuum assisted biopsy and 1 was subjected to MRI-guided coil localization. One pa- tient had a non-mass lesion close to the chest wall deemed non-amenable to MRI-guided biopsy. The patient’s preference was follow-up with MRI and mammography. The lesion remained stable after 2 rounds of breast MRI at 6 and 14 months. In one CEUS negative patient, targeted US had revealed only a slight nonspecific architectural distortion at the site of MRI enhancement not deemed to be morphologically concordant with confidence. Nonetheless, it was biopsied and coil marked after the negative CEUS and subsequently proved to be malignant. More detailed patient and lesion char- acteristics are presented in Table 2. After coiling, a mammogram was acquired in two orthogonal views to confirm the localization of the coil. Seven coil-marked lesions were surgi- cally removed after stereotactic or US-guidance wire localization of the coil. One patient with negative CEUS and malignant MRI-guided vacu- um biopsy is treated in another hospital and her A B C A B C D E Radiol Oncol 2017; 51(4): 386-392. Nykänen A et al. / Contrast-enhanced ultrasound and MRI only visible breast lesions390 Tumor-induced angiogenesis results in imma- ture and dysfunctional vessels with different levels of vascular permeabilities and these are also visual- ized in dynamic gadolinium enhanced breast MRI. Microvascular density is linked to tumor growth and prognosis. As MRI contrast readily leaks out of the vasculature especially in malignant tumors, it obscures the structures of possible microvessels and complicates the assessment of their density.26 Nevertheless, it is believed permissible to link the morphology of MRI enhancement to the microvas- cularity pattern.27 Furthermore, according to an an- imal model study conducted by Jansen et al., it has been speculated that gadolinium penetrates and collects inside the dilated DCIS ducts i.e. gadolin- ium accumulation was observed within the intra- ductal neoplastic space 2 minutes after the admin- istration of the contrast agent. In contrast, CEUS specifically reveals only the microvascular struc- tures without any interfering leakage. Intuitively, it can be assumed that invasive cancers have a denser microvascular network and are thus more amenable to CEUS visualization. Interestingly, in our study, we were not able to visualize some of the more aggressive DCIS and invasive lesions, in- dicating that the vascular density of a malignant le- sion is not necessarily related to either lesion mor- phology or histopathology. Therefore, a negative CEUS cannot be used to rule out malignancy and further evaluation with MRI-guided interventions should be deemed necessary. This observation will need to be confirmed in a larger patient popula- tion. The use of CEUS also requires experience and state of the art US scanners with contrast applica- tions. Biopsy and clip-marking of CEUS visualized lesions, although feasible as demonstrated in our study, can prove to be a challenging procedure. It could therefore be speculated that in cases of negative second look targeted US, MRI-guided biopsy would be a straightforward next step pro- cedure. Nevertheless, non-visualization after contrast agent administration does occur also on MRI-guided biopsy in about 8–13% cases requiring TABLE 2. Patients, indications for MRI and MRI-only detected lesions’ characteristics, interventions and histopathological diagnosis Case Age yrs Indication for MRI Occult-lesion characteristics on MRI Occult lesion size CEUS visualization Intervention CB and final histopathological diagnosis and size of lesion 1 71 Preoperative local staging Mass, round, circumscribed, washout kinetic curve 6 mm Positive CEUS- guided CB CB: Low grade DCIS Final: Low grade DCIS. 3 mm 2 78 Problem solving Mass, oval, circumscribed, washout kinetic curve 7 mm Negative MRI-guided localization Final: Papilloma. 5 mm 3 64 Incidental breast lesion on CT NME, focal, heterogeneous 20 mm Negative US-guided CB CB: high grade DCIS. Final: high grade DCIS.16 mm 4 54 Preoperative local staging Mass, round, circumscribed, heterogeneous, washout kinetic curve 7 mm Positive CEUS-guided clip placement Final: Intermediate grade IC NST. 5 mm 5 54 Axillary metastasis from an occult breast cancer Mass, irregular, not circumscribed, plateau kinetic curve 12 mm Negative MRI-guided CB VAB: Carcinoma with medullary feature Final: Carcinoma with medullary feature and high grade DCIS.10 mm 6 66 Problem solving Mass, round, circumscribed, ring like enhancement, persistent kinetic curve 5 mm Positive CEUS-guided CB. Follow-up CB: Fibrocystic lesion, liponecrosis 7 30 Problem solving NME, focal, heterogeneous 10 mm Negative Follow-up -- 8 61 Preoperative local staging Mass, oval, irregular, washout kinetic curve 10 mm Positive CEUS- guided CB CB: Low grade DCIS Final: low grade IC NST 6 mm and Low grade DCIS 5mm 9 53 Preoperative local staging Mass, oval, irregular, washout kinetic curve 16 mm Positive CEUS- guided CB CB: High grade IC NST Final: High grade IC NST. 13 mm 10 65 Axillary recurrence Mass, oval, circumscribed, homogeneous, persistent kinetic curve 9 mm Negative MRI-guided VAB VAB: High grade DCIS + suspected microinvasion CB = Core biopsy; CEUS = Contrast-enhanced ultrasound; CT = Computed tomography; DCIS = Ductal carcinoma in situ; IC NST = Invasive carcinoma no special type; MRI = Magnetic resonance imaging; NME = Non-mass-enhancing; US = Ultrasound; VAB = Vacuum assisted biopsy Radiol Oncol 2017; 51(4): 386-392. Nykänen A et al. / Contrast-enhanced ultrasound and MRI only visible breast lesions 391 short term follow-up.28 Furthermore, the technical aspects of MRI-guidance have additional weak- nesses including the use of a compression grid system which limits access to areas between grid holes and makes it difficult to localize lesions in the retromamillary region, near the chest wall and in axillary tail. Even though compression grid free freehand MRI biopsy technique is not subject to localization limitations, it might prolong examina- tion time due to repeated imaging to confirm prop- er needle placement.29 Nonetheless, regardless of all the previously mentioned difficulties, we be- lieve that CEUS confers considerable added value in the immediate evaluation of occult lesions and therefore more investigations into its applicability are warranted. The small number of patients in this study is a major limitation and therefore no definitive solid conclusions can be made about the diagnostic per- formance and cost-effectiveness of CEUS in this indication. Nevertheless, our study can be consid- ered as a pilot report, clearly showing that some small US occult lesions do enhance with CEUS and therefore this procedure is feasible and further in- vestigations on this issue are well-founded. In conclusion, CEUS is a feasible and more con- venient alternative to MRI-guided biopsy for some MRI only visible lesions, resulting in a more cost- effective and less time-consuming practice. CEUS has the potential to be included in the diagnostic algorithm for the evaluation of MRI only visible breast lesions. Based on our initial experience, fur- ther studies are warranted to confirm our findings and possibly to define which lesions benefit most from this technique. 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Korean J Radiol 2013; 14: 171-8. doi: 10.3348/kjr.2013.14.2.171 Radiol Oncol 2017; 51(4): 393-400. doi: 10.1515/raon-2017-0030 393 research article Baseline tumor Lipiodol uptake after transarterial chemoembolization for hepatocellular carcinoma: identification of a threshold value predicting tumor recurrence Yusuke Matsui1,2, Masahiro Horikawa1, Younes Jahangiri Noudeh1, John A. Kaufman1, Kenneth J. Kolbeck1, Khashayar Farsad1 1 Dotter Interventional Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd, Portland, USA 2 Department of Radiology, Okayama University Medical School, 2-5-1 Sikata-cho, Kita-ku, Okayama 700-8558, Japan Radiol Oncol 2017; 51(4): 393-400. Received 16 April 2017 Accepted 30 June 2017 Correspondence to: Yusuke Matsui, Department of Radiology, Okayama University Medical School, 2-5-1 Sikata-cho, Kita-ku, Okayama 700-8558, Japan. Phone: +81 86 235 7313; Fax: +81 86 235 7316; E-mail: y-matsui@okayama-u.ac.jp Disclosure: Financial activities related to the present article: Khashayar Farsad received a grant from Guerbet for this study. Financial activi- ties not related to the present article: Yusuke Matsui reports a grant from Japan Radiological Society supported by Bayer Yakuhin, a consul- tancy fee from Stryker Japan, a lecturer’s fee from Terumo Corporation; John A. Kaufman reports grants from NIH, WL Gore, and BTG, per- sonal fees from VIVA Physicians, Delcath, MarrowStim and COOK Medical, book royalties and journal editor stipend from Elsevier, section edi- tor stipend from ARRS/AJR, stock ownership in Hatch Medical, Vu Medi, Endoshape, and Veniti, stock options in Javelin Medical, AV Medical, and BIO2 Medical; Kenneth J. Kolbeck reports a grant from Guerbet. A summary of this work was presented at the Society of Interventional Radiology (SIR) 2016 annual meeting (http://dx.doi.org/10.1016/j.jvir.2015.12.227). Background. The aim of the study was to evaluate the association between baseline Lipiodol uptake in hepatocel- lular carcinoma (HCC) after transarterial chemoembolization (TACE) with early tumor recurrence, and to identify a threshold baseline uptake value predicting tumor response. Patients and methods. A single-institution retrospective database of HCC treated with Lipiodol-TACE was reviewed. Forty-six tumors in 30 patients treated with a Lipiodol-chemotherapy emulsion and no additional particle embolization were included. Baseline Lipiodol uptake was measured as the mean Hounsfield units (HU) on a CT within one week after TACE. Washout rate was calculated dividing the difference in HU between the baseline CT and follow-up CT by time (HU/month). Cox proportional hazard models were used to correlate baseline Lipiodol uptake and other vari- ables with tumor response. A receiver operating characteristic (ROC) curve was used to identify the optimal threshold for baseline Lipiodol uptake predicting tumor response. Results. During the follow-up period (mean 5.6 months), 19 (41.3%) tumors recurred (mean time to recurrence = 3.6 months). In a multivariate model, low baseline Lipiodol uptake and higher washout rate were significant predictors of early tumor recurrence (P = 0.001 and < 0.0001, respectively). On ROC analysis, a threshold Lipiodol uptake of 270.2 HU was significantly associated with tumor response (95% sensitivity, 93% specificity). Conclusions. Baseline Lipiodol uptake and washout rate on follow-up were independent predictors of early tumor recurrence. A threshold value of baseline Lipiodol uptake > 270.2 HU was highly sensitive and specific for tumor re- sponse. These findings may prove useful for determining subsequent treatment strategies after Lipiodol TACE. Key words: transarterial chemoembolization; hepatocellular carcinoma; Lipiodol, tumor response, threshold Introduction Transarterial chemoembolization (TACE) has been proven effective for treatment of intermediate stage hepatocellular carcinoma (HCC).1,2 Lipiodol, a di- iodinated ethyl ester derivative of poppy seed oil, has been widely used in the standard TACE pro- cedure.3 When administered selectively via the he- patic artery, Lipiodol accumulates preferentially in HCC and is retained for a longer period than in the Radiol Oncol 2017; 51(4): 393-400. Matsui Y et al. / Baseline Lipiodol uptake predicting recurrence after TACE for HCC394 background liver parenchyma.4-6 Because Lipiodol demonstrates trans-sinusoidal penetration, arterial injection of Lipiodol can temporarily achieve dual arterial and portal embolization, unlike other par- ticulate embolic materials such as microspheres.3,7 An association between intratumoral Lipiodol ac- cumulation, pathological tumor necrosis8-12 and lo- cal recurrence rate13-15 has been shown in retrospec- tive analyses; however, no study has investigated a specific value of baseline Lipiodol uptake, i.e., the Lipiodol uptake immediately after TACE, predict- ing tumor recurrence. If a specific baseline Lipiodol uptake value proves useful to predict tumor recur- rence, it should have significant meaning for sub- sequent clinical management of HCC patients. This study examined whether baseline Lipiodol uptake in HCC was associated with tumor response, and whether a cutoff value of Lipiodol uptake could be identified predicting tumor recurrence. Patients and methods Study population Retrospective single institution database review was performed of patients with primary HCC who underwent Lipiodol-TACE from April 2007 to August 2013. Institutional review board approval was obtained. Inclusion criteria were i) document- ed non-contrast computed tomography (CT) of the liver within one week after TACE, ii) at least one documented triphasic contrast CT including non- contrast, arterial and delayed phase imaging on follow-up for evaluation of tumor recurrence, iii) lesion diameter ≥ 10 mm, and iv) lesions stained entirely and homogeneously with Lipiodol af- ter TACE. Exclusion criteria were i) patients with more than 6 lesions, ii) lesions stained partially or heterogeneously with Lipiodol; those lesions were not suitable for the Lipiodol uptake measurement because of the nonuniform Lipiodol concentration and possibility of incomplete treatment, iii) lesions treated with additional particulate embolic mate- rials, iv) lesions for which a second TACE or any additional treatment including surgery, radiofre- quency ablation (RFA), radiation therapy, or sys- temic therapy (e.g., sorafenib) was performed be- fore the initiation of follow up. Forty-six tumors in 30 patients meeting criteria were evaluated. TACE procedure Selective catheterization of lobar, segmental or sub- segmental hepatic arteries supplying tumor was performed under standard fluoroscopic guidance using 5–6 Fr base catheters in the first order aortic branch (celiac or superior mesenteric artery) fol- lowed by microcatheter selection of higher order branches as appropriate. A Lipiodol-chemotherapy emulsion (doxorubicin or epirubicin, 50mg) was prepared by admixture in a 1:1 or 2:1 ratio using a three-way stopcock and injected into selected he- patic arteries through the microcatheter. The end- point of injection was stasis of blood flow in the feeding artery or visualization of trans-sinusoidal portal branches. The volume of Lipiodol injected ranged from 1 to 10 mL per lesion. Lipiodol was used as the sole embolic material; no additional em- bolization with other particulate embolic materials was performed. Although this method corresponds to “chemo-lipiodolization” in European guide- lines16 or “Lip-TAI (transcatheter arterial infusion)” in Japanese guidelines17, “TACE” was employed as a unified term in this study. The embolic particles were not routinely used in our institutional practice because our institutional data (not shown) demon- strated patients’ survival outcome was not signifi- cantly different using particulate embolization. Data collection and image evaluation Patient data including age, sex, etiology of liver disease, Child-Pugh classification, and Barcelona Clinic Liver Cancer (BCLC) staging were collect- ed from the electronic medical record. Procedural details recorded included selectivity of treatment (lobar vs. subsegmental/segmental), volume of Lipiodol delivered and amount of chemothera- peutic delivered. Image evaluation was performed using a Digital Imaging and Communication in Medicine (DICOM) viewer (IMPAX; Agfa, Mortsel, Belgium). Lipiodol uptake was measured using the Hounsfield units (HU) of the target lesion on a non- contrast CT taken within one week after TACE and on every follow-up CT. The HUs on the CT with- in one week after TACE represented the baseline Lipiodol uptake. The follow-up interval varied case by case. The HUs were measured by hand drawing a region of interest (ROI) along the lesion periphery at the axial slice with the tumor maximum diam- eter, using the contrast-enhanced pre-procedure diagnostic scan as a reference. ROI analysis was performed with consistent contrast window level (WL) and width (WW) (WL/WW = 50/350). The diameter of the ROI on the baseline CT was used as the lesion size, since only lesions demonstrating entire and homogeneous uptake were included in the analysis. Lipiodol washout was defined as de- Radiol Oncol 2017; 51(4): 393-400. Matsui Y et al. / Baseline Lipiodol uptake predicting recurrence after TACE for HCC 395 creased density of Lipiodol in a part of the tumor or uniformly within the entire tumor, and evaluated using HU analysis on every available follow-up CT. The degree of washout was quantitatively evalu- ated by calculating the HU difference between the baseline CT and the follow-up CT at which wash- out was initially detected. If the HUs within the tu- mor on the last follow-up CT were greater than at baseline CT, as was occasionally seen with contract- ing tumors after treatment, the quantitative wash- out was recorded as 1.0 HU. The washout rate was calculated by dividing the quantitative washout by the follow up time period (HU/month). Tumor re- currence was defined as the appearance of new ar- terial enhancement within or along the margins of a completely treated lesion on the follow-up triphasic contrast CT. Evaluation was terminated when addi- tional treatment was performed or when recurrence was identified. Two board-certified radiologists in- dependently performed all image evaluation, with discrepancies decided by consensus with a third ra- diologist. Mean values of two measurements were used in all analyses. Statistical analysis Numerical data were summarized as mean ± standard deviation (SD) and the categorical vari- ables were shown as frequency (percentage). Inter- observer reliability of measurements was assessed by Cronbach’s alpha18, and standard error of the measurement was calculated using the formula: , where SEM is standard er- ror of measurement, SD is standard deviation and rxx demonstrates the reliability of the test repre- sented by Cronbach’s alpha19. Analyses for pre- dictors of tumor response and estimation of the cutoff lipiodol uptake value predicting tumor re- sponse were performed on lesion-by-lesion basis. Uni- and multivariate Cox proportional hazard models were created to evaluate the predictability of baseline Lipiodol uptake for tumor response. In addition to the baseline Lipiodol uptake, other variables such as lesion size, treatment selectivity and washout rate were subjected to the univari- ate model. Variables with a P value < 0.200 were entered in the multivariable model. Independent samples t-test was used to compare the mean base- line Lipiodol uptake between the tumors with and without recurrence, as well as between the degrees of treatment selectivity. The median recurrence- free interval was defined, and tumor recurrence before the median recurrence-free interval was defined as early recurrence. Baseline Lipiodol up- take and washout rates were compared between tumors with and without early recurrence using the independent samples t-test. Receiver operat- ing characteristic (ROC) curve analysis was used to determine the optimal cutoff point for baseline Lipiodol uptake predicting tumor recurrence, and for Lipiodol washout rates predicting early tumor recurrence. Natural logarithm values of the numer- ical exposure variables were used in Cox regres- sion models, and P values < 0.05 were considered statistically significant. All statistical analyses were done using Stata IC version 13.1 for Windows. Results The study population is summarized in Table 1. The mean follow-up period was 5.6 months (range, TABLE 1. Summary of the study population Characteristics Patients Age (years) 59.9 ± 7.9 Gender Female 12 (40.0%) Male 18 (60.0%) Liver disease etiology HCV 13 (43.3%) HCV + Alcohol 7 (23.3%) Alcohol 4 (13.3%) HBV 1 (3.3%) Other 5 (16.7%) Child-Pugh class A 10 (33.3%) B 20 (66.7%) BCLC stage A 17 (56.7%) B 13 (43.3%) Tumors Baseline tumor size (mm) 20.3 ± 10.1 Treatment selectivity Lobar 16 (34.8%) Segmental 22 (47.8%) Sub-segmental 8 (17.4%) Baseline Lipiodol uptake (HU) 328.3 ± 185.2 Lipiodol uptake at final evaluation (HU) 205.0 ± 219.5 Washout (HU) 136.9 ± 127.6 Follow-up time (months) 5.6 ± 6.2 Tumor recurrence Yes 19 (41.3%) No 27 (58.7%) The numerical data were summarized as mean ± standard deviation and the categorical data were shown as frequency (percentage). BCLC = Barcelona clinic liver cancer; HBV = hepatitis B virus; HCV = hepatitis C virus; HU = Hounsfield unit Radiol Oncol 2017; 51(4): 393-400. Matsui Y et al. / Baseline Lipiodol uptake predicting recurrence after TACE for HCC396 1.1–37.6). Nineteen (41.3%) lesions recurred, with a mean time to recurrence of 3.6 months (range, 1.1– 15.1) and a median recurrence-free interval of 3.0 months. The mean baseline Lipiodol uptake was 328.3 ± 185.2 HU. The mean quantitative washout of Lipiodol during the entire follow-up period was 136.9 ± 127.6 HU (58.4 ± 89.5 HU per month). Inter- observer reliability was high amongst the two in- dependent radiologists for measuring Lipiodol uptake (Cronbach’s alpha = 0.994), with a standard error of measurement of 14.3 HU. Predictors of tumor response No recurrences were seen in tumors with uni- formly retained Lipiodol at follow up imaging. Of the 29 tumors with visible washout on follow-up imaging, 19 (65.5%) demonstrated recurrence. In univariate analysis, lower baseline Lipiodol uptake and greater Lipiodol washout rate were statisti- cally significant predictors of earlier time to tumor recurrence (Table 2, P = 0.002 and < 0.0001, respec- tively). Performing a non-selective (lobar) TACE was also significantly associated with earlier time to recurrence compared to selective (segmental/ subsegmental) TACE (Table 2, P = 0.001). In mul- tivariate Cox regression analysis, lower baseline Lipiodol uptake and greater Lipiodol washout rate were both significant predictors of earlier time to tumor recurrence (Table 2, P = 0.001 and < 0.0001, respectively). The mean baseline Lipiodol uptake of tumors without recurrence was significantly higher than in tumors demonstrating recurrence (Figure 1; 426.3 ± 148.8 HU vs. 189.1±137.5 HU, P < 0.0001). Tumors treated with selective TACE demonstrated signifi- cantly higher baseline Lipiodol uptake compared with tumors treated with lobar TACE (Figure 1; 401.6 ± 32.4 HU vs. 190.9 ± 26.4 HU, P < 0.0001). Baseline Lipiodol uptake was significantly lower in TABLE 2. Results of cox regression analysis to predict factors for tumor recurrence Predicting factor Univariate model Multivariate model* Hazard ratio (CI) p Value Hazard ratio (CI) p Value Lesion size (mm) 0.70 (0.24–2.09) 0.527 - - Treatment selectivity (Segmental or subsegmental vs. lobar) 0.19 (0.07–0.51) 0.001 3.05 (0.62–15.05) 0.171 Baseline Lipiodol uptake (HU) 0.37 (0.19–0.70) 0.002 0.002 (0.0–0.087) 0.001 Presence of Lipiodol washout NA** NA - - Washout rate (HU/month) 3.37 (1.78–6.36) < 0.0001 149.03 (11.20–1983.54) < 0.0001 * Variables with a p value less than 0.200 were entered in the multivariate model. ** Not calculated because no tumors without Lipiodol washout recurred while 65.5% (19/29) of the tumors with Lipiodol washout recurred. CI = confidence interval; HU = Hounsfield unit FIGURE 1. Baseline Lipiodol uptake after TACE for HCC. Significantly higher Lipiodol uptake was noted in tumors without recurrence and in tumors treated selectively with TACE. FIGURE 2. Baseline Lipiodol uptake and washout rate in relation to early tumor recurrence. Early recurring tumors demonstrated significantly lower baseline Lipiodol uptake and higher Lipiodol washout rates relative to tumors recurring after the median recurrence free interval (= 3 months) and tumors with no recurrence. Radiol Oncol 2017; 51(4): 393-400. Matsui Y et al. / Baseline Lipiodol uptake predicting recurrence after TACE for HCC 397 tumors recurring before the median recurrence-free interval (e.g., early tumor recurrence) compared to tumors recurring after the median recurrence-free interval and tumors without recurrence (Figure 2; 208.5 ± 154.8 HU vs. 373.2 ± 200.3 HU, P = 0.013). By comparison, the rate of Lipiodol washout at follow up was significantly greater in early tumor recur- rence compared to tumors recurring after the me- dian recurrence-free interval and tumors without recurrence (Figure 2; 103.1 ± 129.1 HU/month vs. 25.7 ± 26.6 HU/month, P=0.044). Estimating the cutoff Lipiodol uptake value predicting tumor recurrence Using the area under the ROC curve for post-TACE baseline Lipiodol uptake, an estimated threshold Lipiodol uptake value of 270.2 HU was identified below which there was a high probability of tumor recurrence and above which there was a high prob- ability of tumor response over the mean follow-up time of 5.6 months with a sensitivity of 95% and specificity of 93% (Figure 3). Comparison of the cumulative hazards of tumor recurrence over the follow-up period also showed significantly higher hazards of recurrence in tumors with Lipiodol up- take < 270.2HU after TACE (Figure 4; P < 0.0001). ROC curve analysis for follow up Lipiodol wash- out rate revealed a value of 37.8 HU per month above which there was a high probability of early tumor recurrence with a sensitivity of 78% and specificity of 74% (Figure 5). The cumulative haz- ards for tumor recurrence were significantly higher in tumors with a washout rate > 37.8 HU per month (Figure 6; P = 0.001). Discussion In this study, we found a strong correlation be- tween baseline Lipiodol uptake after TACE for HCC and tumor response. The baseline uptake was that defined by the density of Lipiodol tumor stain on non-contrast CT imaging within the first week after TACE. The critical threshold value for base- line Lipiodol uptake predicting tumor recurrence vs. response in this cohort was 270.2 HU with a high degree of sensitivity and specificity (95% and 93%, respectively) over the roughly 6 months mean follow up interval. Post-TACE Lipiodol uptake has been shown to predict necrosis in HCC lesions.8-12 Moreover, low or incomplete Lipiodol uptake has been shown to predict tumor recurrence.13-15 The present study contributes to this body of knowl- edge by focusing on the baseline uptake at the time of TACE, and identifies a threshold value of 270.2 HU on baseline post-TACE non-contrast CT imag- ing as a predictor of tumor recurrence. Moreover, our cohort involved TACE using Lipiodol as the sole embolic agent, therefore demonstrating the ef- fect of Lipiodol accumulation independently from additional embolic agent effects. We found only one previous study discussing a critical cutoff point of post-TACE Lipiodol uptake FIGURE 3. ROC curve showing the area under curve and optimal cutoff value of baseline Lipiodol uptake after TACE to predict tumor recurrence. FIGURE 4. Comparison of the cumulative hazards of tumor recurrence over the follow-up period between baseline Lipiodol uptake above and below the identified threshold. Tumors with Lipiodol uptake < 270.2 HU demonstrated significantly higher hazards of recurrence after TACE. Radiol Oncol 2017; 51(4): 393-400. Matsui Y et al. / Baseline Lipiodol uptake predicting recurrence after TACE for HCC398 associated with tumor necrosis.12 Imaeda et al.12 evaluated the histological necrosis level in 12 autop- sies and 23 surgical resection specimens from pa- tients with HCC who had undergone TACE. They reported that uniform dense retention of Lipiodol was observed on CT 3–4 weeks after TACE in 12 out of 15 cases with massive (≥ 97%) histological necrosis and in only one out of 20 cases with non- massive necrosis (P < 0.01), showing a Lipiodol up- take value of 365 HU as a cutoff value for massive necrosis with 89% sensitivity and 73% specificity.12 The cutoff value identified in our study is less than that found in this study, which may be related to the differences in TACE technique, study methods and outcomes. For example, we used the baseline Lipiodol uptake (within one week of treatment) as the input variable, and tumor recurrence by CT as the main outcome variable. Furthermore, we used follow-up data from larger number of lesions (46 tumors in 30 patients). We have observed that le- sion contraction over time can create a greater den- sity of Lipiodol stain compared to baseline values in tumors responding to therapy, which may par- tially explain the higher threshold value observed by Imaeda et al. when evaluating scans obtained approximately one month after TACE. We found the Lipiodol washout rate, defined as loss of HU per month, was also a strong and in- dependent predictor of early tumor recurrence. A similar finding has been observed in another study after evaluation of 42 HCC patients who were scheduled for liver transplantation.20 The authors subjectively classified Lipiodol washout on CT into complete, strong, moderate, and poor, and found that a moderate or strong Lipiodol washout was correlated with lower degrees (≤ 75%) of tumor ne- crosis.20 In the present study, we identified a wash- out rate of 37.8 HU above which there was a high risk of early tumor recurrence over the follow up period in our cohort. Lipiodol can pass through arterio-portal com- munications such as the peribiliary plexus, ter- minal arterial-sinusoidal branches, portal vaso vasori, and direct arterioportal anastomoses.3,7 Consequently, transarterial delivery of Lipiodol re- sults in embolization of feeding vessels from both the arterial and portal sides of the tumor.3,7 Another role of Lipiodol is that of a carrier of the chemo- therapeutic agent.7 In this regard, higher Lipiodol uptake may represent enhanced embolization of tumor blood supply and also contribute to a higher local concentration of chemotherapeutic agents in the tumor. Cone-beam CT (CBCT) has been shown to dem- onstrate tumor Lipiodol accumulation with TACE, and HUs measured on CBCT have been shown to be comparable to multidetector CT.21 The use of intraprocedural CBCT and the threshold Lipiodol uptake value shown in our study may enable in- traprocedural assessment of TACE endpoints. This may prove beneficial in deciding when addition- al therapy is needed and to facilitate a treatment strategy following TACE, such as early repeat in- tervention or treatment using a different modality (i.e. ablation or Y90 radioembolization). FIGURE 5. ROC curve showing the area under curve and optimal cutoff value of Lipiodol washout rate after TACE to predict early tumor recurrence. FIGURE 6. Comparison of the cumulative hazards of tumor recurrence over the follow-up period between Lipiodol washout rates above and below the identified threshold. Tumors with Lipiodol washout rates ≥ 37.8 HU/month demonstrated significantly higher hazards of recurrence after TACE. Radiol Oncol 2017; 51(4): 393-400. Matsui Y et al. / Baseline Lipiodol uptake predicting recurrence after TACE for HCC 399 There are a few limitations in this study. Because of the retrospective nature and strict inclusion/ex- clusion criteria of this study to rigorously detect a baseline Lipiodol threshold uptake value, the size of study population was limited and the follow-up period was relatively short. Although the baseline Lipiodol uptake was defined as HUs on the CT within one week after TACE, the HUs might vary by the timing of CT even during one week; e.g., the HUs on the 1st and the 7th day after TACE might be different. The concentration of the chemothera- peutic agents and the volume of Lipiodol injected was different by the case, which might potentially affect the treatment outcome, although the pur- pose of this study was simply to evaluate the as- sociation of the baseline Lipiodol uptake, which was obtained as the results of our practice, with the tumor recurrence. In addition, we excluded le- sions treated with additional embolic materials. As mentioned above, we do not use the embolic par- ticles routinely because we did not find a survival advantage with the use of particulate embolization (data not shown). This practice would be differ- ent from so-called conventional TACE, in which chemo-Lipiodol injection is usually followed by injection of additional embolic particles such as gelatin sponge. A recent randomized trial by Shi et al. showed no difference in survival outcomes be- tween TACE with and without additional embolic materials following chemo-Lipiodol injection.22 On the contrary, a large cohort study by Takayasu et al. showed that TACE with gelatin sponge embo- lization was associated with a higher survival rate than was a therapy without the particulate embo- lization.23 Thus, the significance of the particulate embolization in TACE seems still controversial and needs further investigation. Nevertheless, in the present study, the cohort of Lipiodol only TACE without embolic particles allowed us to analyze significance of Lipiodol uptake without being in- fluenced by the embolic effect of other materials. This practice and our findings, however, may not extrapolate to a population treated with TACE fol- lowed by additional particulate embolization. Conclusions In conclusion, we demonstrated that Lipiodol uptake at baseline and Lipiodol washout rates at follow-up independently predicted tumor re- sponse after TACE for HCC. The critical threshold for baseline Lipiodol uptake predicting tumor re- currence was 270.2 HU. The association between baseline Lipiodol uptake and tumor response may prove useful for determining subsequent treatment strategies after Lipiodol TACE. Acknowledgments Khashayar Farsad received a grant from Guerbet for this study. References 1. Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. 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AJR Am J Roentgenol 2010; 194: 830-7. doi: 10.2214/AJR.09.3308 Radiol Oncol 2017; 51(4): 401-406. doi: 10.1515/raon-2017-0053 401 research article The relationship between chondromalacia patella, medial meniscal tear and medial periarticular bursitis in patients with osteoarthritis Mustafa Resorlu1, Davut Doner2, Ozan Karatag1, Canan Akgun Toprak1 1 Department of Radiology, Canakkale Onsekiz Mart University, Faculty of Medicine, Canakkale, Turkey 2 Department of Physical Medicine and Rehabilitation, Canakkale Onsekiz Mart University, Faculty of Medicine, Canakkale, Turkey Radiol Oncol 2017; 51(4): 401-406. Received 9 May 2017 Accepted 26 September 2017 Correspondence to: Mustafa Resorlu, M.D., Canakkale Onsekiz Mart University, Terzioğlu Yerleşkesi, Barbaros Mh, 17100, Canakkale, Turkey. Phone: +90 505 454 8722; Fax: +90 286 2180393;.E-mail: mustafaresorlu77@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. This study investigated the presence of bursitis in the medial compartment of the knee (pes anserine, semimembranosus-tibial collateral ligament, and medial collateral ligament bursa) in osteoarthritis, chondromalacia patella and medial meniscal tears. Patients and methods. Radiological findings of 100 patients undergoing magnetic resonance imaging with a preliminary diagnosis of knee pain were retrospectively evaluated by two radiologists. The first radiologist assessed all patients in terms of osteoarthritis, chondromalacia patella and medial meniscal tear. The second radiologist was blinded to these results and assessed the presence of bursitis in all patients. Results. Mild osteoarthritis (grade I and II) was determined in 55 patients and severe osteoarthritis (grade III and IV) in 45 cases. At retropatellar cartilage evaluation, 25 patients were assessed as normal, while 29 patients were diagnosed with mild chondromalacia patella (grade I and II) and 46 with severe chondromalacia patella (grade III and IV). Medial meniscus tear was determined in 51 patients. Severe osteoarthritis and chondromalacia patella were positively correlated with meniscal tear (p < 0.001 and p = 0.018, respectively). Significant correlation was observed between medial meniscal tear and bursitis in the medial compartment (p = 0.038). Presence of medial periarticular bursitis was positively correlated with severity of osteoarthritis but exhibited no correlation with chondromalacia patella (p = 0.023 and p = 0.479, respectively). Evaluation of lateral compartment bursae revealed lateral collateral ligament bursitis in 2 patients and iliotibial bursitis in 5 patients. Conclusions. We observed a greater prevalence of bursitis in the medial compartment of the knee in patients with severe osteoarthritis and medial meniscus tear. Key words: medial periarticular bursitis; medial meniscal tear; osteoarthritis Introduction Although osteoarthritis (OA), a common joint dis- ease, is not fatal it compromises quality of life. It has become more important in terms of public health with the increase in the average life span associated with advances in the medical sphere. The process begins with progressive loss of joint cartilage and gradually causes meniscal, muscle, bone tissue, lig- ament and bursa pathologies.1 Various factors are implicated in the link between aging and OA. The most important of these are decreased cartilage perfusion, impaired joint morphology, increased ligament laxity, decreased anabolic response and Radiol Oncol 2017; 51(4): 401-406. Resorlu M et al. / Chondromalacia patella, medial meniscal tear and medial periarticular bursitis402 thinning in the cartilage plate.2 Female gender and obesity are other important risk factors in addition to aging. Hormonal changes in particular occurring in women in the postmenopausal period are also a significant predisposing factor.3 Obesity also leads to osteoarthritis, not only through its mechanical effect but also due to its metabolic consequences, and has an adverse effect on clinical findings.1,3 Since it results in a mechanical disadvantage for lower extremity functions, pain is more frequent and more severe.4 Another clinical condition fre- quently encountered together with aging is chon- dromalacia patella (CP). Vascular insufficiency, patellar variations, trauma, dislocation, fracture, rheumatological diseases and impairment of me- chanical stress balance on the joint are implicated in the etiology.5 The principal changes in cartilage tissue are softening, swelling, fissuring or ulcera- tion.5 There are several superficially or deeply lo- cated bursae in the knee joint, their principal func- tion being to reduce friction between the surfaces. Pes anserine (PA) bursa, semimembranosus-tibial collateral ligament (SM-TCL) bursa and medial col- lateral ligament (MCL) bursa inflammations may cause to medial knee pain. Similarly, inflammation in the iliotibial or lateral collateral ligament (LCL) bursae in the lateral of knee also lead to knee pain. Etiological factors such as recurring trauma, im- paired joint stability and joint overloading play a role in the development of bursitis.6 Magnetic reso- nance imaging (MRI) is the gold standard radiolog- ical technique in the assessment of pathologies of the ligament, meniscus, cartilage, bursa and bone marrow in the knee joint.7 This study investigated the relationship between pathologies, such as me- niscal tear, OA and CP, affecting knee mechanics and bursitis in medial compartment of knee Patients and methods Patients MR images from 100 patients with OA of the knee were evaluated retrospectively. The patients in- cluded in our study group were selected from among individuals presenting to our hospital in 2014–2016. Individuals aged 65 or over were in- cluded in the study group, irrespective of gender. Patient selection was based on the order of patients on the work station. Patients with OA determined with conventional radiography were included. Exclusion criteria included presence of acute trau- ma or fracture, inadequate image quality, rheuma- tological disease or history of knee surgery. Imaging protocol All patients were evaluated with a routine knee imaging protocol on a 1.5 Tesla MRI unit (Signa Excite; GE Medical Systems, Wisconsin, USA). Section thicknesses were 3.5 mm in sagittal im- ages and 4 mm in axial and coronal images. The fluid-sensitive sequences were performed with fat suppression. The imaging protocol consisted of a sagittal T1 weighted spin-echo sequence (540 ms/11.3 ms, repetetion time [TR]/echo time [TE], 320x224 matrix, number of excitations [NEX] 1.0), sagittal T2 weighted spin-echo (4480 ms/85 ms, TR/ TE; 256x256 matrix; NEX,1.0) axial proton density (3300 ms/34.7 ms, [TR]/ [TE]; [NEX], 1.0; 320x224 matrix), sagittal proton density sequence (4480 ms/45 ms, TR/TE; NEX,1.0; 320x224 matrix) and a coronal proton density sequence (3620 ms/31.8 ms, TR/TE; 320x224 matrix; NEX,1.0). Image assessment The radiological findings of 100 patients with OA of the knee undergoing MRI with a preliminary diagnosis of knee pain were retrospectively evalu- ated by two radiologists. The first radiologist as- sessed all patients in terms of OA, CP, medial meniscal damage and anterior and posterior cruci- ate ligament pathologies. The second radiologist, blinded to the results, investigated the presence of bursitis in all patients. An approval was obtained from Canakkale Onsekiz Mart University Ethics Committee and written informed consent was waived because of the retrospective nature of the study. Presence of osteophytes, narrowing in the articular space and sclerotic changes were assessed with radiography. OA grading was based on radi- ography and the Kellgren-Lawrence system grad- ing scale.8 Grades I and II were regarded as mild and grades III and IV as severe. The Noyes Chondral Injury Classification sys- tem was used to classify CP. Grade 0 was regarded as normal, grade I as focal signal increase in carti- lage, grade II as fissure or ulcer in cartilage of up to 50%, grade III as greater than 50% loss in cartilage thickness and grade IV as full thickness cartilage defect and subchondral edema. Grade I and II were regarded as mild and grade III and IV as severe. Meniscal damage was classified under three groups - Group 1; normal, Group 2; degenerative signal increase without tear, and Group 3; tear opening onto at least one articular surface. Enlargement in the bursa, fluid deposition, wall thickening and accompanying edema in close proximity were in- Radiol Oncol 2017; 51(4): 401-406. Resorlu M et al. / Chondromalacia patella, medial meniscal tear and medial periarticular bursitis 403 terpreted in favor of bursitis.9 Hypointense signal changes on T1W images and hyperintense signal changes on TW2 images reflecting increased con- centration of compounds containing H+ ions in the bone medulla were interpreted in favor of bone marrow edema.10 Statistical analysis Statistical analysis was performed on SPSS 20.0 (SPSS Inc., Chicago, IL, USA) software. The Kolmogorov-Smirnov/Shapiro-Wilk tests were used to assess normal distribution of variables. Descriptive data were expressed as mean, standard deviation and minimum and maximum values, fre- quency and percentage values. The t test was used to compare means in independent groups and the chi square test in the analysis of categoric variables. Relations between constant variables were investi- gated using Kendall’s correlation analysis. P < 0.05 was regarded as statistically significant. Results Gender distribution was 54 female (54%) and 46 male (46%), and the mean age was 70.47 ± 5.35 years (min 65, max 84). Mild OA (grade I and II) was determined in 55 patients and severe OA (grade III and grade IV) in 45. There was no differ- ence between male and female patients in terms of severity of OA (p = 0.170). At evaluation of the ret- ropatellar cartilage, 25 patients were normal, while mild CP (grade I and II) was determined in 25 and severe CP (grade III-IV) in 46. Since lateral meniscal tear was determined in only six patients, this could not be subjected to sta- tistical analysis. At radiological examination of the medial meniscus, 4 patients were normal, while degenerative signal increase was present in 45 pa- tients and meniscal tear in 51. Meniscal tear was present in 36 of the 45 patients with severe OA and in 15 of the 55 patients with mild OA, and the cor- relation between severity of OA and meniscal tear was statistically significant (p < 0.001). Similarly, significant correlation was determined between CP and meniscal tear (p = 0.018). Bursitis in the medial compartment was present in 26 of the 51 patients with medial meniscal tear, and in 15 of the 49 pa- tients with no tear (p = 0.038). Bone marrow edema in the tibia or femur was determined in 45 patients. Presence of bone marrow edema was positively correlated with severity of OA, but not with CP (p < 0.001 and p = 0.93, respectively). Patients’ demo- TABLE 1. Patients’ demographic characteristics and radiological findings Mean age, years 70.47±5.5 Gender, no. Female 54 Male 46 Osteoarthritis, n Mild (Grade I and II) 55 Severe (Grade III and IV) 45 Chondromalacia patella, n Normal 25 Mild (Grade I and II) 29 Severe (Grade III and IV) 46 Medial meniscal tear, no Normal 4 Degeneration 45 Tear 51 Bone marrow edema, no 45 Others Enchondroma 4 Synovial chondromatosis 4 Osteochondroma 1 FIGURE 1. A 70 year-old patient presented with knee pain. Axial MR image shows sartorius (thin arrow) and gracilis tendon (thick arrow) in the patient with pes anserine bursitis (curved arrow). graphic characteristics and radiological findings are shown in Table 1. Bursitis was determined in at least one of the medial periarticular bursae in 41 patients, the most common form being PA bursitis (Figure 1). Bursitis cases determined at MRI are shown in Table 2. Radiol Oncol 2017; 51(4): 401-406. Resorlu M et al. / Chondromalacia patella, medial meniscal tear and medial periarticular bursitis404 partment is shown in Table 3. Baker’s cyst was de- termined in 36 patients, the prevalence increasing with severity of OA but was not correlated with CP (p = 0.044 and p = 0.264, respectively). Positive correlation was present between sever- ity of OA and CP (p = 0.03). Evaluation of lateral compartment bursae revealed lateral collateral bursitis in two patients and iliotibial bursitis in five (Figure 2 and 3). Prepatellar bursitis in the anterior compartment was determined in four patients, in- frapatellar bursitis in four and suprapatellar bursi- tis in three. Additionally, synovial enchondroma- tosis was present in four patients, enchondroma in four and osteochondroma in one. Discussion The knee joint is frequently subjected to trau- ma, and degenerative changes occur with aging. Trauma, degenerative changes, morphological var- iations, mechanical stress impairments and aging are factors involved in the etiology of OA, CP, bur- sitis and medial meniscal tear.3,11 Common risk fac- tors are one of the reasons why pathologies in the knee joint affect more than one anatomical struc- ture, rather than remaining local. Moreover, pa- thologies that begin locally induce morphological and functional changes in other anatomical struc- tures by compromising biomechanics.11,12 Burger et al. investigated the development of OA as a result of inadequate meniscal tear repair in an animal study.12 They reported that OA developed in the medial tibiofemoral and patellofemoral joint due to insufficient meniscal tear repair. Cicuttini et al. showed gradual loss of cartilage in patients under- FIGURE 2. Coronal (A) and axial (B) proton-density fat-suppressed images of knee show LCL bursitis in a 70 years old patient. FIGURE 3. A 80 year-old patient presented with pain and tenderness in knee joint. Axial (A) and coronal (B) images demonstrate a fluid collection in the dilated iliotibial bursa. TABLE 2. Locations of bursitis determined in the knee joint Bursae No. PA bursitis 26 MCL bursitis 13 SM-TCL bursitis 12 Iliotibial bursitis 5 LCL bursitis 2 Prepatellar bursitis 4 Infrapatellar bursitis 4 Suprapatellar bursitis 3 Baker’s cyst 36 LCL = lateral collateral ligament; MCL = medial collateral ligament; PA = Pes Anserine; SM-TCL = semimembranosus–tibial collateral ligament Presence of bursitis in the medial compartment was positively correlated with severity of OA, but was not correlated with CP (p = 0.023 and p = 0.479, respectively). The relation between medial menis- cal tear, OA and CP and bursitis in the medial com- TABLE 3. The relation between osteoarthritis, chondromalacia patella, meniscal tear and medial periarticular bursitis Medial compartment bursitis Yes No p Osteoarthritis Mild (n = 55) 17 38 0.023 Severe (n = 45) 24 21 Chondromalacia Patella Normal (n = 25) 8 17 Mild (n = 29) 14 15 0.479 Severe (n = 46) 19 27 Meniscal Tear No (n = 49) 15 34 0.038 Yes (n = 51) 26 25 A A B B Radiol Oncol 2017; 51(4): 401-406. Resorlu M et al. / Chondromalacia patella, medial meniscal tear and medial periarticular bursitis 405 going partial meniscectomy.13 The significant cor- relation observed in our study between both OA and CP with meniscal tear (p < 0.001 and p = 0.018) was compatible with these findings. Additionally, the correlation between severity of OA and CP (p = 0.030) also supports the previous literature.12,14 These findings all suggest that these pathologies induce one another as a result of changes in knee biomechanics and morphology. Furthermore, com- mon risk factors such as advanced age and degen- eration reinforce this association. Inflammation occurring in the PA, SM-TCL (Figure 4) and MCL bursa in the medial of knee is an important cause of pain. Obesity, degenerative joint disease, valgus deformity, trauma, impaired joint stability, excessive loading on the joint and sporting activity are important causes of inflam- mation in the bursae.15 Some authors have suggest- ed that the structure of the female pelvis creates a predisposition to bursitis.16 We determined no rela- tion between gender and the prevalence of bursitis (p = 0.954). Toktas et al. investigated the prevalence of pes anserine bursitis in patients with OA.17 They determined a greater pes anserine thickness not only in OA patients with bursitis, but also in OA patients without bursitis compared to the control group. We selected all our cases from OA patients and took the OA grade (mild and severe) as our reference points. We also investigated the presence of SM-TCL and MCL bursitis in addition to PA bursitis. We observed higher prevalence of medial periarticular bursitis in patients with severe OA compared to those with mild OA (p = 0.023). Our results are compatible with recent studies reveal- ing as association between OA and pes anserine bursitis.16 Another important finding of this study was a positive correlation between medial meniscal tear and medial compartment bursitis. Bursitis was present in 26 of the 51 patients in whom medial meniscal tear was determined, and in 15 of the 49 patients with no tear. The difference was statisti- cally significant (p = 0.038). Various factors affect the positive relation between prevalence of bursi- tis and severity of OA and medial meniscal tear. The first are similar etiological factors. Second are structural changes associated with OA and menis- cal tear and impairment of joint stability.16 Despite the common etiological factors and the effect on joint biomechanics, we determined no relation between CP and medial compartment bursitis. We think that this finding was affected by patel- lar and trochlear morphology. Variations such as patella alta and baja influence the development of CP. Additionally, it has been suggested that a short medial facet increases mechanical stress on carti- lage and causes CP.5,18 One recent study proposed the sulcus angle/trochlear depth ratio as a power- ful predictor.11 One limitation of our study is that patellar and trochlear morphology were not evalu- ated. Another limitation is that due to its retrospec- tive nature patients were not assessed in terms of diabetes during the study. The prevalence of Baker’s cyst was 36%, ilioti- bial bursitis 5% and LCL bursitis 2%. In their study of 163 patients, Hayashi et al. reported prevalences of popliteal cyst of 40% and of iliotibial bursitis of 1%.19 Baker’s cyst was the most common lesion in both studies, and the results were compatible. A relation was determined between Baker’s cyst and severity of OA in our study, but there was no cor- relation between bursitis in the medial compart- ment and Baker’s cyst (p = 0.044 and p = 0.954, respectively). The positive correlation between Baker’s cyst and OA is compatible with the previ- ous literature.20 Increased joint effusion and intra- articular pressure in OA may be involved in this relationship. We observed prepatellar (4%), infra- patellar (4%) and suprapatellar bursitis (3%) in the anterior of joint. Hayashi et al. reported the preva- lence of prepatellar and infrapatellar bursitis at 2%. The mean age of their study population was 52 ± 6 years, and patients without OA constituted 32% of their cases.19 The higher mean age in our study and all our patients having OA may explain the differ- ence in the findings. FIGURE 4. The axial fat saturated proton density weighted image demonstrates a fluid collection in the SM-TCL bursa. Radiol Oncol 2017; 51(4): 401-406. Resorlu M et al. / Chondromalacia patella, medial meniscal tear and medial periarticular bursitis406 In conclusion, we observed a correlation be- tween the prevalence of bursitis in the medial compartment and the severity of OA and medial meniscal tear. We determined no relation between severity of CP and bursitis. References 1. Ayral X, Pickering EH, Woodworth TG, Mackillop N, Dougados M. Synovitis: A potential predictive factor of structural progression of medial tibi- ofemoral knee osteoarthritis e results of a 1 year longitudinal arthroscopic study in 422 patients. Osteoarthr Cartil 2005; 13: 361-7. doi: 10.1016/j. joca.2005.01.005 2. Arden N, Nevitt MC. Osteoarthritis: epidemiology. Best Pract Res Clin Rheumatol 2006; 20: 3-25. doi: 10.1016/j.berh.2005.09.007 3. Creamer P, Lethbridge-Cejku M, Hochberg MC. Factors associated with functional impairment in symptomatic knee osteoarthritis. Rheumatology (Oxford) 2000; 39: 490-6. 4. de Miguel Mendieta E, Cobo Ibáñez T, Usón Jaeger J, Bonilla Hernan G, Martın Mola E. Clinical and ultrasonographic findings related to knee pain in osteoarthritis. Osteoarthritis Cartilage 2006; 14: 540. doi: 10.1016/j. joca.2005.12.012 5. Tanamas SK, Teichtahl AJ, Wluka AE, Wang Y, Davies-Tuck M, Urquhart DM, et al. The associations between indices of patellofemoral geometry and knee pain and patella cartilage volume: a crosssectional study. BMC Musculoskelet Disord 2010; 11: 87. doi: 10.1186/1471-2474-11-87 6. Alvarez-Nemegyei J. Risk factors for pes anserinus tendinitis/bursitis syn- drome: a case control study. J Clin Rheumatol 2007; 13: 63-5. doi: 10.1097/01.rhu.0000262082.84624.37 7. Nenezic D, Kocijancic I. The value of the sagittal-oblique MRI technique for injuries of the anterior cruciate ligament in the knee. Radiol Oncol 2013; 47: 19-25. doi: 10.2478/raon-2013-0006 8. Kellgren JH, Lawrence JS. Radiological assessment of osteoarthritis. Ann Rheum Dis 1957; 16: 499-502. 9. Hirji Z, Hunjun JS, Choudur HN. Imaging of the bursae. J Clin Imaging Sci 2011; 1: 22. doi: 10.4103/2156-7514.80374 10. Oztekin O, Calli C, Kitis O, Adibelli ZH, Eren CS, Apaydin M, et al. Reliability of diffusion weighted MR imaging in differentiating degenerative and infectious end plate changes. Radiol Oncol 2010; 44: 97-102. doi: 10.2478/ v10019-010-0006-z 11. Resorlu H, Zateri C, Nusran G, Goksel F, Aylanc N. The relation between chondromalacia patella and meniscal tear and the sulcus angle/trochlear depth ratio as a powerful predictor. J Back Musculoskelet Rehabil 2017; 30: 603-8. doi: 10.3233/BMR-160536. 12. Burger C, Kabir K, Mueller M, Rangger C, Mınor T, Tolba RH. Reropatellar chondromalacia associated with medial osteoarthritis after meniscus in- jury. One year of observations in sheep. Eur Surg Res 2006; 38: 102-8. doi: 10.1159/000093281 13. Cicuttini FM, Forbes A, Yuanyuan W, Rush G, Stuckey SL. Rate of knee carti- lage loss after partial meniscectomy. J Rheumatol 2002; 29: 1954-6. 14. Kalichman L, Zhang Y, Niu J, Goggins J, Gale D, Felson DT, et al. The asso- ciation between patellar alignment and patellofemoral joint osteoarthritis features-an MRI study. Rheumatology (Oxford) 2007; 46: 1303-8. doi: 10.1093/rheumatology/kem095 15. Saggini R, Di Stefano A, Dodaj I, Scarcello L, Bellomo RG. Pes anserine bursi- tis in symptomatic osteoarthritis patiens: A mesotehrapy Treatment study. J Altern Complement Med 2015; 21: 480-4. doi: 10.1089/acm.2015.0007 16. Helfenstein M Jr, Kuromoto J. Anserine syndrome. Rev Bras Reumatol 2010; 50: 313-27. doi: 10.1590/S0482-50042010000300011 17. Toktas H, Dundar U, Adar S, Solak O, Ulaslı AM. Ultrasonographic assess- ment of pes anserinus tendon and pes anseinus tendinitis bursitis syndrome in patients with knee osteoarthritis. Mod Rheumatol 2015; 25: 128-33. doi: 10.3109/14397595.2014.931909 18. Endo Y, Schweitzer ME, Bordalo-Rodrigues M, Rokito AS, Babb JS. MRI quantiative morphologic analysis of patellofemoral region: lack of correla- tion with chondromalacia patellaet surgery. AJR Am J Roentgenol 2007; 189: 1165-8. doi: 10.2214/AJR.07.2236 19. Hayashi D, Roemer FW, Dhina Z, Kwoh CK, Hannon MJ, Moore C, et al. Longitudinal assessment of cyst-like lesions of the knee and their relation to radiographic osteoartjritis and MRI-detected effusion and synovitis in pa- tients with knee pain. Arthitis Res Ther 2010; 12: R172. doi: 10.1186/ar3132 20. Hill CL, Gale DG, Chaisson CE, Skinner K, Kazis L, Gale ME, et al. Knee effu- sions, popliteal cysts, and synovial thickening: association with knee pain in osteoarthritis. J Rheumatol 2001; 28: 1330-7. Radiol Oncol 2017; 51(4): 407-414. doi: 10.1515/raon-2017-0048 407 research article Phytotherapeutics oridonin and ponicidin show additive effects combined with irradiation in pancreatic cancer in vitro Jakob Liermann1, Patrick Naumann1, Franco Fortunato2, Thomas E. Schmid3, Klaus-Josef Weber1, Jürgen Debus3, Stephanie E. Combs3,4 1 Department of Radiation Oncology, Heidelberg University Hospital, Heidelberg, Germany 2 Heidelberg University Hospital, Section Surgical Research, Heidelberg, Germany 3 Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany 4 Institute of Innovative Radiotherapy (iRT), Department of Radiation Sciences (DRS), Helmholtz Zentrum München, Neuherberg, Germany Radiol Oncol 2017; 51(4): 407-414. Received 28 June 2017 Accepted 13 October 2017 Correspondence to: Dr. Jakob Liermann, Department of Radiation Oncology, Heidelberg University Hospital, INF 400, 69120 Heidelberg, Germany. E-mail: jakob.liermann@med.uni-heidelberg.de Disclosure: No potential conflicts of interest were disclosed. *J Liermann and P Naumann contributed equally to this work. Background. Chemoradiation of locally advanced non-metastatic pancreatic cancer can lead to secondary oper- ability by tumor mass reduction. Here, we analyzed radiomodulating effects of oridonin and ponicidin in pancreatic cancer in vitro. Both agents are ent-kaurane diterpenoids, extracted from Isodon rubescens, a plant that is well known in Traditional Chinese Medicine. Cytotoxic effects have recently been shown in different tumor entities for both agents. Materials and methods. Pancreatic cancer cell lines AsPC-1, BxPC-3, Panc-1 and MIA PaCa-2 were pretreated with oridonin or ponicidin and irradiated with 2 Gy to 6 Gy. Long-term survival was determined by clonogenic assay. Cell cycle effects and intensity of γH2AX as indicator for DNA double-strand breaks were investigated by flow cytom- etry. Western blotting was used to study the DNA double-strand break repair proteins Ku70, Ku80 and XRCC4. Results. Oridonin and ponicidin lead to a dose-dependent reduction of clonogenic survival and an increase in γH2AX. Combined with irradiation we observed additive effects and a prolonged G2/M-arrest. No relevant changes in the levels of the DNA double-strand break repair proteins were detected. Conclusions. Pretreatment with oridonin or ponicidin followed by irradiation lead to an additional reduction in sur- vival of pancreatic cancer cells in vitro, presumably explained by an induced prolonged G2/M-arrest. Both agents seem to induce DNA double-strand breaks but do not interact with the non-homologous end joining (NHEJ) pathway. Key words: oridonin; ponicidin; irradiation; pancreatic cancer; γH2AX; radiosensitivity Introduction Pancreatic cancer is the fourth leading cause of can- cer related deaths in the United States.1 Surgical re- section is still the most important treatment modal- ity and prognostic factor, but in the late stage of the disease, only 10–20% of all primary tumours are still operable.2 Beside primary metastasized and thus palliative stage IV disease 30-40% of all cases are locally advanced but non-metastasized tumors that could benefit from neoadjuvant treatment.3 We recently reported that approximately one third of these locally advanced tumors become resectable by a neoadjuvant combined chemoradiation and thus regain a curative therapy option.4 To further increase the efficacy of radiotherapy in pancreatic cancer, new chemotherapeutics, that can sensitize pancreatic cancer tissue to radiation, are needed. Radiol Oncol 2017; 51(4): 407-414. Liermann J et al. / Oridonin and ponicidin in pancreatic cancer408 The diterpenoids oridonin and ponicidin are both extracts from Isodon rubescens, a plant used in Traditional Chinese Medicine that is known for its anti-inflammatory and antitumor properties in the treatment of esophageal and cardiac cancer.5 Recently, antineoplastic properties of oridonin and ponicidin have been published.6-8 In pancre- atic cancer oridonin induces apoptosis and leads to an arrest in the G2/M-phase of the cell cycle.9-12 Further, oridonin inhibits nuclear factor-kB.13 Ponicidin is also known to induce apoptosis14-17 but to our knowledge, there is no data for ponicidin in pancreatic cancer. Potential radiosensitization could be observed in chinese-hamster-V79 cells by oridonin.18 Apart from this study we found no publication concern- ing oridonin or ponicidin induced radiomodula- tion. As natural occurring products for both substanc- es no serious side effects in humans are known which qualifies them as potential new chemothera- peutic drugs. Data of an in vivo trial for oridonin used in acute leukemia seems to confirm this hy- pothesis.19 Here, we analyzed radiomodulating ef- fects of oridonin and ponicidin in pancreatic cancer cells in vitro by clonogenic assay, cell cycle analy- sis, γH2AX expression and DNA repair proteins expression analysis. Material and methods Cell culture and irradiation The pancreatic cancer cell lines AsPC-1, BxPC-3 and MIA PaCa-2 were cultured in RPMI-1640 me- dium, the cell line Panc-1 in Dulbecco’s modified Eagle’s Medium (DMEM). Media included 10% FBS and 1% Penicillin/Streptomycin. The cells were cultured at 37°C in 5% CO2. AsPC-1 and Panc-1 cell lines were purchased from CLS cell line ser- vice GmbH, Germany and BxPC-3 as well as MIA PaCa-2 cells from LGC Standards GmbH (ATCC), Germany. Oridonin was obtained from Sigma- Aldrich Corporation, USA and ponicidin from Shanghai Zhanshu Chemical Technology Co., Ltd., China. Both reagents were dissolved in DMSO and stored at -80°C. Cells were X-irradiated (X-RAD 320, Precision X-Ray, Inc., USA) operated at 320 kV and 12.5 mA. The average dose rate was 1.1 Gy/min. The used filter consisted of 1.5 mm Al, 0.25 mm Cu and 0.75 mm Sn. The tissue culture flasks, in which the cells were cultured, were placed on a PMMA-plate of 1 cm height. Clonogenic assay To assess long-term cell survival, a defined number of cells were seeded in 25 cm2 tissue culture flasks and incubated for 24 hours. Cell counts were ad- justed for the different treatment schemes. Cell lines AsPC-1, BxPC-3 and Panc-1 were treated with ori- donin, ponicidin or DMSO (control) for 24 hours as indicated and irradiated afterwards once with 2 Gy or 6 Gy. The dose of each substance and the inten- sity of irradiation were established through pre-ex- periments to achieve a survival rate of 20-70% after single treatment alone. MIA PaCa-2 cells were pre- treated with lower doses of oridonin and ponicidin due to higher sensitivity to the reagents. 8-9 days (time differs among cell lines) after the treatment cells were fixed in 70% ethanol and stained with 0.2% methylenblue. Colonies, defined as contain- ing at least 50 cells, were counted manually under a light microscope and the plating efficiency, de- fined as the quotient of counted colonies to plated cells, was determined. The surviving fraction was defined as the ratio of each experiment’s plating ef- ficiency to its control. Cells were plated in triplets to compensate for pipetting errors and at least three independent experiments were performed. Determination of radiosensitivity To analyze combined treatments, surviving frac- tions were normalized to the values of correspond- ing single reagent treatment using a ratio of the combined treatment’s plating efficiency to the sin- gle reagent treatment one’s. Dose-response curves were created using the linear-quadratic model.20 Combination effects were analyzed defining additivity according to Steel and Peckham.21 For this purpose a dose-response curve was calculated that represents the course of the measured control curve at a pre-effect similar to the single reagent treatment’s effect („theoretical control-curve“). Additive effects were defined as area between the measured control curve and the theoretical one. Supraadditive effects were defined as beneath the theoretical control curve and radioprotective effects as above the measured control curve. Independent toxicity was defined as identical curve to the meas- ured control curve (for further information see Suppl. Figure 1). Cell cycle analysis Since impact on cell cycle distribution was regress- ing 24 hours after pretreatment a shorter exposi- Radiol Oncol 2017; 51(4): 407-414. Liermann J et al. / Oridonin and ponicidin in pancreatic cancer 409 tion time to oridonin and ponicidin compared to the other experiments was chosen. Simultaneously the dosage was augmented to compensate for shortage in exposition time. Cell lines Panc-1 and MIA PaCa-2 were pretreated for two hours with oridonin, ponicidin or DMSO (control) and irra- diated as indicated. To induce a less lethal dam- age than one would expect of 6 Gy, an irradiation dose of 4 Gy was chosen. Cells were then fixed in ice-cold ethanol and RNA-strands were cleaved by RNase A (AppliChem GmbH, Germany) at the fol- lowing time points: beginning of the treatment, be- fore irradiation, 12 and 24 hours after irradiation. Then cells were stained with propidium iodide (Sigma-Aldrich Corporation, USA) and the amount of propidium iodide per cell was quantified in a flow cytometer (FACScan, Becton Dickinson and Company, USA). At least 104 ungated cells were analyzed by BD CellQuest Pro 4.0.2 (Becton Dickinson and Company, USA) and the different phases of the cell cycle were determined using the software ModFit LT 3.0 (Verity Software House, Inc., USA). At least two independent experiments were performed. γH2AX-quantification Since clonogenic assays revealed different sensitiv- ity between AsPC-1, BxPC-3 and Panc-1 cells on the one hand and MIA PaCa-2 cells on the other hand further experiments were done with the cell lines Panc-1 and MIA PaCa-2. To quantify the induc- tion of DNA double-strand breaks Panc-1 and MIA PaCa-2 cells were pretreated with oridonin, poni- cidin or DMSO (control) for 24 hours and were ir- radiated once as indicated. At different time points after irradiation MIA PaCa-2 and Panc-1 cells were fixed using 1% PFA and ice-cold ethanol. Cells FIGURE 1. Clonogenic survival of Panc-1 cells treated with either irradiation and/or oridonin (ORI) and ponicidin (PON) as indicated. (A) survival curves and (B) radiosensitivity to different dosages with measured and theoretical control curve, according to Steel and Peckham for Panc-1 cells. Results are presented as mean values +/- standard deviation. At least three independent experiments were performed. A B Radiol Oncol 2017; 51(4): 407-414. Liermann J et al. / Oridonin and ponicidin in pancreatic cancer410 ence between the compared results. *P < 0.05, **P < 0.005, ***P < 0.001. Results Oridonin and ponicidin reduce clonogenic survival and show additive effects when combined with irradiation To investigate long-term survival cells were treated with oridonin or ponicidin in a low dosage of 0.5 µg/ml and high dosage of 2.5 µg/ml (Figure 1A). Since MIA PaCa-2 cells exposed a higher sensitiv- ity to both oridonin and ponicidin, we used 0.1 µg/ ml and 0.5 µg/ml as the low and the high dosage in these cells, respectively. MIA PaCa-2 cells showed a significant reduction in clonogenic survival to a fraction of 51% when treated with the lower dos- age of oridonin and thus were much more sensi- tive than the other cell lines, with a mean surviv- ing fraction of 88% in AsPC-1, BxPC-3 and Panc-1 cells (Suppl. Figure 2). Interestingly, ponicidin was more effective than oridonin: 2.5 µg/ml of ponici- din lead to a mean surviving fraction of 12%. The same amount of oridonin showed an almost three- fold higher surviving fraction of 32% in AsPC-1-, BxPC-3- and Panc-1 cells (Suppl. Figure 3). A further dose-dependent reduction in survival could be observed by combination with irradia- tion. The mean surviving fraction of 88% after sin- gle treatment with low dosage of oridonin could be further attenuated by additional irradiation with 2 Gy or 6 Gy to mean 63% and 13%, respectively. Using these results we investigated the radio- sensitivity according to the model of Steel and Peckham as described in the methods section.21 The higher dose of oridonin and ponicidin lead to addi- tive effects in almost all investigated cell lines when combined with 2 Gy or 6 Gy irradiation, whereas the lower dose just showed independent toxicity (Figure 1B and Suppl. Figure 4). Radioprotectivity as well as supraadditivity could not be observed. G2/M-arrest after combined treatment of radiation and oridonin or ponicidin To determine changes in cell cycle, the four pancre- atic cancer cell lines were treated with oridonin or ponicidin alone as well as in combination with irra- diation. Cell cycle phases were analyzed 12 and 24 hours after irradiation. Pretreatment with oridonin (Figure 2A) or ponicidin (Figure 2B) induced a clear G2/M-arrest of the cell lines AsPC-1 and Panc-1 af- ter 12 hours with up to 36% (Figure 2 and Suppl. were then stained with a γH2AX-antibody (art. no. 16-202A, Merck KGaA, Darmstadt, Germany) and pan-nuclear intensity was measured using a flow cytometer. Results were normalized by creating a ratio of the measured intensity to the intensity of the control probes. At least three independent ex- periments were performed. Western Blot Cell lines Panc-1 and MIA PaCa-2 were treated with oridonin, ponicidin or DMSO (control) for 24 hours. Then lysates of the cells were gained using lysis buffer (10 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT) and inhibitors (PMSF, Pepstatin, Leupeptin, Aprotinin). The protein concentration was determined in a Bradford protein assay and 20 µg to 30 µg Protein were used for Western Blots. A SDS-polyacrylamide-gel-electrophoresis was run using 12% polyacrylamide-gels with a voltage of 160 V and amperage of 250 mA. A wet transfer was performed (80 V, 60 mA) to transfer the proteins on a polyvinylidenfluoride membrane. The membrane was blocked using Roti-Block (Carl Roth. GmbH & Co. KG, Germany). b-Actin (art. no. 4967), GAPDH (art. no. 2118), Ku70 (art. no. 4588) and Ku80 (art. no 2753) primary antibodies were purchased from Cell signaling Technology Inc., USA. XRCC4 (art. no. sc-271087) and secondary antibodies (art. no. sc-2380, sc-2379) were obtained from Santa Cruz Biotechnology, Inc., USA. Dilution was done ac- cording to manufacturer’s instructions. X-Ray films were scanned and the density of the protein bands were determined with the software Image J 1.47V (National Institute of Health, USA). Density was normalized by creating a ratio of protein band to house-keeping protein’s one to avoid minimal differences in the amount of protein load. Then another ratio of the treatment’s value to its control was calculated and allowed to quantify densities and treatment differences. At least two independ- ent experiments were performed. Statistical analysis The results of each experiment were generated as described above. Statistical analysis of the results was done with an unpaired two-tailed t-test using Microsoft® Excel® 2008 for Mac 12.3.6 (Microsoft Corporation, USA). The dose control curves and the figures were calculated and generated with SigmaPlot 12.0 (Systat Software Inc., USA). Results are presented as mean values +/- standard devia- tion. P < 0.05 was considered as a significant differ- Radiol Oncol 2017; 51(4): 407-414. Liermann J et al. / Oridonin and ponicidin in pancreatic cancer 411 Figure 5). The G2/M- fraction of combined treat- ments was not as high as the one observed after irra- diation alone but was augmented in time leading to the fact that 24 hours after irradiation there still was a clear G2/M-arrest in the cell lines AsPC-1, BcPC-3 and Panc-1 when treated with the combined thera- py. MIA PaCa-2 cells showed a clear G2/M-arrest 24 hours after combined treatment with ponicidin but not with oridonin (Suppl. Figure 5). We did not observe a sub-G1 fraction as surrogate of apoptosis. Oridonin and poncidin induce an increased pan-nuclear γH2AX-intensity The potential influence of oridonin or ponicidin on the DNA double-strand break repair was analyzed by measuring the pan-nuclear γH2AX-intensity of pretreated cells via flow cytometry. Both oridonin (Figure 3A) and poncidin (Figure 3B) lead to an approximately 2.5-fold increase of the γH2AX- intensity shortly after treatment, independent of the particular dose level (7.5 µg/mL vs. 15 µg/mL). There was an increase of γH2AX after irradiation alone but highest γH2AX-intensities could be ob- served in combined treatments. The Intensity of γH2AX already declined 10 hours posttreatment. We could not observe a difference in the relative reduction of the γH2AX-intensity between irradia- tion alone and combined treatments. Oridonin and ponicidin do not influence the expression of DNA repair proteins related to the non-homologous end joining (NHEJ) pathway To determine the influence of oridoinin or poni- cidin on the repair of DNA double-strand breaks, Panc-1- and MIA PaCa-2-cells were treated with either agent and three main proteins of the NHEJ pathway, namely XRCC4, Ku70 and Ku80 were detected by Western Blots. Pretreatment with ori- donin or ponicidin lead to a slight reduction of XRCC4-expression but there was no considerable change in Ku70 and Ku80 protein expression in MIA PaCa-2 cells. Panc-1 cells showed no stringent effect in XRCC4 protein expression but a dose-de- pendent increase of Ku70 and Ku80 after pretreat- ment with oridonin or ponicidin (Figure 4). Discussion The aim of this study was to investigate the two phytotherapeutics oridonin and ponicidin for their A A B B FIGURE 2. Cell cycle assay of Panc-1 cells treated with irradiation and/or (A) oridonin (ORI) and (B) ponicidin (PON) as indicated. Results are presented as mean values +/- standard deviation. *P<0.05, **P<0.005, asterisk referring to the amount of cells in G2/M-phase. At least two independent experiments were performed. FIGURE 3. γH2AX-intensity of Panc-1 and MIA PaCa-2 cells treated with irradiation and/or (A) oridonin (ORI) and (B) ponicidin (PON) as indicated. Results are presented as mean values +/- standard deviation. *P<0.05, **P<0.005, ***P<0.001, n.s.=not significant. At least three independent experiments were performed. Radiol Oncol 2017; 51(4): 407-414. Liermann J et al. / Oridonin and ponicidin in pancreatic cancer412 capability to enhance effects of irradiation in pan- creatic cancer cells. Compared to other tumor en- tities pancreatic cancer cells are more resistant to chemo- and radiotherapy and thus radiosensitiz- ing agents are highly clinically relevant. To investigate treatment efficacy long-term survival was determined by clonogenic assays. Changes in cell cycle distribution and intensity of γH2AX as indicator for DNA double-strand breaks were investigated by flow cytometry, since effects of radiotherapy base predominantly on induction of DNA double-strand breaks.22 Western blotting was used to study key proteins of the NHEJ path- way of the DNA double-strand break repair. To our knowledge we are the first who tested ponicidin in combination with irradiation whereas potential ra- diosensitzing effects of oridonin could already be observed in chinese-hamster-V79 cells.18 After treatment with either oridonin or ponici- din we observed a dose-dependent reduction of clonogenic survival in all tested pancreatic cancer cell lines AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 after treatment with either oridonin or ponicidin. Compared to oridonin, equal doses of ponicidin were more effective in reducing clonogenic surviv- al. Similar results were observed by Hsieh et al. in clonogenic survival assays of the breast cancer cell line MCF-7, who also tested both agents in prolif- eration assays with comparable results of oridonin and ponicidin.23 Based on these data we infer that ponicidin might be a more potent cytostatic agent than oridonin. After combined treatment with irradiation and different concentrations of oridonin or ponicidin we observed an increased reduction of clonogen- ic survival. The analysis of potential interactions revealed a trend towards additive effects for the higher concentration of oridonin as well as poni- cidin. Supraadditive effects that provide evidence for actual radiosensitization could not be observed. Cell cycle assays of the present study showed that both oridonin and ponicidin lead to an accu- mulation of cells in the G2/M-phase of the cell cy- cle. The combination of irradiation and oridonin or ponicidin leads to an even prolonged G2/M-arrest. Cells that are stuck in this phase usually present DNA-lesions, such as DNA double-strand breaks or blocked DNA replication forks, which have to be repaired before passing the G2/M-checkpoint.24 An oridonin-induced G2/M-arrest was previously reported by Qi et al. for the pancreatic cancer cell line Panc-1 along with a suppression of cell cycle regulating cyclin B1 and cdc2.10 In our study, we focused on DNA double-strand breaks as underly- ing mechanism especially after combination with irradiation. Therefore we did not investigate any changes in proteins of the cell cycle regulation but further experiments should pinpoint the precise mode G2/M-phase arrest induced by oridonin and ponicidin. Since induction of apoptosis was re- ported for both oridonin and ponicidin9-12,14-17, we expected a sub-G1 peak in cell cycle analysis but could not observe any peak in the sub-G1 noise. Interestingly, the cell cycle assays of Qi et al. for PANC-1 cells treated with oridonin also did not show any sub-G1 peak.10 The analyzed pan-nuclear γH2AX-quantification revealed a two-fold higher γH2AX-intensity after treatment with oridonin or ponicidin. This increase in γH2AX level was independent of the particular dose level of oridonin and ponicidin. Combined with irradiation both oridonin and ponicidn lead to an even higher amount of γH2AX. Pan-nuclear determination of γH2AX is just an indirect indica- tor of DNA double-strand break induction, but it is comparable to the quantification of γH2AX-foci25 which is known to be highly suspect for an induc- tion of DNA double-strand breaks.26 Accordingly, oridonin and ponicidin as well as irradiation seem to induce DNA double-strand breaks. Beside possi- ble effects on cell cycle phase proteins as suggested by Qi et al.10 the induction of DNA double-strand breaks are a good explanation for the observed G2/M-arrest. An already described augmentation A B FIGURE 4. Western Blots of Panc-1 and MIA PaCa-2 cells treated with either (A) oridonin (ORI) and (B) ponicidin (PON) as indicated. One representative Western Blot is shown. At least two independent experiments were performed. NHEJ = non- homologous end-joining. Radiol Oncol 2017; 51(4): 407-414. Liermann J et al. / Oridonin and ponicidin in pancreatic cancer 413 of γH2AX in oridonin-treated BxPC-3 cells and an increased γH2AX-concentration in breast cancer cells exposed to irradiation are in line with our pre- sent data.9,27 By comparison of γH2AX-intensities at different time points the level of γH2AX of the combined treatment schemes decreased quite similar to that of the irradiation treatment alone. We therefore have to conclude that oridonin and ponicidin do not significantly lower the DNA double-strand break repair capacity. To verify this, proteins of the DNA double-strand break repair were detected by Western Blot. Since pancreatic cancer cells are known to show only weak activity of the homolo- gous recombination pathway28, we concentrated on the key proteins of the NHEJ. There were no relevant changes in the amount of Ku70, Ku80 or XRCC4 in oridonin- or ponicidin-treated cells sup- porting the hypothesis that oridonin and ponicidin do not reduce the DNA double-strand break repair mechanisms. Further studies should investigate if oridonin or ponicidin interfere with proteins of the homologous recombination pathway of DNA dou- ble-strand break repair, such as RAD51 or MRE11/ RAD50/NBS1 complex. Recently, Wang et al. could show synergistic effects of an oridonin derivative and paclitaxel through increased reactive oxygen species (ROS) levels and subsequent apoptosis induction.29 Cao et al. observed a synergistic effect of cetuximab and oridonin through increased ROS levels and epi- dermal growth factor receptor (EGFR) inhibition.30 Activation of ROS is known to be one of the main effects of irradiation in cancer treatment. These findings strengthen the idea of combined treat- ment schemes with oridonin and irradiation. In the next step, both substances should be test- ed in combination with the recent standard chemo- therapies such as gemcitabine or FOLFIRINOX. Finally, in vivo data are needed to reveal if efficacy of irradiation in pancreatic cancer patients can be improved by oridonin and ponicidin. Conclusions The current data demonstrate new aspects of the two interesting phytotherapeutics oridonin and poncidin. In vitro both substances are highly effec- tive against pancreatic cancer cells especially when combined with irradiation. Therefore both ori- donin and ponicidin are promising agents in pan- creatic cancer therapy. Our data warrant further in vivo investigations especially in combination with irradiation or standard chemotherapy such as FOLFIRINOX or gemcitabine and nab-paclitaxel. Acknowledgements We thank Lena Orschiedt, Sigrid Daffinger, Sylvia Trinh and Ludmilla Frick for the excellent technical assistance with the experiments. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. JL and PN designed and performed the experi- ments. JL and PN wrote the manuscript. TES per- formed the γH2AX-quantification. FF, SEC, KJW and JD helped finalizing the manuscript. All au- thors read and approved the final manuscript. 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Preliminary results on differential conductivity Clara Pañella1, Quim Castellví2, Xavier Moll3, Rita Quesada1, Alberto Villanueva4, Mar Iglesias5, Dolores Naranjo5, Patricia Sánchez-Velázquez1, Anna Andaluz3, Luís Grande1, Antoni Ivorra2 and Fernando Burdío1 1 General Surgery Department, Hospital del Mar Medical Research Institute (IMIM), Universitat Pompeu Fabra, Barcelona, Spain 2 Department of Informatics and Communication Technologies, Universitat Pompeu Fabra, Barcelona, Spain 3 Department of Pathological Anatomy, Hospital del Mar Medical Research Insitute (IMIM), Universitat Pompeu Fabra, Barcelona, Spain 4 Chemoresistance and Predictive Factors Group, Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat, Barcelona, Spain 5 Medical and Surgery Animal Department, Faculty of Veterinary, Universitat Autònoma de Barcelona3, Cerdanyola del Vallès, Barcelona, Spain Radiol Oncol 2017; 51(4): 415-421. Received 2 July 2017 Accepted 22 October 2017 Correspondence to: Fernando Burdío M.D., Clara Pañella M.D., Unidad de Cirugía Hepática. Servicio de Cirugía General y Digestiva, Hospital del Mar de Barcelona, Passeig Maritim 25-29, 08003 Barcelona, Spain. E-mail: fburdio@hotmail.com, cpvilamu@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Spread hepatic tumours are not suitable for treatment either by surgery or conventional ablation methods. The aim of this study was to evaluate feasibility and safety of selectively increasing the healthy hepatic conductivity by the hypersaline infusion (HI) through the portal vein. We hypothesize this will allow simultaneous safe treatment of all nodules by irreversible electroporation (IRE) when applied in a transhepatic fashion. Material and methods. Sprague Dawley (Group A, n = 10) and Athymic rats with implanted hepatic tumour (Group B, n = 8) were employed. HI was performed (NaCl 20%, 3.8 mL/Kg) by trans-splenic puncture. Deionized serum (40 mL/ Kg) and furosemide (2 mL/Kg) were simultaneously infused through the jugular vein to compensate hypernatremia. Changes in conductivity were monitored in the hepatic and tumour tissue. The period in which hepatic conductivity was higher than tumour conductivity was defined as the therapeutic window (TW). Animals were monitored during 1-month follow-up. The animals were sacrificed and selective samples were used for histological analysis. Results. The overall survival rate was 82.4% after the HI protocol. The mean maximum hepatic conductivity after HI was 2.7 and 3.5 times higher than the baseline value, in group A and B, respectively. The mean maximum hepatic conductivity after HI was 1.4 times higher than tumour tissue in group B creating a TW to implement selective IRE. Conclusions. HI through the portal vein is safe when the hypersaline overload is compensated with deionized serum and it may provide a TW for focused IRE treatment on tumour nodules. Key words: irreversible electroporation; liver tumour; electrical conductivity Introduction Electroporation (EP) is the phenomenon by which the cell membrane permeability is increased when the cell is subjected to high electric fields. Such in- crease in membrane permeability can irreversibly alter cell homeostasis and lead to cell death, either by necrosis or apoptosis. In this case the term irre- Radiol Oncol 2017; 51(4): 415-421. Pañella C et al. / Increasing hepatic conductivity by hypersaline through the portal vein416 Without hypersaline infusion through the portal vein With hypersaline infusion through the portal vein (NaCl 20%) 1000 900 800 700 600 500 400 300 200 100 0 1000 900 800 700 600 500 400 300 200 100 0 Electric Field (V/cm) Electric Field (V/cm) A B C D liver liverσ σ = 0.12 S/m = 1.2 S/m versible electroporation (IRE) is used.1-5 IRE is typi- cally performed by inserting thin needle electrodes into the targeted tissue and delivering a number (8-100) of short (10-100 µs) high-voltages pulses across the electrodes, thereby producing field mag- nitudes in the tissue in the order of 1000 V/cm or more.3 The ideal ablation system for multiple hepatic nodules should treat the tumoural tissue while preserving the remaining healthy hepatic tissue. Unfortunately, the IRE as it is used today, does not allow the preservation of the surrounding healthy tissue. Although, it is a non-thermal technique and is credited with sparing the extracellular matrix, IRE leads to the apoptosis of either healthy or tu- moural hepatic tissue where the electric field is ap- plied.5-10 In fact, this technique is highly dependent on tissue conductivity11-13 and tumours have higher dielectric properties (and higher conductivities) than normal healthy tissue because of the higher sodium and water content of cancer cells.14 This higher conductivity may lead to a lower electric field in the tumoural tissue than in the surround- ing healthy tissue.12 Our group recently studied the long-term effec- tiveness of treating a large amount of healthy he- patic tissue harbouring an implanted tumour with parallel transhepatic electrodes.15 In this study we showed that even with high electrical fields (up to 2000 V/cm) complete eradication of the tumour was infrequent even when massive destruction of nearby healthy hepatic tissue was present (ex- pressed by life-threating ionic imbalances).16 In this context, we recently used a mathemati- cal model aiming to assess the electrical conduc- tivity of the liver after introducing hypertonic so- lution (20% NaCl) through the portal vasculature (Figure 1). This model was further validated with an in vivo pilot safety study on pigs, which demon- strated that this infusion could increase the mean basal conductivity by up to 4 times.17 Interestingly, the tumour nodules lack of sinusoids means they are only supplied with blood from the hepatic ar- tery.18-20 In fact, this characteristic is currently used for identifying tumours by injecting contrast agents with CT or MRI. Hypersaline infusion through the portal vein would therefore raise the electrical conductivity of the healthy hepatic tissue, so that, when a potential difference is applied between the opposite sides of the liver, the electrical field magnitude in the tumours would be significantly larger than in the rest of the healthy tissue. Thus it would make possible to produce electroporation in tumour cells and without affecting healthy hepatic tissue (Figure 1).18 The aim of this study was to evaluate feasibil- ity and safety of selectively increasing of healthy hepatic conductivity by HI through the portal vein as a means to increase the electric field in scattered tumoural nodules with transhepatic IRE. Materials and methods All aspects of this study had the approval of the Ethics Committee on Animal Research of the Government of Catalonia (FBP-13-1474P2 proce- dure, DAAM: 7016). This animal experimental study was conducted following directives 2010/63/ EU of the European Parliament and Council of 22 September 2010, for the protection of animals for experimental and scientific purposes. Animal model Two animal models were considered in the study: Group A (n = 10) Sprague Dawley rats (Charles River Laboratories, Kingston, NY, USA) and Group B D FIGURE 1. IRE application model with plate electrodes, in scattered tumoural liver without (A) or with (B) hypersaline infusion through the portal vein and its corresponding numerical simulation (C and D) obtained from the received electric field in healthy and tumoural tissue – own results obtained from our previous research (17, 18). (C) Without hypersaline infusion through the portal vein, there is not selective effect on scattered tumoural nodules. Nevertheless, in (D) with hypersaline infusion through the portal vein, there is an increase on the electric field on scattered tumours (meaning a preferential ablative IRE effect on these nodules) because of the increase in healthy tissue conductivity (σ). Radiol Oncol 2017; 51(4): 415-421. Pañella C et al. / Increasing hepatic conductivity by hypersaline through the portal vein 417 B (n = 8) Athymic nude rats Hsd:RH-Foxn1mu (Harlan laboratories, Indianapolis, IN, USA). Eighteen six-week-old males were used and all of them were maintained under standard conditions with a laboratory animals diet and water ad libitum. We supervised all the animals following Morton and Griffiths’ guidelines on the recognition of pain, distress and discomfort in experimental animals.21 Group A was used to develop the HI protocol and show that it had no influence on survival rate; and group B was conceived for tumour implanta- tion and conductivity test on changes between he- patic and tumour tissue. Tumour implantation technique Primary colorectal metastases were obtained from Bellvitge Hospital (HUB) and the Catalan Institute of Oncology (ICO) with approval by the Ethical Committee, and ethical and legal protection guidelines of human subjects, including informed consent, were followed. Primary metastasis was implanted orthotopically in the liver of Crl:NU- FOXn1 nu mice, and when the orthoxenograft or patient derived orthotopic xenograft (PDOX) growth, the mice were sacrificed. Then, the tumour was minced and re-implanted in the left hepatic lobe of group B, after a quarantine period at the age of six weeks, by midline laparotomy under general anaesthesia (mixture of isofluorane and inhaled oxygen). Tumour pieces of measuring 5-8 mm2 were sutured with non-reabsorbable prolene 6/0 on the surface of the left hepatic lobe. The mid- line laparotomy closure was made up of a running suture within the muscle layer with reabsorbable monocryl 5/0 and skin with simple silk 5/0 stitches. The tumour grew for a mean of 20 days to allow adequate hepatic implantation. All the animals developed a tumour on the left hepatic lobe, ap- propriate for conductivity measurement after HI through the portal vein. Hypersaline infusion protocol HI was applied by means of a hypersaline infusion at 20% (NaCl), which is an aqueous solution with high conductivity (204 S/m).22,23 Once anaesthetised by inhalation anaesthesia (isoflurane 4% for induction and 2.5% for mainte- nance with O2 0.8 L/minute), subcutaneous anal- gesia was administered (buprenorphine 0.05 mg/ Kg). The abdomen and anterior cervical area were shaved and prepared aseptically with iodine. The liver was exposed by iterative abdominal incision and dressing pad underneath, after 7 days after the tumour implantation in group A and after mean of A B FIGURE 2. General setting of the hypersaline infusion protocol through the portal vein in Sprague Dawley rats (A) and in Athimic rats with implanted tumour (B). After exposing spleen at abdominal midline, hypersaline infusion was performed by trans-splenic puncture with a pump. Hyposaline infusion through the jugular vein was performed to compensate the systemic hypernatremia. Two tetrapolar setup electrodes measured tumour and healthy hepatic conductivity and data were collected with a multi- frecuency device and gathered in a computer. Radiol Oncol 2017; 51(4): 415-421. Pañella C et al. / Increasing hepatic conductivity by hypersaline through the portal vein418 0.1 0.2 0.3 0.4 TW 4 5 6 7 8 9 10 11 12 13 0 0.1 0.2 0.3 0.4 Group A Group B Liver conductivity (S/m) Tumour conductivity (S/m) Time (min)Time (min) C on d uc tiv ity (S /m ) C on d uc tiv ity (S /m ) 0 1 2 3 0 1 2 3 4 5 6 7 8 9 20 days of tumour growth in group B. The anterior vertical cervicotomy approach allowed anterior jugular vein dissection and catheterization with a flexible plastic tube (Tygon® ND 100-80 tubing, USP corporation, USA) for deionised serum infu- sion (DiH2O). The infusion of hyposaline serum, DiH2O, (40 mL/Kg)18, through the anterior jugular vein, avoids the HI side effects, such as systemic hypernatremia. Prior to the HI protocol, the ani- mals were pre-hydrated with a quarter of the total hyposaline serum (DiH2O), calculated by weight, at the same infusion rate as the HI. NaCl 20% (3.8 mL/Kg) was then injected into the portal vein by trans-splenic puncture (27G needle)18, during hy- posaline infusion. The HI rate was set up to admin- ister the dose in 1 minute. Trans-splenic puncture was used for the HI protocol, since splenic vein is a part of the portomesenteric system and infusion is more feasible than dissection of the portal vein (Figure 2). Intravenous infusion of furosemide (2 mL/Kg) was administered before and after HI protocol in order to compensate the fluid overload after intra- venous administration of hyposaline serum (via jugular) and hypersaline serum (via trans-splenic).18 Electrical conductivity measurement Hepatic and tumour conductivity were measured using the tetrapolar method following the setup employed in24. This consists of four stainless steel needles (0.25 mm of diameter and 3.5 mm of expo- sure) placed in a straight line with 0.5 mm between the centres of the needles. Tumour electrode was inserted at the centre of the tumour, with four- needle shape, in a way that it favoured its stability. Measurements were performed at ten-excitation frequencies from 5 kHz to 1 MHz at a rate of one sweep per second. Signal generation and the acqui- sition of both current and voltage across the sam- ple were performed by a Red Pitaya board and a front-end.25 Post-surgical management During the surgical procedure and during the immediate postoperative period, a heat lamp or heat pad controlled the risk of hypothermia. Administered analgesia was buprenorphine 0.05 mg/Kg s.c. and meloxicam 1 mg/Kg s.c., both every 24 hours for 3 days. An oral prophylactic antibiotic was also supplied: enrofloxacin in drinking water, 500 mg/Kg, for the first 3 days. Weight was moni- tored periodically as an objective sign of positive outcome after catheterization and the HI protocol. Histopathological samples Animals were sacrificed according to modified Morton and Griffiths criteria21 if signs of suffer- ing or distress were observed. Animals of group A were sacrificed 1 month after the procedure while animals of group B were sacrificed in case of clini- cal evidence of tumour dissemination (for instance, subcutaneous tumour implants). At necropsy, liver and spleen were collected from both groups. Tissue samples were fixed on formaldehyde and kept in paraffin blocks. Sections of 3 µm were prepared for routine haematoxylin-eosin staining (HEOS) slides. Statistical analyses All the statistics were processed by the SPSS sta- tistical software package (SPSS, version 21, IBM, Armonk, NY, USA) and expressed as mean ± stand- ard deviation. Since the sample was small, the Wilcoxon test was used for non-parametric data to make pairwise comparisons of weight and conduc- tivity of healthy tissue before and after HI, in both groups. A P-value of < 0.05 was considered statisti- cally significant. Results Hypersaline infusion through the portal vein is safe when compensated The HI protocol was reproduced in 94.4% (17/18) of cases in group A and B. One animal died from an- aesthetic problems before the HI protocol. Overall A B FIGURE 3. Conductivity changes after hypersaline infusion protocol in a representative case of group A (Sprague Dawley) and B (Athimic rats with implanted tumour). In both groups, a sharp increase and a posterior slowing decrease of conductivity in healthy hepatic tissue was observed. On the contrary, conductivity of the tumoural tissue (Group B) was stable. Thus, it created a therapeutic window (TW), the safety period where IRE it would be applied. Radiol Oncol 2017; 51(4): 415-421. Pañella C et al. / Increasing hepatic conductivity by hypersaline through the portal vein 419 technical-related survival rate was 82.4% (14/17) af- ter the HI protocol for both groups and there were 3 technical-related deaths (one animal in group A and 2 in group B). In all of these technical-related deaths, extra HI was administered after observ- ing recurrent reflux of the HI through the trans- splenic puncture and no correct compensation of the HI overload was possible in these cases. Two died shortly after the HI protocol and the other was sacrificed two days after when it was seen to be le- thargic (see comments on this animal in histology section). During the postoperative period of the an- imals with HI protocol, a mean increase of weight of 100 grams was observed in the other animals in group A (from 366.5±51.6 grams to 466.5±71.9 grams at the necropsy date). Implantation and tumour development The polyclonal human colon adenocarcinoma tu- mour model was well implanted with a correct growth after 60-90 days and with no evidence of peritoneal carcinomatosis at necropsy in all cas- es. Mean tumour volume, at implantation, was 122.5±84.3 mm3 and it remained stable at the time of the HI protocol, being 121±65.11 mm3. Mean tumour volume at the necropsy was 692.8±347.3 mm3. As expected in this tumoural model, the mean weight of the animals in group B initially increased after tumour implantation (from 158±7.33 grams to a mean maximum weight of 217.34±9.19 grams), but then decreased (156.06±39.68 grams, at nec- ropsy) (Table 1). Hypersaline infusion increases conductivity in healthy hepatic parenchyma In all animals increased hepatic tissue conductiv- ity was observed after the HI protocol. In group A, the mean baseline conductivity before administer- ing HI was 0.10±0.02 S/m and the mean maximum conductivity peaked at 0.27±0.75 S/m. Likewise, in group B, the mean baseline hepatic conductivity was 0.11±0.02 S/m and the mean maximum hepat- ic conductivity after HI was 0.38±0.16 S/m (range 0.19-0.66 S/m), which was around 3.5 times higher than the baseline value (Figure 3). Hypersaline infusion through the portal vein does not change tumoural conductivity As expected, tumoural conductivity in group B was not influenced by HI (mean value: 0.24±0.03 S/m). The higher conductivity in healthy hepatic tissue created a mean TW of 175±115.23 seconds (Figure 3B), where the mean maximum hepatic TABLE 1. Comparison between two groups A (Sprague Dawley) and B (Athimic). *PO: Postoperative GROUP A p-value GROUP B p-value Administration of HI protocol n= 9 n=8 Weight increase during PO* (grams) 100±30.57 0.012 -1.94±37.81 0.386 Baseline Conductivity in healthy tissue (S/m) 0.10±0.02 0.008 0.13±0.02 0.008 Conductivity after HI in healthy tissue (S/m) 0.27±0.75 0.49±0.17 Baseline Conductivity in tumour tissue (S/m) NA 0.24±0.03 Therapeutic window (sec) NA 175±115 A B FIGURE 4. These were representative livers from group A (Sprague Dawley) and group B (Athimic rats with implanted tumour) after HI protocol at the 4th week of postoperative. Histological analysis revealed the indemnity of the architecture and morphology of healthy hepatocytes (H) of both groups A and B. In group B, histological samples showed that HI protocol did not interfere in tumour (T) evolution. Radiol Oncol 2017; 51(4): 415-421. Pañella C et al. / Increasing hepatic conductivity by hypersaline through the portal vein420 conductivity after HI was 1.4 times higher (range 1.04-1.76) than tumour tissue. In this TW, elec- troporation could therefore be advantageously ap- plied. Histopathological evaluation In group A, hepatic parenchyma exhibited nor- mal architecture, with polygonal hepatocytes and preserved porta trials (Figure 4A). Congestion in vessels was seldom scattered throughout healthy hepatic tissue. No changes were present in cellular component or architecture after HI. Similar results to group A were found in group B, with no architectural or morphological damage after the HI protocol. However, the animal that was sacrificed two days after two extra doses of HI showed patchy areas of coagulation necrosis in the liver especially around the central veins of acini. No damage in neoplastic tissue was observed (Figure 4B). Discussion The preliminary results show that the HI employed in this study selectively increases the conductivity of healthy tissue, so that when the electric pulses are applied to a whole hepatic section, the magni- tude of the electric field in the tumours would be significantly higher than in the rest of the healthy tissue. The results also show that this infusion could be safe when it is correctly compensated as we previously demonstrated in the pilot study on pigs.17 Concerning the safety of the protocol, it should be remembered that hypersaline solutions has been extensively used to resuscitate patients with traumatic brain injury.26 It is currently assumed that the maximum dosage at 20% is 1.8 ml/Kg. Twice this dose was used in the present study (3.8 ml/Kg) combined with measures to compensate the corresponding hypernatremia (doses of hy- posaline serum through the anterior jugular vein and furosemide). With this protocol all the animals survived the HI protocol, with the appropriate increase in their body weight and no histological damage being found at necropsy. The three animal deaths were related to extra HI doses (adminis- tered after observing recurrent reflux of the infu- sion through the trans-splenic puncture) without correct compensation for the overload. As regards the potential translation of these conductivity results, the basal hepatic and tu- moural conductivity observed are similar to those described in the literature for humans and in the tumour itself can vary up to 20%.27 For example, Laufer et al. found 0.12 S/m and 0.27 S/m for normal and tumoural liver tissue, respectively.24 Although little data is available in the literature on increas- ing conductivity by administering HI through the portal vein, in our previous study on pigs, with the same HI protocol, similar peak conductivity results were obtained (range 0.21-0.37 S/m)17, which fur- ther supports the consistency of the present data despite the small sample size. Finally, our actual TW (175±115.2 sec) appears to be enough to imple- ment a conventional IRE protocol. Although the application protocols are varied, most studies use between 70 and 90 pulses for duration of 70-100 µs.28-30 For instance, our previous IRE protocol with plate electrodes lasted for 100 seconds (90-100 puls- es at a frequency of 1 Hz).16,31 Our protocol IRE last- ed 100 seconds, without pauses. However, pauses can be reduced in case the TW values obtained in this preliminary study are set in this range of time, in future essays with bigger sample size. In other words, the IRE protocol can be adjusted according to the average TW obtained. In conclusion, the presented preliminary results provide evidence in favour of safety of administer- ing HI through the portal vein when compensated and show that it may provide a therapeutic win- dow for focused IRE treatment on tumour nodules. Further studies should be carried out to confirm these results, giving that this study is a preliminary research. 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IEEE Trans Biomed Eng 2012; 59: 604-7. doi: 10.1109/ TBME.2011.2180722 Radiol Oncol 2017; 51(4): 422-430. doi: 10.1515/raon-2017-0043 422 research article Minimally invasive electrochemotherapy procedure for treating nasal duct tumors in dogs using a single needle electrode Felipe Maglietti1,2,4, Matías Tellado3, Nahuel Olaiz1,2,4, Sebastian Michinski1,2,4, Guillermo Marshall*1,2,4 1 Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Física, Buenos Aires, Argentina 2 CONICET-Universidad de Buenos Aires, Instituto de Física del Plasma (INFIP), Buenos Aires, Argentina 3 Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Buenos Aires, Argentina 4 CONICET-Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Computación, Laboratorio de Sistemas Complejos, Buenos Aires, Argentina Radiol Oncol 2017; 51(4): 422-430. Received 1 August 2017 Accepted 2 October 2017 Correspondence to: Prof. Guillermo Marshall, Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Física, Buenos Aires, Argentina. E-mail: marshall.guillermo@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Nasal cavity tumors are usually diagnosed late, when they already have infiltrated adjacent tissues thus requiring very aggressive treatments with serious side effects. Here we use electrochemotherapy (ECT), a well demonstrated treatment modality for superficial tumors. Materials and methods. In the case of deep-seated tumors, the main limitation of ECT is reaching the tumor with an appropriate electric field. To overcome this limitation we introduce the single needle electrode (SiNE), a minimally invasive device that can deliver an appropriate electric field with a simple procedure. Twenty-one canine patients with spontaneous tumors were selected, eleven were treated using the SiNE with ECT, and ten with surgery plus adju- vant chemotherapy as a control group. Results. In the SiNE group, 27% achieved a complete response, 64% had a partial response, and 9% had a stable disease. This means that 91% of objective responses were obtained. The mean overall survival was 16.86 months (4–32 months, median 16.5 months), with a survival rate significantly higher (p = 0.0008) when compared with control group. The only side effect observed was the inflammation of the treated nasal passage, which was controlled with corticos- teroid therapy for one week. One year after the treatment, 60% of the canine of the SiNE group vs. 10% of the control group remained alive, and after the 32 months follow-up, the survival rate were 30% and 0%, respectively. Conclusions. ECT with the SiNE can be safely used in canine to treat nasal tumors with encouraging results. Key words: electrochemotherapy; nasal cavity; canine; cancer; electrochemotherapy Introduction Spontaneous tumors of the nasal cavity and para- nasal sinuses in canine patients account for 1% of all neoplasms. Their etiology is unknown, but ani- mals living in urban areas are at an increased risk, due to the nasal filtering of pollutants. Also, expo- sure to tobacco smoke and fossil fuel combustion products increase the risk of this type of cancer. The average age of dogs with nasal cancer is around 10 years, predominantly in medium and large breeds. Common clinical signs include epistaxis, mucopu- rulent nasal discharge, facial deformity, sneezing, dyspnea, local pain, stertorous breathing, exoph- thalmos, and ocular discharge.1 The diagnostic procedures are the same as in hu- man patients and involve high-resolution imaging procedures such as Computed Tomography (CT) scans or Magnetic Resonance Imaging (MRI) to de- termine the extension of the tumor and evaluating Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors 423 the involvement of neighboring structures such as the palate, the retro-orbital area, the sinuses, and the cranial vault.1 The most common histologies are adenocarcinoma, squamous cell carcinoma and sarcomas.1,2 Metastases are rare at the time of pres- entation, yet they are present at the time of death in around 40–50% of the cases. Lungs and lymph nodes are common metastatic sites.1 Electrochemotherapy (ECT) is a new treatment modality which has been gaining territory in on- cology since the Standard Operating Procedures were published in 2006.3,4,5 ECT consists of the ap- plication of an electric field to a tumor in order to increase the uptake of bleomycin that was previ- ously administered (locally or intravenously) at a very low dose6; alternatively, cisplatin can be used locally with equally good results.7 ECT is mainly in- dicated for cutaneous and subcutaneous tumors of any histology. As cell membrane permeabilization is produced by a physical phenomenon known as electroporation, it affects all cells, regardless of the histology of the tumor. Recently, a meta-analysis of ECT clinical studies in human oncology showed that the overall objective response (OR) rate vary from 62.6% to 82.2% depending also on the route of administration of the drug, being either intra- venous or intratumoural.8 Great efforts are being made to extend ECT to non-cutaneous locations such as the liver9, the brain10 and the bones.11 Moreover, an endoscopic electrode was developed to treat the colon.12 For deep-seated tumors, ECT is performed in an intraoperative manner, thus re- quiring the patient to be exposed to the risks and complications of an open surgery. Here we explore ECT for treating nasal cavity tumors by means of the Single Needle Electrode (SiNE). The aim of this work is to perform a validation study of the treatment of nasal cavity tumors using the SiNE with ECT in dogs and compare the over- all survival with the standard treatment available in our setting, i.e.: surgery plus adjuvant chemo- therapy. Materials and methods Consent was obtained from the dog’s owner to per- form the treatment and for using the dog’s image in this scientific work. In all cases, all recommen- dations from the Consejo Profesional de Médicos Veterinarios de Buenos Aires (Buenos Aires Veterinary Council) were observed, as well as the relevant local legislation in Argentina, Act No. 14072 which governs Veterinary Medicine prac- tice. Recommendations from Campana et al.13 for reporting clinical studies on ECT were followed. The SiNE pictured in Figure 1 is an electrode specifically designed in our lab for deep-seated tu- mor treatment with ECT. It consists of an insulated needle with an anode in one side and a cathode in the other. This design provides an electric field of cylindrical shape around it, strong enough to pro- duce the electroporation of the cells. Twenty-one patients were selected, eleven for the SiNE group and ten for the control group. The patients for the SiNE group were selected accord- ing to the following inclusion criteria: • Canine patient with confirmed diagnosis of na- sal duct tumor. • Life expectancy of more than 1 month. • Surgery refusal by the owner. The control group was formed with ten patients whose owners accepted surgery plus adjuvant chemotherapy. In the SiNE group, rhinoscopy was performed using a flexible or a rigid endoscope of variable di- ameters according to patient size; nasal neoplasia was diagnosed on the basis of histological find- ings. A complete blood count, serum biochemical profile, coagulation profile, abdominal ultrasound, and 3-view thoracic radiographs were performed. A CT/MRI was performed to determine the size and extension of the tumor. The patients were staged using Adam’s staging system (Table 1). FIGURE 1. The SiNE. In (A) the prototype, in (B) the scheme of the tip, zoomed from the squared area in A. The electrode consists of a needle isolated surrounded by two parallel plaques at each side. A B TABLE 1. Adam’s modified staging system Stage Tumor characteristics T1 Confined to one nasal passage, paranasal sinus, or frontal sinus with no bony involvement. T2 Any bony involvement, but no evidence of orbital, subcutaneous, or submucosal mass. T3 Involvement of orbit or subcutaneous or submucosal mass. T4 Tumor extension into nasopharynx or cribriform plate. Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors424 In this set of patients (Table 2), 7 were males and 4 females. Four of the males were neutered, and 3 of the females were spayed. The median age of the dogs starting ECT treatment was 9 years (range of 6–13 years). The mean weight was 26 kg (range of 3.5–48 kg). Labrador Retriever (n = 3) and Golden Retriever (n = 3) were the most common breeds in this study. The other breeds were Collie, Old English Mastiff, Miniature Pincher, Spanish Breton, and a crossbreed dog. The clinical findings re- vealed sneezing in 11 dogs (100%), nasal discharge in 9 dogs (81%), epistaxis in 6 dogs (55%), exoph- thalmos in 2 dogs (18%), neurological symptoms in 2 dogs (18%) and facial deformity in 1 dog (9%). Regarding clinical Adam´s staging, there was 1 dog with T1 disease (9%), 6 dogs with T2 disease (55%), 2 dogs with T3 disease (18%) and 2 dogs with T4 disease (18%). No dogs had regional lymph nodes or distant metastasis at initial presentation. None of the patients had previous treatments. The treatment was performed under gen- eral anesthesia with premedication with intra- muscular administration of xylazine (Xylazine 100®, Richmond, Buenos Aires, Argentina) 0.5 mg/kg and tramadol (Tramadol®, John Martin, Buenos Aires, Argentina) 2 mg/kg. The induc- tion was made with intravenous administration of propofol (Propofol Gemepe®, Gemepe, Buenos Aires, Argentina) 3 mg/kg. The maintenance was performed with inhaled isofluorane (Zuflax®, Richmond, Buenos Aires, Argentina) 2–3% and in- travenous fentanyl (Fentanyl Gemepe®, Gemepe, Buenos Aires, Argentina) 2 mcg/kg. This anesthe- sia scheme provided adequate comfort during the treatment. Prophylactic antibiotics and meloxicam (Meloxivet®, John Martin, Buenos Aires, Argentina) 0.2 mg/kg were administered orally for analgesia after the treatment according to the needs of each patient. The ECT procedure consisted of an intravenous bolus of bleomycin (Blocamicina®, Gador, Buenos Aires, Argentina) at a dose of 15 U/m2 of body surface area in 30–45 seconds, followed by the de- livery of the electric pulses eight minutes later to allow drug distribution according to the Standard Operating Procedures published by Mir et al. [14]. The SiNE was inserted deep into the nasal fossa and a train of pulses was delivered, see Figure 2. For that purpose, a BTX ECM 830 Harvard Apparatus (Holliston, MA, USA) was used. The electric pulses consisted of thirty-two square pulses of 300V, 100 µs long at 1 Hz. The electrode was rotated 90 de- grees clockwise and the pulses were repeated. After the rotation, the electrode was removed 2 cm and the whole procedure was repeated until com- plete coverage of the nasal duct. It was not possible TABLE 2. SiNE group patient details and treatment outcome Patient Breed Histology Adam’s stage Response Clinical Evaluation 1 Golden Retriever Adenocarcinoma grade 3 T2 PR Partial reduction of clinical signs, with a persistent minimum nasal discharge. 2 Miniature Pinscher Adenocarcinoma grade 2 T2 CR Complete resolution of clinical signs. 3 Labrador Retriever Solid carcinoma grade 3 T3 PR Partial reduction of clinical signs, with persistence of ocular discharge. Death by euthanasia due to distal radius metastasis. 4 Old English Mastiff Adenocarcinoma grade 2 T4 PR Complete Resolution of clinical signs. 5 Crossbreed Squamous cell carcinoma T2 PR Partial reduction of clinical signs with persistence of minimum nasal discharge. 6 Labrador Retriever Round cell sarcoma T3 PR Complete resolution of clinical signs. 7 Collie Sticker sarcoma T1 CR Complete resolution of clinical signs. 8 Golden Retriever Squamous cell carcinoma T2 PR Partial reduction of clinical signs, sneezing remains every day with occasional nasal discharge. Death by euthanasia based on the owner’s decision. 9 Spanish Breton Adenocarcinoma grade 2 T4 SD No reduction of clinical signs was observed, sneezing remained every day with continuous nasal discharge and persistent inflammation of the subcutaneous tissue and paranasal sinuses. Death by euthanasia based on poor quality of life. 10 Labrador Retriever Squamous cell carcinoma T2 PR Partial reduction of clinical symptoms with occasional sneezing. 11 Golden Retriever Fibrosarcoma T2 CR Complete resolution of clinical signs. Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors 425 to treat safety margins due to the location of the tumors. In order to prevent the SiNE from inadvertently passing through the cribriform plate during the insertion, it was marked to show the maximum insertion depth (following CT/MRI measurements performed before the treatment). A single ECT treatment session was performed, and no other oncological treatment was indicated previously, concomitantly or afterward. For the assessment of treatment results, follow- up was performed within 7, 15 and 30 days; dur- ing each visit, a complete clinical examination was performed. A second CT/MRI was obtained 60 days after the treatment to determine results. The patients were followed up for 32 months and re- sponses reported according to WHO’s Criteria.15 The control group consisted of 10 cases, 5 males, and 5 females, whose owners accepted surgery as treatment (Table 3). One of the males and three of the females were neutered. The median age of the dogs was 10 years (range of 6–14 years). The mean weight was 22.7 kg (range of 5.6–34 kg). Crossbreed (n = 4) was the most common breed, other breeds were Puddle, Akita Inu, Boxer, Labrador Retriever, Fox Terrier and Cocker Spaniel. The clinical find- ings revealed sneezing in 10 dogs (100%), weight loss in 7 dogs (70%), epistaxis in 6 dogs (60%), hy- porexia in 6 dogs (60%), facial deformity in 4 dogs (40%) and neurological symptoms in 1 dog (10%). Regarding Adam’s staging, there was 1 dog with T1 disease (10%), 5 dogs with T2 disease (50%), 3 dogs with T3 disease (30%) and 1 dog with T4 dis- ease (10%). No dog had regional lymph nodes or distant metastases. Surgery plus adjuvant chemotherapy was per- formed since it is the standard treatment available in our setting. The surgical procedure consisted of a dorsal/ventral uni/bilateral rhinotomy depending on the surgeon’s decision, and according to tumor localization and extension. The anesthesia proce- dure was identical to the one mentioned above. The patients remained in observation for 24 hours with intravenous butorphanol (Torbutrol Plus®, Alpha Farma, Buenos Aires, Argentina) 0.1 mg/kg every 4 hours for one day and oral meloxicam (Meloxivet®, John Martin, Buenos Aires, Argentina) 0.2 mg/kg every 24 hours for 10 days for analgesia. The adju- vant chemotherapy consisted of intravenous carbo- FIGURE 2. Picture of patient 9 during the treatment. The SiNE is inserted in the nasal fossa. TABLE 3. Control group patient details Patient Breed Histology Adam’s stage Cause of death 1 Puddle Tubulopapillary carcinoma T1 Euthanasia due to local progression. 2 Crossbreed Carcinoma T2 Euthanasia due to local progression. 3 Akita Inu Chondrosarcoma T2 Euthanasia due to local progression to central nervous system 4 Crossbreed Adenocarcinoma T3 Death due to local progression. 5 Boxer Carcinoma T4 Death due to lung metastasis. 6 Crossbreed Squamous cell carcinoma T2 Euthanasia due to breathing difficulty. 7 Crossbreed Carcinoma T2 Euthanasia. 8 Labrador Retriever Carcinoma T3 Euthanasia due to uncontrollable pain. 9 Fox Terrier Solid carcinoma T2 Euthanasia. 10 Cocker Spaniel Adenocarcinoma T3 Euthanasia due to local progression and pain. Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors426 platin (Carboplatino Delta Farma®, Sidus, Buenos Aires, Argentina) 300 mg/m2 of body surface area every 21 days. Regarding the statistical analysis, Kaplan–Meier curves for survival were compared by the log-rank test with an alpha of 0.05. Age and body weight were compared between groups with the Mann- Whitney U test, with an alpha of 0.05. Results From the 21 patients selected, 11 were treated us- ing the SiNE with ECT (SiNE group), and 10 were treated with surgery plus adjuvant chemotherapy (control group). The SiNE and control groups did not show significant differences in age or body- weight (p = 0.46 and p = 0.13, respectively). In the SiNE group, out of the 11 patients: 27% (3/11) achieved a complete response (CR), 64% (7/11) had a partial response (PR), and 9% (1/11) had a stable disease (SD). In the nasal duct treated, tumor reduction remained stable and the lesions did not grow again during the follow-up period. In the first two patients only one nasal duct was treated, in the non-treated duct, the tumor contin- ued growing according to its natural history. For this reason and because of the highly satisfactory results obtained in the treated duct, both nasal pas- sages were treated from patient number 3 onwards. The only side effect observed was the inflamma- tion of the treated nasal passage, which was very well controlled with corticosteroid therapy for one week. The bleeding through the nasal fossa ceased immediately after the procedure. The patients remained in follow-up for 32 months, with no relapses in the treated area. This means that 91% objective responses (OR) were ob- tained. Figure 3, pictures the CT scan of patient 11 showing a complete response obtained with the treatment. The Kaplan-Meier graph for overall survival in both groups can be seen in Figure 4. The overall survival of the SiNE group was significantly higher (p = 0.0008) than in the control group, with a mean survival time of 16.86 months (4–32 months, me- dian 16.5 months). In the control group, a mean survival time of 5.3 months (3–10 months, median 5.5 months) was obtained. One year after the treat- ment, 60% of the patients of the SiNE group vs. 10% of the control group remained alive, and after the 32 months follow-up, the survival rate for the SiNE group is 30% vs. 0% for the control group. Discussion In an effort to provide a satisfactory treatment mo- dality to canine patients with nasal tumors, and ad- vance in the area of deep-seated tumor treatment using ECT, we developed the SiNE and a protocol for its implementation. We compared the results of this new treatment modality with the standard treatment modality in our setting: surgery plus chemotherapy. When treating deep-seated tumors, the standard ECT procedure consists of inserting multiple nee- dles to cover the whole tumor with an appropri- ate electric field. This procedure is very complex and prone to fail because the misplacement of the A B FIGURE 3. CT image of patient 11 before and after the treatment. (A) The tumor can be seen occupying the lateral inferior part of the left nasal passage (white arrow). (B) Two months after a single treatment a CR was obtained; as readily observed, the tumor tissue is absent leaving a defect in the nasal wall (white arrow). FIGURE 4. Kaplan-Meier graph of the outcome of dogs with nasal tumors treated using the SiNE with ECT procedure (SiNE group) versus the dogs treated with surgery plus adjuvant chemotherapy (control group). Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors 427 needles leaves areas of the tumor untreated. In some cases, open surgery is needed for placing the needles correctly16, such as in the case reported by Suzuki et al.17, where the results were very good though the procedure required open surgery and the filling of the nasal cavity with gel to improve conductivity. These was not needed in our case since the electrode is inserted through the nasal or- ifice and, secretions and blood present in the nasal duct replaced conductive gel. The most important advantage of the SiNE against standard electrodes is that only a single needle is inserted, thus reduc- ing tissue intrusion and simplifying treatment planning and its guidance by imaging procedures. The disadvantage is that it is restricted to small tu- mors when compared with multiple needles. A high percentage of objective responses was ob- tained. Most of them were partial responses (64%), mainly because these tumors are highly infiltra- tive, making them very difficult to treat. In these cases, tumor tissue was still seen in CT/MRI scans. This could be due to the fact that the electric field was not enough to cover all the tumor, leaving it untreated, or more likely because those tumor cells are quiescent. In the latter case, bleomycin already acted on those cells by cleaving the DNA strands, and they will die when cell division is attempted. If the electric field would not have been strong enough, we would have seen a rapid growth of the residual tumoral tissue, at least, comparable to the non-treated passage, which did not hap- pen. Although a fraction of the tumor remained, a considerable improvement in the quality of life was achieved due to the reduction in the clinical symptoms. The patients who achieved a complete response (27%), had a complete palliation of the symptoms as was expected. Regarding short-term side effects, the inflamma- tion and pain of the treated nasal passage proved to be non-problematic and easily manageable with medication. Regarding the bleeding during the procedure it ceased immediately after the treat- ment was completed, probably because of the “vascular lock” phenomenon, which consists of the sudden stop of blood flow in the area exposed to the electric field used in ECT procedures.3 We ob- served no long-term side effects. The gold standard treatment is surgery plus ad- juvant radiotherapy.18 Although surgery is usually rejected by the owner due to mutilating results, fre- quently it is impossible to perform, and even pro- cedures such as rhinotomy, can result in acute and chronic morbidity. Hypofractionated or orthovolt- age radiation therapy have many side effects such as oral mucositis, rhinitis, moist desquamation, keratoconjunctivitis, and blepharitis. These symp- toms can last for up to 8 weeks after therapy, even with medication, but if they persist, esophagos- tomy or gastrostomy tubes may be needed in the short term for an adequate nourishment of the animal. Long-term side effects include cataracts, keratoconjunctivitis, anterior uvea hemorrhage, brain necrosis, seizures, optic nerve degeneration, osteonecrosis, and fibrosis. Some of these compli- cations are generally non-treatable.19,20 Our results show that mean survival with SiNE group was 16.86 months (4–32 months) with 60% of survival at 1 year and a 30% at 2 years. This is very encouraging since it is better when compared with the standard treatment option in our setting (surgery plus chemotherapy). Also, it is similar to the best treatments described in the literature. As mentioned before, the treatment of choice for nasal tumors is surgery plus radiotherapy. Unfortunately, due to the facilities and resources required, this option is not locally available for vet- erinary patients. In fact, this situation is replicated in the vast majority of centers around the world. In relation to the data available for this treatment in the literature we found that for radiotherapy alone in its different modalities, the median survival re- ported is around 8 months.21,22 The combination of radiation therapy followed by surgery when it is possible seems to have better results in the overall median survival (47.7 months).23 The new modali- ties like intensity-modulated radiation therapy (IMRT) report a median survival of 446 d (14.9 months) with 50% of survival at 1 year and a 25% at 2 years.24 The prolonged survival in our study may be par- tially due to the lack of need for euthanasia as the general condition of the patients greatly improved. Further research is needed to confirm this hypoth- esis. It remains to be seen whether the combination of treatments can improve outcomes. The combina- tion of ECT using the SiNE with radiotherapy or systemic chemotherapy may enable the reduction of the dose in later treatments, reducing their side effects.25,26 Canine patients with spontaneous tumors are excellent models for preclinical validation. In par- ticular, nasal tumors in dogs behave in a similar way to nasal tumors in humans.27 Dogs are ex- posed to similar environmental carcinogens. They develop tumors with the same histologies having the same interactions between tumor and stroma to those found in human patients; thus, the tumor grows in the context of an intact immune system Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors428 that has been developed during its lifetime and exposed to different antigens. They show sponta- neous metastasis and resistance to different forms of therapy. They have similar druggable targets, angiogenic pathways, and mechanisms of apop- tosis. This enables a more accurate assessment of therapeutic approaches compared to rodent mod- els. Other advantages of dogs, particularly for the purpose of this work, is that their size allows the use of the typical imaging procedures performed in humans; there is no need for miniaturization of the electrodes, and it is possible to repeat tissue and fluid sampling over time with more tissue for analysis than that available in rodent models. Also, typical forms of treatment used in humans can be performed in dogs to compare the results of the treatment.27,28 In human patients the situation is similar to canine patients, nasal tumors are a complex pa- thology in which current treatments do not often provide satisfactory results. The variety of tumor types, its proximity to critical anatomical struc- tures and the late development of symptoms make malignant tumors of the paranasal sinuses and nasal cavity a complex problem. Diagnostic proce- dures and treatment modalities are also very simi- lar to canine patients.29 Therefore, new therapeutic approaches that improve outcomes and minimize adverse effects are required. These tumors are ra- re and patients are often asymptomatic until late in the course of the disease. In the United States, nasal tumors account for only 0.2–0.8% of all can- cers diagnosed annually. The incidence reported is 0.3–1 per 100,000 populations. They occur most commonly in the fifth decade of life, although they can occur at any age.29 These tumors are usually diagnosed when airway obstruction symptoms are present and the disease is at an advanced stage. Early and late symptoms are similar to those in canine patients.30 These regions are affected by a wide variety of tumor types31, with very similar histologies.32,33,34 In a study conducted by Dulguerov et al. the 5-year survival rate was 40%, and the local control rate was 59%. The prognosis depends on tumor histology, localization, tumor stage, and treatment modality.35 There is a consensus that complete sur- gical resection should be performed if possible. Surgery and postoperative radiation therapy may result in improved local control, absolute survival and complications when compared with radiation therapy alone.36 Chen et al. have not found im- provements in disease control or overall survival for patients treated with radiotherapy in the last five decades.37 Nevertheless, new forms of ra- diotherapy reduce complications38, and intensity- modulated radiation therapy particularly promises better outcomes when compared to conventional radiotherapy.39 The role of chemotherapy in the neoadjuvant setting and/or postoperatively with irradiation is not clearly defined, but it is generally used for the most advanced cases and requires fur- ther investigation.34,40 In such a sensitive region, the proximity of criti- cal normal anatomical structures restricts in many cases the possibility to adequately perform surgery and apply effective radiotherapy.38 Sometimes a tumor is resecable, but the patient’s clinical status may preclude surgery or, very often, patients reject it.41 For these reasons, also in human patients, new approaches for treating nasal tumors are needed. Besides obvious differences between canine and human nasal tumors, this work shows that nasal tumors can be effectively treated with ECT and fur- ther studies should be conducted to validate this treatment in human patients. Treating nasal tumors in human or canine pa- tients using the ECT with the SiNE could be use- ful in selected cases where first-line treatments are not possible. It can be especially used to palliate symptoms in patients with short life expectancy, or a poor clinical condition. In any case, it can be performed as a cytoreductive treatment to sim- plify surgery and does not preclude other treat- ments.42,43,44 Therefore, it remains to be seen wheth- er the use of the SiNE followed by surgery will improve survival, as happens with radiotherapy. In conclusion, deep nasal tumors in canine pa- tients can be successfully treated by ECT, and it can be safely and effectively performed using the SiNE, especially in tumors that are difficult or impossible to reach with conventional electrodes. Acknowledgements F Maglietti holds a scholarship from CONICET, N Olaiz and G Marshall are researchers from CONICET, S Michinski is a CPA-CONICET. This work is supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas, www.conicet.gov.ar, (PIP 379/2012/2016 and STAN 599/12) and Universidad de Buenos Aires, www. uba.ar, (UBACyT 2014/2017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu- script. This was proofread by YasminTranslations. com. Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors 429 Author contributions FM developed the electrode, worked on the pro- duction of the prototype, treated the patients and worked in the preparation of this manuscript. MT selected and treated the patients and collaborated on the preparation of the manuscript. NO was in charge of provision of study material and equip- ment setup. SM collaborated on the treatment of the patients and the preparation of the manuscript. GM coordinated the group and collaborated on the preparation of the manuscript. References 1. Withrow SJ, Vail DM, Page R. Withrow and MacEwen’s small animal clinical oncology. Elsevier Health Sciences; 2013. doi: 10.1111/avj.12086 2. Madewell B, Priester W, Gillette E, Snyder S. Neoplasms of the nasal pas- sages and paranasal sinuses in domesticated animals as reported by 13 veterinary colleges. Am J Vet Res 1976; 37: 851-6. 3. Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. 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Abstract SY28-01: Spontaneous cancer in dogs: opportunities for preclinical evaluation of novel therapies. Cancer Res 2011; 71 (8 Suppl): SY28–01. doi: 10.1158/1538-7445.AM2011-SY28-01 29. Holland JF, Hong WK. Holland-Frei cancer medicine 8. Hong WK, Bast R, Hait WN, Kufe DW, Pollock RE, Weichselbaum RR, et al, editors. Shelton: People’s Medical Publishing House; 2010. 30. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, et al. AJCC cancer staging manual. Vol. 649. New York: Springer; 2010. doi: 10.1001/ jama.2010.1525 31. Iezzoni JC, Mills SE. “Undifferentiated” small round cell tumors of the sinon- asal tract: differential diagnosis update. Am J Clin Pathol 2005; 124(Suppl 1): S110-21. doi: 10.1309/59RBT2RK6LQE4YHB 32. Marcus DM, Marcus RP, Prabhu RS, Owonikoko TK, Lawson DH, Switchenko J, et al. Rising incidence of mucosal melanoma of the head and neck in the United States. J Skin Cancer 2012; 2012: 231693. doi: 10.1155/2012/231693.S 33. Smith SR, Som P, Fahmy A, Lawson W, Sacks S, Brandwein M. A clinico- pathological study of sinonasal neuroendocrine carcinoma and sinona- sal undifferentiated carcinoma. Laryngoscope 2000; 110: 1617-22. doi: 10.1097/00005537-200010000-00007 Radiol Oncol 2017; 51(4): 422-430. Maglietti F et al. / Electrochemotherapy of nasal duct tumors430 34. Mourad WF, Hauerstock D, Shourbaji RA, Hu KS, Culliney B, Li Z, et al. Trimodality management of sinonasal undifferentiated carcinoma and review of the literature. Am J Clin Oncol 2013; 36: 584-8. doi: 10.1097/ COC.0b013e31825eb3a5 35. Dulguerov P, Jacobsen MS, Allal AS, Lehmann W, Calcaterra T. Nasal and paranasal sinus carcinoma: are we making progress? Cancer 2001; 92: 3012- 29. doi: 10.1002/1097-0142(20011215)92:12 36. Katz TS, Mendenhall WM, Morris CG, Amdur RJ, Hinerman RW, Villaret DB. Malignant tumors of the nasal cavity and paranasal sinuses. Head & Neck 2002; 24: 821-9. doi: 10.1002/hed.10143 37. Chen AM, Daly ME, Bucci MK, Xia P, Akazawa C, Quivey JM, et al. Carcinomas of the paranasal sinuses and nasal cavity treated with radiotherapy at a sin- gle institution over five decades: are we making improvement? Int J Rad Oncol Biol Phys 2007; 9: 141-7. doi: 10.1016/j.ijrobp.2007.02.031 38. Porceddu S, Martin J, Shanker G, Weih L, Russell C, Rischin D, et al. Paranasal sinus tumors: Peter MacCallum Cancer Institute experience. Head Neck 2004; 26: 322-30. doi: 10.1002/hed.10388 39. Dirix P, Vanstraelen B, Jorissen M, Vander Poorten V, Nuyts S. Intensity- modulated radiotherapy for sinonasal cancer: improved outcome com- pared to conventional radiotherapy. Int J Radiol Oncol Biol Phys 2010; 78: 998-1004. doi: 10.1016/j.ijrobp.2009.09.067 40. Sohrabi S, Drabick JJ, Crist H, Goldenberg D, Sheehan JM, Mackley HB. Neoadjuvant concurrent chemoradiation for advanced esthesioneuro- blastoma: a case series and review of the literature. J Clin Oncol 2011; 29: e358–61. doi: 10.1200/JCO.2010.30.9278 41. Pfister DG, Ang KK, Brizel DM, Burtness BA, Busse PM, Caudell JJ, et al. Head and neck cancers, version 2. 2013. J Natl Compr Canc Netw 2013; 11: 917- 23. doi: 10.6004/jnccn.2013.0113 42. Spugnini EP, Citro G, Baldi A. Adjuvant electrochemotherapy in veterinary patients: a model for the planning of future therapies in humans. J Exp Clin Cancer Res 2009; 28: 1. doi: 10.1186/1756-9966-28-114 43. Lowe R, Gavazza A, Impellizeri J, Soden D, Lubas G. The treatment of canine mast cell tumours with electrochemotherapy with or without surgical exci- sion. Vet Comp Oncol 2017; 15: 775-84. doi: 10.1111/vco.12217 44. Spugnini E, Vincenzi B, Citro G, Dotsinsky I, Mudrov T, Baldi A. Evaluation of Cisplatin as an electrochemotherapy agent for the treatment of incom- pletely excised mast cell tumors in dogs. J Vet Int Med 2011; 25: 407–411. doi: 10.1111/j.1939-1676.2011.0678.x Radiol Oncol 2017; 51(4): 431-437. doi: 10.1515/raon-2017-0042 431 research article Metformin enhanced in vitro radiosensitivity associates with G2/M cell cycle arrest and elevated adenosine-5′-monophosphate- activated protein kinase levels in glioblastoma Sebastian Adeberg1-4, Denise Bernhardt1,3,4, Semi B. Harrabi1,3,4, Nils H. Nicolay1-4, Juliane Hörner-Rieber1,3,4, Laila König1,3,4, Michael Repka5, Angela Mohr1,3,4, Amir Abdollahi1,3,4,6, Klaus-Josef Weber1,4, Juergen Debus1-4, Stefan Rieken1,3,4 1 University Hospital of Heidelberg, Department of Radiation Oncology, Heidelberg, Germany 2 Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Heidelberg Ion-Beam Therapy Center (HIT), Heidelberg, Germany 4 Heidelberg Institute for Radiation Oncology, University Hospital of Heidelberg, Heidelberg, Germany 5 Department of Radiation Medicine, Georgetown University Hospital, Washington DC, USA 6 Tanslational Radiation Oncology, German Cancer Consortium (DKTK), National Center for Tumor Diseases German Cancer Research Center (DKFZ), Heidelberg, Germany Radiol Oncol 2017; 51(4): 431-437. Received 12 February 2017 Accepted 30 September 2017 Presented in part at the Annual Meeting of the German Society of Radiation Oncology (DEGRO), 15th - 18th June 2017, Berlin, Germany Correspondence to: Sebastian Adeberg, M.D., University Hospital of Heidelberg, Department of Radiation Oncology, Im Neuenheimer Feld 400, 69120 Heidelberg,Germany. Phone: +49 6221 5635654; Fax: +49 6221 56565353; Email: sebastian.adeberg@med.uni-heidelberg.de Disclosure: No potential conflicts of interest were disclosed. Background. It is hypothesized that metabolism plays a strong role in cancer cell regulation. We have recently dem- onstrated improved progression-free survival in patients with glioblastoma who received metformin as an antidiabetic substance during chemoradiation. Although metformin is well-established in clinical use the influence of metformin in glioblastoma is far from being understood especially in combination with other treatment modalities such as radiation and temozolomide. Materials and methods. In this study, we examined the influence of metformin in combinations with radiation and temozolomide on cell survival (clonogenic survival), cell cycle (routine flow cytometric analysis, FACScan), and phosphorylated Adenosine-5′-monophosphate-activated protein kinase (AMPK) (Phopho-AMPKalpha1 - ELISA) levels in glioblastoma cell lines LN18 and LN229. Results. Metformin and temozolomide enhanced the effectiveness of photon irradiation in glioblastoma cells. Cell toxicity was more pronounced in O6-methylguanine DNA methyltransferase (MGMT) promoter non-methylated LN18 cells. Induction of a G2/M phase cell cycle block through metformin and combined treatments was observed up to 72 h. These findings were associated with elevated levels of activated AMPK levels in LN229 cells but not in LN18 cells after irradiation, metformin, and temozolomide treatment. Conclusions. Radiosensitizing effects of metformin on glioblastoma cells treated with irradiation and temozolomide in vitro coincided with G2/M arrest and changes in pAMPK levels. Key words: metformin; glycolysis; metabolism; gliomas; proliferation; cell cycle Introduction The relationship of tumorigenesis, and tumor cell metabolism by an increased glycolysis was first described in the early 20th century by Otto Warburg.1,2 Multiple modifications in cancer cell metabolism have subsequently been detected, but the influence on alterations in signaling path- Radiol Oncol 2017; 51(4): 431-437. Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells432 ways, cell growth, and therapy response is not yet understood. Nonetheless, tumor cell metabolism represents an intriguing potential target in the multidisciplinary treatment of cancer. The addi- tion of metabolically active substances, particular- ly those with limited toxicity, with chemotherapy and radiation is an area of active research. In this context, the antidiabetic medication metformin is of particular interest as it demonstrated prolonged progression-free survival in a retrospective study in diabetic patients with glioblastoma (GBM).3 The primary glucoregulatory effects of metformin are predominantly explained through reduced hepatic glucose production and increased glucose uptake in the periphery.4 These effects lead to decreased mitochondrial-dependent ATP production and cell proliferation, increased glycolytic ATP production, induction of cell cycle arrest, autophagy, and apop- totic processes through activation of adenosine-5′- monophosphate-activated protein kinase (AMPK)5 and inhibition of the mTOR (mammalian target of rapamycin) pathway in glioblastoma cells.6,7 AMPK is a serine/threonine kinase that func- tions as a cellular energy sensor. AMPK is an ob- ligate heterotrimer, consisting of one catalytic subunit (α) and two regulatory subunits (β and γ).8 Under cellular stress conditions, AMPK is acti- vated by increased AMP-to-ATP ratios to promote catabolism and inhibit anabolism.8 High cellular activated AMPK levels, particularly by phospho- rylation (pAMPK), seems to be associated with tu- mor cell growth and cell survival.9,10 Furthermore, increased AMPK phosphorylation has been ob- served in cells following radiation-induced DNA damage in several studies.7,11,12 The activation of AMPK is hypothesized to regulate irradiation- induced metabolism changes and might be a key determinant of cell survival after exposure to ion- izing radiation.7 The AMPK pathway is therefore a potential objective for targeted therapies. Although the concomitant application of metformin with te- mozolomide (TMZ) and this effect is not well un- derstood. In this study, we investigated the effects of metformin effect in combination with current standard of care therapy in glioblastoma cell lines. Materials and methods Cell lines and culture conditions Two representative human GBM cell lines (ATCC; Manassas, VA, USA) were utilized in this study. LN18 is a GBM cell line with a mutant tumor sup- pressor protein 53 (p53mut) and an un-methyl- ated O6-methylguanine DNA methyltransferase (MGMT) promoter. LN229 is a GBM cell line with both mutant and wild type p53 (p53 mut/WT) and a methylated MGMT promoter. Both cell lines were cultured in Dulbeccos’s modified Eagle medium (Biochrom, Berlin, Germany) supplemented with 10% Fetal Calf Serum (FCS) superior (Biochrom AG) and 1% penicillin/streptomycin (Gibco, Darmstadt, Germany). Cultures were maintained in exponential monolayer at incubator conditions with 37°C, 5% CO2, and 95% humidity. Drug treatment and irradiation We performed clonogenic assays to evaluate treat- ment effects on cell survival. Low passage cells were plated in T25 flasks (Becton, Dickinson, Heidelberg, Germany) with 5 ml medium as described previously.13 TMZ was obtained by Schering-Plough (Kenilworth, NJ, USA) and dis- solved in dimethyl sulfoxide (DMSO) (Sigma- Aldrich, Deisenhofen, Germany) at concentrations < 0.5%. Cell samples were incubated with 10 μM or 50 μM TMZ for 4 hours prior to irradiation. 1,1-Dimethylbiguanide hydrochloride (metform- in; 97%) was provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and applied in con- centrations of 1 mM or 20 mM for 24 hours prior to irradiation. Immediately prior to irradiation, all samples were rinsed twice with phosphate buffered saline and fresh medium was added. Adherent cells were irradiated using a 6MV pho- ton linear accelerator (XRAD 320 Precision X-ray Inc., N.Bradford, USA) with single photon doses up to 6 Gy. Cells were fixed with 70% ethanol and stained with methylene blue. Colonies of > 50 were counted. Experiments were repeated in triplicate at least three times. Cell cycle analyses To evaluate cell cycle distributions, cells were har- vested, washed and fixed in ice-cold 70% ethanol after 24, 48 or 72 hours and stained with propidium iodide (Sigma-Aldrich). 10.000 events were count- ed for each experimental setup with routine flow cytometric analysis (FACScan, Becton-Dickinson, Heidelberg, Germany). Histograms were cre- ated and analyzed using ModFit software (Verity Software House, Topsham, ME, USA). Each experi- ment was repeated at least three times on different days for validation. Radiol Oncol 2017; 51(4): 431-437. Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells 433 Phopho-AMPKα1 – ELISA ELISA for pAMPK levels in LN229 and LN18 cell lysates was performed by commercially available DuoSet ELISA development kits (R&D Systems, Minneapolis, USA) according to the manufacturer’s instruction using goat anti-human AMPKα1 (T183) as primary antibody and biotinylated rabbit anti- human phospho-AMPKα1 (T183) as secondary antibody. Recombinant human phospo-AMPKα1 (T183) was utilized as standard. The results were presented as relative values to the control value, which was set as 1. Data analysis was carried out using the Infinite® F50/Robotic ELISA plate read- er (absorbance at 450 nm, correction wavelength at 570 nm) and Magellan for F50 software (Tecan Group Ltd, Crailsheim, Germany). Measurements were repeated at least three times on three differ- ent days. Statistical analysis Clonogenic survival was calculated from the measured plating efficiencies. With the data of the combined treatment approaches survival curves were generated with the linear quadratic (LQ) - model as described earlier.14 Sigma Plot’s (Systat Software GmbH, Erkrath, Germany) non-linear least-squares regression option was used to fit the calculated survival curves. Clonogenic survival was calculated using the sensitizer enhancement ratio (SER), comparing the radiation dose at 20% cell survival, due to the high efficiency of TMZ and metformin on cellular killing. The Student’s two-sided t-test was used for comparison of cell survival curves, differences in cell cycle analysis distribution and pAMPK levels. Data are shown as mean values ± standard devia- tion. Statistical significance was set at p < 0.05. Results Metformin enhances the effectiveness of irradiation in glioblastoma cells Metformin sensitivity of glioblastoma cell lines LN18 and LN229 was investigated by clonogenic survival assays. Increasing concentrations of met- formin (1 mM and 20 mM) and TMZ (10 μM and 50 μM) were chosen for all baseline experiments. Representative concentrations (20 mM metform- in and 50 μM TMZ) were chosen for subsequent combined experiments. Clonogenic survival of the MGMT promoter methylated LN229 cell line was significantly reduced after treatment with TMZ compared to the untreated control and LN18 cell lines. Combined treatment approaches with irra- A B C D FIGURE 1. (A,B) Clonogenic survival assays of LN229 and LN18 glioblastoma cell lines after treatment with photon irradiation. (C,D) Clonogenic survival assays after comparing LN229 and LN18 cell lines after combined treatment with TMZ (C) or metformin (D). Error bars represent standard deviations. * shows a statistical significance (P <0.05). In LN229 enhanced cell kill was observed by TMZ and in LN18 cells lines metformin treatment resulted in increased cell toxicity compared to the control, as well as the combination of both agents itself. Combined bimodal and trimodal treatment results in superior cell toxicity. TABLE 1. Sensitizer enhancement ratio (SER 20%) for LN229 and LN18 cells after treatment with metformin, temozolomide (TMZ) and combined treatment with 2Gy irradiation SER (20% survival) / 2 Gy Cell line LN18 LN229 50 μM TMZ 1.11 2.15 1mM Metformin 0.67 0.74 20 mM Metformin 3.13 0.61 50 μM TMZ + 20mM Metformin 2.57 2.69 SER values (ranging from 0.67–3.13) depending on cell line, chemotherapeutic agent and dose Radiol Oncol 2017; 51(4): 431-437. Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells434 diation, TMZ and metformin showed additive cell toxicity compared to the control (P < 0.05, Student’s t-test) (Figure 1A,B and Table 1). Furthermore, clonogenic survival after metformin exposure was reduced in LN18 cells when compared to the untreated control and to LN229 cells (P < 0.05) (Figure 1C,D and Table 1). Additionally, additive cell toxicity could be reached in LN229 cells add- ing ionizing irradiation (2 Gy and 6 Gy) to TMZ and Metformin, whereas additional effects with ionizing irradiation could only be reached for met- formin in LN18 cells. Metformin induces a G2/M phase block in combination with irradiation Cell cycle assessment of glioblastoma cell lines was carried out using FACS analyses. Exposure to met- formin (20 mM) resulted in accumulation at G2/M phases after 72 hours to a higher degree in LN18 cells (G2 phase cells: ctrl vs 20mM metformin: 19.9% vs 43.8%). However, the results did not reach statistical significance. Combined treatment ap- proaches with irradiation and metformin resulted in a more pronounced G2/M block after 72 hours (P < 0.05, Student’s t-test) (Table 2). Accumulations in G2/M phases after 72 hours were even more marked when using higher radiation doses (6 Gy) (Table 2) and trimodal approaches with irra- diation, 50 μM TMZ, and 20 mM metformin (P < 0.05, Student’s t-test) (Table 2). Analysis of sub-G1 populations indicating apoptosis did not show any measurable results. Irradiation, TMZ and metformin enhance activated AMPK levels in glioblastoma cells Phosyphorylated serine/threonine kinase AMPK levels induced by irradiation, TMZ, and metformin exposure were investigated as a potential mecha- nism for the described metformin sensitivity of glioblastoma cell lines. Increased pAMPK levels were demonstrated in LN229 cells after treatment with the following regimens: 2 Gy + 50 μM TMZ, 2 Gy + 20 mM metformin, 2 Gy + 50 μM TMZ + 20 mM metformin, 6 Gy, 6 Gy + 50 μM TMZ, 6 Gy + 20 mM metformin, and 6 Gy + 50 μM TMZ + 20 mM metformin (P<0.05, Student’s t-test) (Figure 2A). All other treatment approaches showed a trend to- wards higher pAMPK levels in LN229 cells (P<0.1, Student’s t-test). Higher radiation doses were as- sociated with increased pAMPK levels (P<0.05, Student’s t-test) (Figure 2A). Interestingly, LN229 TABLE 2. Cell cycle distribution into G1, S, and G2/M phase of LN18 and LN229 cells after various treatments. Measurements were performed after 72h. * shows a statistical significance (P <0.05) of the treatment compared to the control Ln18 24h 72h G1 (% ) ± std.dev. S (%) ± std.dev. G2/M (%) ± std.dev. Ctrl 74.5 ± 8 5.6 ± 0 19.9 ± 1 50μM TMZ 66.0 ± 8 9.5 ± 1 24.6 ± 3 1mM Metformin 71.0 ± 5 6.3 ± 1 22.7 ± 0 20mM Metformin 46.9 ± 3 9.3 ± 2 43.8 ± 2* 50μM TMZ + 1mM Metformin 64.5 ± 0 8.6 ± 4 26.9 ± 4 50μM TMZ + 20mM Metformin 41.2 ± 0 19.4 ± 7 39.4 ± 2 2Gy 71.7 ± 1 7.5 ± 1 20.8 ± 4 6Gy 56.2 ± 5 8.0 ± 1 35.7 ± 1 2Gy + 50μM TMZ 62.0 ± 5 6.6 ± 5 31.4 ± 4 2Gy + 1mM Metformin 68.2 ± 7 10.3 ± 1 21.5 ± 2 2 Gy + 20mM Metformin 41.3 ± 0 16.1 ± 1 42.6 ± 4* 2Gy + 50μM TMZ + 1mM Metformin 60.1 ± 7 8.6 ± 1 31.3 ± 0 2Gy + 50μM TMZ + 20mM Metformin 33.5 ± 2 20.0 ± 3 46.5 ± 4* 6Gy + 50μM TMZ 47.1 ± 7 11.6 ± 3 41.3 ± 3* 6Gy + 1mM Metformin 51.3 ± 2 10.4 ± 0 38.2 ± 3 6Gy + 20mM Metformin 36.1 ± 3 14.2 ± 0 49.7 ± 3* 6Gy + 50μM TMZ + 1mM Metformin 46.3 ± 4 10.7 ± 1 42.9 ± 2* 6Gy + 50μM TMZ + 20mM Metformin 27.9 ± 1 14.1 ± 3 58.0 ± 2* Ln229 24h 72h G1 (%) S (%) G2/M (%) Ctrl 86.0 ± 2 3.6 ± 2 10.4 ± 2 50μM TMZ 37.4 ± 3 8.1 ± 2 54.6 ± 5* 1mM Metformin 87.4 ± 3 3.3 ± 1 9.3 ± 0 20mM Metformin 63.9 ± 10 14.9 ± 1 21.2 ± 9 50μM TMZ + 1mM Metformin 38.9 ± 0 6.1 ± 2 55.0 ± 4* 50μM TMZ + 20mM Metformin 25.2 ± 1 25.4 ± 2 49.4 ± 2* 2Gy 83.5 ± 5 3.3 ± 2 13.2 ± 5 6Gy 67.1 ± 2 6.2 ± 1 26.7 ± 1 2Gy + 50μM TMZ 44.4 ± 1 6.9 ± 1 48.7 ± 4* 2Gy + 1mM Metformin 82.3 ± 1 3.5 ± 1 14.2 ± 2 2 Gy + 20mM Metformin 55.6 ± 2 11.8 ± 2 32.6 ± 1* 2Gy + 50μM TMZ + 1mM Metformin 46.0 ± 1 7.9 ± 1 46.1 ± 0* 2Gy + 50μM TMZ + 20mM Metformin 32.3 ± 3 28.9 ± 7 38.8 ± 5* 6Gy + 50μM TMZ 41.6 ± 2 8.9 ± 3 49.5 ± 3 6Gy + 1mM Metformin 55.5 ± 11 8.8 ± 4 35.7 ± 23 6Gy + 20mM Metformin 56.5 ± 18 7.8 ± 6 35.7 ± 16 6Gy + 50μM TMZ + 1mM Metformin 41.9 ± 2 5.9 ± 2 52.2 ± 2* 6Gy + 50μM TMZ + 20mM Metformin 18.4 ± 1 16.2 ± 3 65.4 ± 3* Radiol Oncol 2017; 51(4): 431-437. Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells 435 demonstrated a twofold higher level of pAMPK af- ter treatment with 6 Gy compared to the untreated control (P<0.05, Student’s t-test). pAMPK measure- ments obtained from LN18 cell lines did not show a significant increase after each treatment combi- nation (Figure 2B). Discussion Standard of care multidisciplinary management of GBM entails surgical resection followed by ra- diotherapy with concomitant and adjuvant TMZ, resulting in overall survival rates of approximately one year. Given this dismal prognosis, the need to improve the efficacy of chemoradiation for these common primary brain tumors is urgent. Recently, we demonstrated improved progression- free survival rates in diabetic patients receiving metformin3, a biguanide that is commonly used and well tolerated in patients with type II diabe- tes. Accordingly, combined approaches target- ing cell metabolism became attractive. Metformin is known to exhibit anticancer effects via LKB1/ AMPK/mTOR/S6K1 pathway blockade15,16, inhi- bition of tumor growth17,18 and induction of au- tophagy and apoptosis19,20 in various cancer cell lines. Accordingly, integration of approaches tar- geting cell metabolism into standard therapy is an attractive area of investigation. In the present study, we examined the interaction of metformin in combination with photon irradiation and the alkylating agent TMZ. Furthermore, we demon- strated that metformin has antitumoral effects and increases sensitivity to ionizing radiation, which was particularly pronounced in a non-MGMT methylated cell line (LN18). Of note, both the LN18 and LN229 cell lines express wildtype for PTEN, which is associated with increased sensitivity to metformin.6 This sus- ceptibility may be explained by opposing PI3K signaling, thus leading to a down-regulated AKT survival pathway and decreased glucose consump- tion.6 Therefore, the higher metformin sensitivity for LN18 cannot solely be explained by the more effective deactivation of AKT in these cells. The au- thors believe the MGMT promoter methylation in metformin sensitive LN18 cells is rather a coinci- dent than the cause of this finding. In fact, glioma cells are known to have a high intrinsic radiation sensitivity caused by several intrinsic factors such as high efficient radiation damage repair, a high ra- tio of hypoxic cell fraction and rapid repopulation following irradiation.21 The intrinsic sensitivity of GBM cells is probably independent of the MGMT promotor methylation status. The mechanistic ef- fects of these findings are beyond the scope of this manuscript and will be evaluated in future ex- periments. An inverse effect was shown for TMZ, where the MGMT promoter-methylated LN229 cells were more sensitive to TMZ compared to MGMT promoter non-methylated LN18 cells. This finding can be explained by the lack of the MGMT DNA-repair protein in LN229 cells which normally removes O6-MG-DNA and counteracts the anti- neoplastic effect of TMZ.22 We performed cell cycle analyses in order to fur- ther investigate the antiproliferative effects of met- formin on glioblastoma cell lines. In PTEN wild- type cells, cytotoxic effects have been described starting at 48 hours.6 However, in LN18 and LN229 cell lines, significant increase of G2/M block rates started delayed, chiefly 72 hours after irradiation. This effect was pronounced in combined treatment approaches with higher radiation doses and TMZ administration. These results indicate that the anti- proliferative effects of metformin on glioblastoma cell lines might be mediated trough cell cycle ar- rest starting at 72 hours post-exposure. These re- sults are in line with results from in vitro data of Yu et al. who observed G2/M cell cycle arrests af- ter TMZ and metformin. A combined treatment showed synergistic effects.23 In a former study G1 arrests were described after metformin exposure, A B FIGURE 2. (A) In LN229 pAMPK measurements were significantly increased after treatment with Metformin, 2 Gy and temozolomide and 6 Gy combined with Metformin compared to the control if not otherwise specified (* p<0.05). (B) No significant increase of pAMPK levels were observed in cell line LN18. Error bars represent standard deviation. Radiol Oncol 2017; 51(4): 431-437. Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells436 whereas mainly G2/M phase arrests were observed in the current study. This effect might be due to the two-fold metformin dose (10 nM vs 20 nM) with varying impact. Furthermore metformin effects were mainly observed after 72 hours but the obser- vation period of Sesen et al. ended after 48 hours.6 Nevertheless, molecular mechanisms of metformin are far from understood and further research to examine its role in tumor cell metabolism is neces- sary. Previous studies have demonstrated that activa- tion of AMPK leads to an inhibition of mTOR6,15,24,25 and is essential for glioma proliferation by pro- moting cell cycle progression in vitro and in vivo.26 These findings are supported by reports indicat- ing a major AMPK phosphorylation and activa- tion through the tumor suppressor LKB1.27-29 Furthermore, AMPK has been associated with p53- dependent apoptosis through p53 phosphoryla- tion30, underlining the potential function of AMPK activation as an “energy checkpoint”.31 This pro- posed mechanism permits proliferation and cell growth in cells with intact AMPK signaling only in favorable metabolic cell conditions. Conversely, cancer cells with deficient AMPK signaling might be capable of receiving a metabolism-independ- ent growth stimulus. In these cases, induction of AMPK activation could present a valuable thera- peutic approach.26 Although the role of AMPK as a metabolic sensor in homeostasis is well described, its function in cancer remains opaque. In vitro stud- ies have shown highly efficient inhibition of tumor cell growth across multiple glioblastoma cell lines with several AMPK agonists, including 5-amino- imidazole-4-carboxamide ribonucleotide (AICAR) and activated AMPK adenovirus.32-35 In this study, we demonstrated a dose-depend- ent increase of AMPK in LN229 glioblastoma cells following radiation in combination with metformin and TMZ. Although AMPK level changes did not show statistically significant changes in the LN18 cell line, the radiosensitizing effect of metformin was more pronounced in these cells. Another rea- son for the absent of significant findings could be attributed to a high standard deviation masking subtle changes (Figure 4B). The authors carefully performed pAMPK level measurements with at least n=3. Even though, future in-depth analyses may help to unmask subtle changes. Interestingly, the failure of LN18 to phosphorylate AMPK com- pared to LN 229 cells could be a reason for the en- hanced radiosensitivity in the latter, since AMPK activation might be a keyregulator for glioma cell proliferation.26 On the other hand, it is likely that metformin exhibits both AMPK dependent and AMPK-independent effects which are contingent on molecular tumor characteristics.6,15 Taken together, the present finding that acti- vated AMPK levels are elevated after treatment with radiation, TMZ, and metformin contributes to the understanding of GBM metabolism follow- ing therapeutic intervention. However, more de- tailed knowledge of the antitumoral effects of met- formin, the role of AMPK, and tumor cell biology is necessary to establish a novel multidisciplinary approach to glioblastoma therapy. We planned to perform mechanistic in vitro metformin experi- ments in the future based on the current baseline results. Additional challenges, including the ability of AMPK activating agents such as AICAR to cross the blood brain barrier more effective, are ongoing. Nonetheless, our results suggest that the develop- ment of an AMPK activating agent with high cen- tral nervous system bioavailabity may be a promis- ing new therapeutic avenue in the treatment of this aggressive malignancy. Conclusions Together with our previously published clinical findings3 and the well-established use of metform- in in clinical practice, these data show that radio- sensitizing effects of metformin on glioblastoma cells treated with irradiation and temozolomide in vitro coincided with G2/M arrest and changes in pAMPK levels. Acknowledgement The authors thank Mrs Lena Orschiedt and Mrs Sigrid Daffinger for excellent technical assistance. References 1. Warburg O, Wind F, Negelein E. The metabolism of tumors in the Body. J Gen Physiol 1927; 8: 519-30. 2. Warburg O. On the origin of cancer cells. Science 1956; 123: 309-14. 3. Adeberg S, Bernhardt D, Harrabi SB, Bostel T, Mohr A, Koelsche C, et al. Metformin influences progression in diabetic glioblastoma patients. Strahlentherapie Onkol 2015; 191: 928-35. doi: 10.1007/s00066-015-0884- 5 4. Goodarzi MO, Bryer-Ash M. Metformin revisited: re-evaluation of its proper- ties and role in the pharmacopoeia of modern antidiabetic agents. Diabetes Obes Metab 2005; 7: 654-65. doi: 10.1111/j.1463-1326.2004.00448 5. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP- activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108: 1167-74. doi: 10.1172/JCI13505 Radiol Oncol 2017; 51(4): 431-437. Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells 437 6. Sesen J, Dahan P, Scotland SJ, Saland E, Dang VT, Lemarie A, et al. Metformin inhibits growth of human glioblastoma cells and enhances therapeutic re- sponse. PLoS One 2015; 10: e0123721. doi: 10.1371/journal.pone.0123721 7. Zannella VE, Cojocari D, Hilgendorf S, Vellanki RN, Chung S, Wouters BG, et al. AMPK regulates metabolism and survival in response to ionizing radia- tion. Radiother Oncol 2011; 99: 293-9. doi: 10.1016/j.radonc.2011.05.049 8. Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Review 2009; 89: 1025-78. doi: 10.1152/physrev.00011.2008 9. Jang T, Calaoagan JM, Kwon E, Samuelsson S, Recht L, Laderoute KR. 5’-AMP-activated protein kinase activity is elevated early during primary brain tumor development in the rat. Int J Cancer 2011; 128: 2230-9. doi: 10.1002/ijc.25558 10. Park HU, Suy S, Danner M, Dailey V, Zhang Y, Li H, et al. AMP-activated pro- tein kinase promotes human prostate cancer cell growth and survival. Mol Cancer Ther 2009; 8: 733-41. doi: 10.1158/1535-7163 11. Sanli T, Rashid A, Liu C, Harding S, Bristow RG, Cutz JC, et al. Ionizing radia- tion activates AMP-activated kinase (AMPK): a target for radiosensitization of human cancer cells. Int J Radiat Oncol Bool Phys 2010; 78: 221-9. doi: 10.1016/j.ijrobp 12. Zhang WB, Wang Z, Shu F, Jin YH, Liu HY, Wang QJ, et al. Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition. J Biol Chem 2010; 285: 40461-71. doi: 10.1074/jbc.M110.164046 13. Bischof M, Abdollahi A, Gong P, Stoffregen C, Lipson KE, Debus JU, et al. Triple combination of irradiation, chemotherapy (pemetrexed), and VEGFR inhibition (SU5416) in human endothelial and tumor cells. Int J Radiat Oncol Biol Phys 2004; 60: 1220-32. doi: 10.1016/j.ijrobp 14. Combs SE, Bohl J, Elsasser T, Weber KJ, Schulz-Ertner D, Debus J, et al. Radiobiological evaluation and correlation with the local effect model (LEM) of carbon ion radiation therapy and temozolomide in glioblastoma cell lines. Int J Radiat Biol 2009; 85: 126-37. doi: 10.1080/09553000802641151 15. Liu X, Chhipa RR, Pooya S, Wortman M, Yachyshin S, Chow LM, et al. Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independ- ent of AMPK. Proc Natl Acad Sci U S A 2014; 111: E435-44. doi: 10.1073/ pnas.1311121111 16. Wurth R, Pattarozzi A, Gatti M, Bajetto A, Corsaro A, Parodi A, et al. Metformin selectively affects human glioblastoma tumor-initiating cell vi- ability: A role for metformin-induced inhibition of Akt. Cell Cycle 2013; 12: 145-56. doi: 10.4161/cc.23050 17. Janjetovic K, Harhaji-Trajkovic L, Misirkic-Marjanovic M, Vucicevic L, Stevanovic D, Zogovic N, et al. In vitro and in vivo anti-melanoma action of metformin. Eur J Pharmacol 2011; 668: 373-82. doi: 10.1016/j.ejphar 18. Scotland S, Saland E, Skuli N, de Toni F, Boutzen H, Micklow E, et al. Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells. Leukemia 2013; 27: 2129- 38. doi: 10.1038/leu 19. Feng Y, Ke C, Tang Q, Dong H, Zheng X, Lin W, et al. Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by down- regulating Stat3 signaling. Cell Death Dis 2014; 5: e1088. doi: 10.1038/cddis 20. Tomic T, Botton T, Cerezo M, Robert G, Luciano F, Puissant A, et al. Metformin inhibits melanoma development through autophagy and apop- tosis mechanisms. Cell Death Dis 2011; 2: e199. doi: 10.1038/cddis 21. Taghian A, Suit H, Pardo F, Gioioso D, Tomkinson K, DuBois W, et al. In vitro intrinsic radiation sensitivity of glioblastoma multiforme. Int J Radiat Oncol Biol Phys 1992; 23: 55-62. 22. Harrabi S, Combs SE, Brons S, Haberer T, Debus J, Weber KJ. Temozolomide in combination with carbon ion or photon irradiation in glioblastoma multi- forme cell lines - does scheduling matter? Int J Radiat Biol 2013; 89: 692-7. doi: 10.3109/09553002 23. Yu Z, Zhao G, Li P, Li Y, Zhou G, Chen Y, et al. Temozolomide in combination with metformin act synergistically to inhibit proliferation and expansion of glioma stem-like cells. Oncol Lett 2016; 11: 2792-800. doi: 10.3892/ ol.2016.4315 24. Shi WY, Xiao D, Wang L, Dong LH, Yan ZX, Shen ZX, et al. Therapeutic met- formin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy. Cell Death Dis 2012; 3: e275. doi: 10.1038/cddis 25. Zheng B, Jeong JH, Asara JM, Yuan YY, Granter SR, Chin L, et al. Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote mela- noma cell proliferation. Mol Cell 2009; 33: 237-47. doi: 10.1016/j.mol- cel.2008.12.026 26. Rios M, Foretz M, Viollet B, Prieto A, Fraga M, Costoya JA, et al. AMPK activation by oncogenesis is required to maintain cancer cell proliferation in astrocytic tumors. Cancer Res 2013; 73: 2628-38. doi: 10.1158/0008-5472 27. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, et al. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2003; 2: 28. doi: 10.1186/1475-4924-2-28 28. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 2004; 101: 3329-35. doi: 10.1073/pnas.0308061100 29. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, et al. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 2003; 13: 2004-8. 30. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMP-activated pro- tein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 2005; 18: 283-93. doi: 10.1016/j.molcel 31. Fogarty S, Hardie DG. Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer. Biochim Biophys Acta 2010; 1804: 581-91. doi: 10.1016/j.bbapap 32. Guo D, Cloughesy TF, Radu CG, Mischel PS. AMPK: A metabolic checkpoint that regulates the growth of EGFR activated glioblastomas. Cell Cycle 2010; 9: 211-2. doi: 10.4161/cc 33. Isakovic A, Harhaji L, Stevanovic D, Markovic Z, Sumarac-Dumanovic M, Starcevic V, et al. Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis. Cell Mol Life Sci 2007; 64: 1290-302. doi: 10.1007/s00018-007-7080-4 34. Kisfalvi K, Eibl G, Sinnett-Smith J, Rozengurt E. Metformin disrupts crosstalk between G protein-coupled receptor and insulin receptor signaling systems and inhibits pancreatic cancer growth. 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Stem Cell Trans Med 2012; 1: 811-24. doi: 10.5966/sctm.2012-0058 Radiol Oncol 2017; 51(4): 438-446. doi: 10.1515/raon-2017-0033 438 research article Evaluation of deformable image registration (DIR) methods for dose accumulation in nasopharyngeal cancer patients during radiotherapy Wannapha Nobnop1,2, Imjai Chitapanarux2, Hudsaleark Neamin1, Somsak Wanwilairat2, Vicharn Lorvidhaya2, Taweap Sanghangthum3 1 Department of Radiologic Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand 2 Division of Radiation Oncology, Department of Radiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 3 Division of Radiation Oncology, Department of Radiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Radiol Oncol 2017; 51(4): 438-446. Received 3 June 2017 Accepted 16 July 2017 Correspondence to: Imjai Chitapanarux, Division of Radiation Oncology, Department of Radiology, Faculty of Medicine, Chiang Mai University, 110 Intavaroros Rd., Sriphum 50200, Chiang Mai, Thailand. Phone: +66 539 354 56; +66 869 133 065; Fax: +66 539 354 91; E-mail: imjai@ hotmail.com Disclosure: No potential conflicts of interest were disclosed. Introduction. Deformable image registration (DIR) is used to modify structures according to anatomical changes for observing the dosimetric effect. In this study, megavoltage computed tomography (MVCT) images were used to generate cumulative doses for nasopharyngeal cancer (NPC) patients by various DIR methods. The performance of the multiple DIR methods was analysed, and the impact of dose accumulation was assessed. Patients and methods. The study consisted of five NPC patients treated with a helical tomotherapy unit. The weekly MVCT images at the 1st, 6th, 11th, 16th, 21st, 26th, and 31st fractions were used to assess the dose accumulation by the four DIR methods. The cumulative dose deviations from the initial treatment plan were analysed, and correlations of these variations with the anatomic changes and DIR methods were explored. Results. The target dose received a slightly different result from the initial plan at the end of the treatment. The organ dose differences increased as the treatment progressed to 6.8% (range: 2.2 to 10.9%), 15.2% (range: -1.7 to 36.3%), and 6.4% (range: -1.6 to 13.2%) for the right parotid, the left parotid, and the spinal cord, respectively. The mean un- certainty values to estimate the accumulated doses for all the DIR methods were 0.21 ± 0.11 Gy (target dose), 1.99 ± 0.76 Gy (right parotid), 1.19 ± 0.24 Gy (left parotid), and 0.41 ± 0.04 Gy (spinal cord). Conclusions. Accuracy of the DIR methods affects the estimation of dose accumulation on both the target dose and the organ dose. The DIR methods provide an adequate dose estimation technique for observation as a result of inter-fractional anatomic changes and are beneficial for adaptive treatment strategies. Key words: deformable image registration; dose accumulation; nasopharyngeal cancer; MVCT; helical tomotherapy Introduction Modern radiation therapy has the ability to utilize multimodality imaging technologies for disease definition, patient setup, and treatment assess- ment.1 Helical megavoltage CT (MVCT), which is a volumetric imaging modality, is adopted with the primary purpose of more accurate target localiza- tion.2 Moreover, with this, information about inter- fractional anatomical variations has become more accessible.3 Some head and neck cancer patients undergo significant anatomical changes, and these may result in unforeseen changes in the delivered dose.3 In ideal practice, when a patient’s anatomy Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation 439 changes, a new adaptive plan must be developed, in accordance with the concept of adaptive radio- therapy (ART).4 These procedures include modifi- cation of the initial plan according to the changes in the target volume or normal organs; manual con- touring can be used to modify deformation in order to evaluate the dosimetric effect.5-7 However, the process of manual contouring is time-consuming. Therefore, a deformable image registration (DIR) can be used to resolve these challenges. By regis- tering multiple daily CTs to the planned CT, the al- gorithm can automatically generate deformed con- tours on daily CTs while creating cumulative doses by tracking the dose to the tissue voxels through- out the course of the radiation therapy.2 As for the application of MVCT in deformable dose accumulation routinely, it would require ac- curate structure deformation even in low contrast regions8 because accuracy of DIR may have a sig- nificant dosimetric impact on radiation treatment planning.9 Nowadays, various deformable image registration algorithms have been developed3 and the accuracy of the deformable image registration naturally depends on the deformation model.10 Therefore, the choice of the deformation algo- rithms and the transformation in the MVCT image application are of great importance in the registra- tion process as it entails important compromise between computational efficiency and richness of description for more accurate results.10 Deformable image registration and adaptive radiotherapy (DIRART)11 is a software suite for DIR plus ART. DIRART is a large set of programs developed using MATLAB. Four DIR methods by two algorithms, Horn & Schunck optical flow and demon with the two transformation frameworks, asymmetric and symmetric transformation, have been considered. In this study, weekly MVCT im- ages from helical tomotherpy were used to gen- erate cumulative doses for nasopharyngeal can- cer (NPC) patients. Different DIR methods from DIRART were used. The weekly cumulative doses were analysed to assess the dosimetric impact of the DIR methods on dose accumulation. The dosi- metric variations from the initial plan were report- ed, and correlations of these variations with ana- tomic changes and DIR methods were explored. Patients and methods Patient characteristics The study population consisted of five NPC pa- tients treated using a helical tomotherapy unit (Tomo-Therapy, Inc., Madison, Wisconsin). All the patients underwent intensity modulated radiother- apy (IMRT) with a planned dose of 70 Gy delivered to the gross disease at 2.12 Gy/fraction for a total of 33 fractions with a simultaneous integrated boost technique (SIB) according to the RTOG 022512, keeping the mean parotid dose as low as could be possibly achieved and respecting the tissue toler- ance of other normal structures. Patient position- ing was ensured by appropriate headrest and a personalized HN and shoulder mask. This study received ethics approval, granted by the institu- tional research committee. Image acquisition The planned kVCT images were acquired on a com- puterized tomography unit (Somatom, SIEMENS, Germany) by using a matrix of 512 × 512 with voxel dimension of 0.976 × 0.976 × 3mm3 for the treat- ment planning process. The 1st day MVCT images were also acquired on the helical tomotherapy unit as source images for deformable investigation on the same day of acquisition of the planned kVCT images. When the radiotherapy treatment was started, the daily MVCT images were acquired on the heli- cal tomotherapy unit prior to each treatment frac- tion used for patient alignment by using a matrix of 512 x512 with voxel dimension of 0.763 × 0.763 × 4 mm3. Typically, the MVCT scan range covers the entirety of the gross tumour volume (GTV), the clinical target volume (CTV), and the parotid glands bilaterally. The weekly MVCT images on the 1st, 6th, 11th, 16th, 21st, 26th, and 31st fractions were used as the target images to assess the dose accu- mulation in this study. Target localization The regions of interest (ROIs) including the target and the organ at risks (OARs) were defined by the radiation oncologist on planned kVCT images for the treatment planning processes. The ROIs on the planned kVCT images were transferred to the first- day MVCT images as the source images for each image set. The same oncologist who localized the target and the OAR for the HT treatment planning process also contoured the GTV, CTV, the bilateral parotid glands, and the spinal cord on the weekly MVCT images as the reference images. These con- tours were compared to the automatic deformed structure generated by the deformable image reg- istration software. Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation440 DIR The popular software suite for DIR and ART, DIRART version 1a developed by Yang (2009) 11, was used to create automatic deformed contours and dose accumulation from the MVCT images of NPC patients. DIRART works complementarily with computational environment for radiotherapy research (CERR) to offer more functions9 and pro- vide the capability of selecting various deformation algorithms, transformation frameworks, and map- ping directions for providing the deformation vector field (DVF) in deformable registration procedures. In this study, the algorithms used were Horn and Schunck (HS) optical flow and demons (DM) combined with the asymmetric (Asy) and symmet- ric (Sym) transformation framework. Therefore, the study was carried out with four DIR methods, including the asymmetric transformation with the Horn and Schunck optical flow (AsyHS), the asym- metric transformation with the demon algorithm (AsyDM), the symmetric transformation with the Horn and Schunck optical flow (SymHS), and the symmetric transformation with the demon algo- rithm (SymDM). A multi-resolution technique was used in this study. For the optimum DIR performance for each algorithm, various parameters were systematically adjusted: four multigrids were used (n = 1, 2, 3, and 4) with 10n to 40n iterations per pass.9 The num- ber of passes for the optical flow algorithm was 6 and the number of passes for the demon algorithm was between 2 and 6. Coarser stages were typically run with a greater number of passes to improve the agreement with the target image prior to resa- mpling at finer resolutions.9 Validation of DIR The objective of the validation technique was to evaluate the accuracy of the automatic deformed contour by four DIR methods on weekly MVCT images, including the terms of the volume-based criterion and the deformation field analysis. Regarding the volume-based criterion, the most common overlap metric is the Dice similarity co- efficient (DSC).13 DSC is the metric that computes the number of pixels that overlaps between two volumes. If the images have no overlap, then the DSC is 0, and as the contours become identical, the DSC approaches the value of 1.13 Zimring et al.14 suggested that the satisfactory volume matching should be 70% (DSC of 0.7) or more for adaptive radiotherapy application. Inverse consistency error (ICE) was used to en- sure that the transformations were physically in- vertible for the deformation field analysis. Optimal transformation is found when the ICE minimizes the distance error.15 Assessment of impact of DIR methods on dose accumulation The dose accumulation process relied on six steps, as illustrated in Figure 1. Firstly, the ROIs from the planned kVCT images were transferred to the 1stday MVCT as source images for registration. The four DIR methods were performed between the 1st day MVCT and the weekly MVCTs (step 2). The deformation vector field was applied for creating the automatic deformed structure of the ROIs and for propagation to the weekly MVCT images (step 3), and the weekly dose distribution was deformed (step 4). The weekly dose deformation values were summed to the accumulated dose (step 5) and compared to the initial planned dose distribution (step 6). To evaluate the effect of dose accumulation from DIR errors, the reference accumulated dose on ROIs was computed by summing the weekly doses corresponding to the weekly MVCTs defined by the radiation oncologist. Moreover, to ensure that the weekly dose summation from the DIRART software was accurate, dose accumulation from independent software, Planned Adaptive software FIGURE 1. The diagram of the study workflow for dose accumulation and dose comparison. Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation 441 (TomoTherapy Inc., Madison, WI) was used to compare. Regarding the evaluation of accumulated dose, for the target volume, the median absorbed dose (D50%), the near-minimum (D98%) absorbed dose, and the near-maximum (D2%) absorbed dose values from each DIR method were assessed, the mean ab- sorbed dose (Dmean) of the bilateral parotid glands and D2% of the spinal cord were compared to the original planned dose for the OARs investigation. The one-way analysis of variance (ANOVA) test and the paired sample t-test were carried out on each set of comparison metrics to determine the statisti- cal significance, with a threshold of p < 0.05; SPSS statistical software version 17 was used to compare and assess the impact of each of the DIR methods. Results ROI volume variations Regarding volume variations during the radio- therapy, the percent ratio to the volume at the initial treatment planning of five NPC patients is illustrated in Figure 2. The averages of the NPC patients for the volume variation in the initial plan were significantly different from the averages after the treatment in 3 weeks for GTV, with p-value = 0.025, as demonstrated in Figure 2A, and for CTV, with p-value = 0.020, as demonstrated in Figure 2B. The volume was observed to have decreased by an average of 29.8% (GTV) and 21.0% (CTV) at the end of the treatment course. As regards the OARs, the right and the left pa- rotid volume variations were significantly differ- ent from those of the initial plan after 5 weeks and 4 weeks of treatment, with p-values of 0.017 and 0.026, respectively. The average volume decreased by 40.3% (right) and 43.6% (left) at the end of the treatment. DIR validation The results of DIR accuracy were consistent be- tween the volume-based criterion, DSC, and the deformation field analysis, ICE. Figure 3A dem- onstrates the histogram of the DSC values for all of the ROIs by four DIR methods. The SymDM methods showed significant difference from other methods by the one-way ANOVA analysis, with p- value = 0.00, with the worst performance in terms of volume-based criterion by mean values of DSC = 0.50 ± 0.30, 0.56 ± 0.34, 0.67 ± 0.19, 0.65 ± 0.28, and 0.69 ± 0.19 for GTV, CTV, right parotid, left parotid, and spinal cord, respectively. The average of DSC value was less than 0.7 for all the ROIs, that repre- sented unsatisfactory volume matching for adap- tive radiotherapy application.14 Regarding the ICE analysis, the results were consistent with DSC val- ue. Figure 3B illustrates the histogram of the ICE values for all of the ROIs by the four DIR methods. The SymDM method also showed the maximum error in the deformation field analysis in terms of A B C D FIGURE 2. The percent ratio to the volume at the initial treatment planning of (A) gross target volume GTV, (B) clinical target volume CTV, (C) right parotid gland, and (D) left parotid gland. Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation442 ICE for all of the ROIs throughout the treatment. The one-way ANOVA analysis showed that the SymDM method was significantly different from other methods, with p-value = 0.00. Therefore, the SymDM failed to adequately register on weekly MVCT for the dose accumulation application in this study. This paper focuses on the three highest performing algorithms in the AsyHS, AsyDM, and SymHS methods for dose accumulation. Accumulated dose variation from initial planned dose As regards target dose variation, the median GTV and CTV doses received at the end of treatment were slightly different from those in the initial plan. They were 0.11% (range: 0–0.29%) lower than the initial planned dose. The median dose varia- tions of the GTV and CTV were significantly dif- ferent from the initial planned dose after 6 weeks of treatment, with p-value = 0.016. Regarding the minimum and the maximum doses, they are rep- resented by near-minimum dose (D98%) and near- maximum dose (D2%), respectively. As for the D98%, they received slightly higher doses than the initial plan deals, with an average variation less than 0.5% (range: 0.29–1.60%). However, the dose at the end of treatment received slightly decreased doses of 0.45% (GTV) and 0.28% (CTV) from the initial dos- es planned for the D2. Regarding organ dose variation, the dose dif- ferences tended to increase as the treatment pro- gressed. For the bilateral parotid gland, the dis- crepancy between the delivered and the planned mean doses was found to have increased by 6.8% (range: 2.2 to 10.9%) for the right parotid and by 15.2% (range: -1.7 to 36.3%) for the left parotid. The average mean parotid dose increased in the ranges of 2.24 ± 0.97 Gy (right) and 5.70 ± 4.12 Gy (left) at the end of the treatment. The mean parotid dose variations were significantly different from the ini- tial plan after 6 weeks (right) and 5 weeks (left) of the treatment, with p-value = 0.049 (right) and p- value = 0.010 (left). The spinal cord dose received increased by 6.4% (range: -1.6 to 13.2%) from the initial plan, with the average near-maximum dose increasing in the range of 1.83 ± 1.5 Gy at the end of the treatment. Impact of DIR methods on weekly dose accumulation For each patient, the running cumulative doses were calculated using the CERR software through the three deformable image registration methods carried out by the DIRART software. Figure 4 dem- onstrates the 1st day MVCT image with original bi- lateral parotid gland (A) and the MVCT image at the 31st fraction with the automatic deformed con- A A B B FIGURE 3. Histogram of (A) the dice similarity coefficients (DSC) for all of the targets and organs at risk and (B) the inverse consistency error (ICE) in each treatment week and by asymmetric Horn and Schunck (AsyHS), asymmetric demon (AsyDM), symmetric Horn and Schunck (SymHS), and symmetric demon (SymDM) deformable image registration (DIR) methods. FIGURE 4. The 1st day MVCT image showing the original bilateral parotid gland (A) and the MVCT image at the 31st fraction showing the automatic deformed contour (B) from the AsyDM method. The initial planned dose distribution on the 1st day MVCT image (C) which was used to compare with the accumulated dose distribution at the end of the treatment (D). C D Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation 443 tour obtained using the AsyDM method (B). The in- itial planned dose distribution on the 1st day MVCT image, as illustrated in Figure 4C, was used for comparison with the accumulated dose distribu- tion at the end of the treatment, as demonstrated in Figure 4D. The variations in the cumulative doses between the delivered dose and the initial planned dose are illustrated in Figure 5 and Figure 6. Figure 5A illustrates the weekly GTV dose vari- ation from the initial plan with three DIR methods. The average of the median dose difference for all methods at the end of the treatment was lower than that in the initial plan, with 0.34 Gy (0.5%), 0.04 Gy (0.1%), and 0.30 Gy (0.4%) for the AsyHS, AsyDm, and SymHS DIR methods, respectively. However, the reference dose of GTV was found to have de- creased by 0.11%, with the accumulated GTV dose at 70.12 Gy (range: 69.9–70.4 Gy), at the end of treat- ment. The median dose variations of the GTV was significantly different from the initial planned dose after 6 weeks of treatment, with p-value = 0.016. Regarding the near-minimum dose and the near-maximum dose, the D98% of GTV in three DIR methods were found to be lower than that in the initial plan, as illustrated in Figure 5B; the aver- age discrepancy of the three DIR methods between the planned dose and the delivered dose was 0.33 Gy (0.5%) at the end of the treatment with the high differences of 1.0% after 5 week for AsyHS method. However, the reference near-minimum dose was found to have increased by 0.3% of the initial planned dose, with 69.2 Gy (range: 68.7–70 Gy). As for the maximum GTV dose considera- tion, the three DIR methods of D2% are presented in Figure 5C with gradually decrease in dose dif- ference from initial plan for all methods when the time increase. The average D2% from the three methods was lower than the initial D2%, with 0.76 Gy (1.1%), at the end of the treatment. The refer- ence D2% was found to have decreased by 0.45% of the initial planned dose, with 71.5 Gy (range: 70.9–72.5 Gy). As regards the median CTV dose, the dose vari- ations tended to be similar to the dose variations of GTV. Figure 5D illustrates the very small me- dian CTV differences of various deformable reg- istration methods from the initial planned dose. The discrepancy at the end of the treatment was lower than that in the initial planned dose, by 0.34 Gy (0.4%), 0.02 Gy (0%), and 0.26 Gy (0.4%) for AsyHS, AsyDm, and SymHS DIR methods, respec- tively. However, the reference median dose was found to have decreased by 0.11%, with 70.12 Gy (range: 69.9–70.4 Gy) at the end of the treatment. FIGURE 5. Cumulative dose comparison, calculated by the asymmetric Horn and Schunck (AsyHS), asymmetric demon (AsyDM), symmetric Horn and Schunck (SymHS), and symmetric demon (SymDM) deformable registration methods of gross tumour volume (GTV) for (A) median dose, D50 (B) near-minimum dose, D98 and (C) near-maximum dose, D2; and clinical tumour volume (CTV) for (D) median dose, D50 (E) near-minimum dose, D98, and (F) near-maximum dose, D2. The reference (Ref) accumulated dose was computed by summing the weekly doses corresponding to the weekly MVCTs defined by the radiation oncologist. A B C D E F Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation444 The median dose variations of the CTV was signifi- cantly different from the initial planned dose after 6 weeks of treatment, with p-value = 0.016. Regarding the D98 dose analysis of CTV, the dis- crepancy between the initial and the delivered dose of the three DIR methods are shown in Figure 5E. The average of the D98% variations from the initial D98% in the three DIR methods was 0.89 Gy (1.2%). Figure 5F shows the D2% of CTV; the average vari- ation from the initial D2% of the three DIR methods was 0.76 Gy (1.1%). As regards organ dose accumulation in the three DIR methods, Figure 6 illustrates the weekly dose difference from the initial plan in the three DIR methods for the bilateral parotid gland and the spinal cord. Overall, the dose differences tended to increase as the treatment progressed. Figure 6A shows the mean right parotid dose (Dmean) to be higher than the initial planned dose, by 5.38 Gy (16.0%), 3.38 Gy (10.1%), and 4.84 Gy (14.4%) for the AsyHS, AsyDm, and SymHS methods, respec- tively. However, the reference mean dose was found to have increased by 2.24 Gy (range: 0.8–3.7 Gy), at 6.82%, at the end of the treatment. For the left parotid mean dose, as illustrated in Figure 6B, these variations were higher after treatment than those for the initial planned dose, and the discrepancy was by 6.88 Gy (18.3%), 4.12 Gy (11.0%), and 6.82 Gy (18.1%) for the AsyHS, AsyDm, and SymHS DIR methods, respectively. However, the reference mean dose was found to have increased by 5.7 Gy (range: -0.6 to12.4 Gy), at 15.2% at the end of the treatment. The mean parot- id dose variations were significantly different from the initial plan after 6 weeks (right parotid) and 5 weeks (left parotid) of the treatment, with p-value of 0.049 and 0.010, respectively. As regards spinal cord weekly dose accumu- lation, the variations tended to increase in all the three DIR methods, by 2.33 Gy (7.9%), 1.46 Gy (4.9%) and 0.60 Gy (5.4%) for AsyHS, AsyDm, and SymHS, respectively. However, the reference cord dose variation was found to have increased by 1.83 Gy (range: -0.5 to 4.0 Gy), at 6.37% at the end of the treatment. DIRART and planned adaptive software for dose accumulation To ensure dose summation on the DIRART software, the independent planned adaptive software on heli- cal tomotherapy treatment planning was used to compare the dose accumulation values. The same data set of all the reference ROIs on weekly MVCTs defined by the radiation oncologist was transferred to the planned adaptive software. The comparison of the weekly accumulated doses between DIRART and planned adaptive is illustrated in Figure 7. The variations in the accumulated median parotid doses of DIRART were not significantly different accord- ing to the planned adaptive software, with p-value = 0.972 for the right parotid gland, as shown in Figure 7A, and p-value = 0.958 for the left parotid gland, as shown in Figure 7B. The consistency in the dose variations between the two independent types of software demonstrates that the dose accumula- tion of the DIRART software can be applied for use in dose accumulation studies. Discussion The validation of DIR was consistent in terms of the volume-based criterion, DSC, and the deforma- A B C FIGURE 6. Cumulative dose comparison, calculated from the asymmetric Horn and Schunck (AsyHS), asymmetric demon (AsyDM), symmetric Horn and Schunck (SymHS), and symmetric demon (SymDM) deformable registration methods in mean dose, Dmean, of (A) right parotid gland and (B) left parotid gland, and near-maximum dose, D2, of (C) spinal cord. Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation 445 tion field analysis, ICE. Accuracy in terms of DSC analysis tended to decrease as the treatment pro- gressed as a result of organs with large-scale de- formation causing reduction in the DIR accuracy.16 The AsyDM method showed the best performance with the highest average DSC value of all ROIs, the mean value of DSC = 0.804 ± 0.07, and also enabled the minimization of the inverse consistency error by the lowest mean value of ICE = 0.006 ± 0.002, as demonstrated in Figure 3. As regards the weekly dose variation from the initial plan, the results demonstrated that the me- dian, the near-minimum dose, and the near-maxi- mum dose of the target slightly varied, by less than 0.5% of the initial plan. However, with regard to organ doses like the bilateral parotid gland, the discrepancy between the planned and the deliv- ered mean doses was 6.8% (right) and 15.8% (left) higher than the initial plan. The accumulated mean parotid dose increased to be in the range of -0.6 to 12.4 Gy. Lee et al.2 analysed the changes in the parotid gland dose with reference to the anatomic changes throughout the course of radiotherapy. The daily parotid mean dose of the 10 patients differed from the planned dose by an average of 15%. At the end of the treatment, 3 of the 10 pa- tients were estimated to have received greater than 10% higher mean parotid dose than in the original plan (range: 13–42%), whereas the remaining 7 pa- tients received doses that differed by less than 10% (range: 6–8%). Regarding the correlation between dose ac- cumulation and DIR accuracy, there was consist- ency between the accuracy of ROI deformation and discrepancy of dose accumulation. The DIR method that yielded the highest DSC value was considered the best method for dose accumula- tion with the lowest variation from the reference dose. The AsyDM method demonstrated the best performance for target deformation with the high- est mean DSC value of 0.802 ± 0.08, as presented in Figure 3, and showed the best agreement for target dose accumulation with the lowest average varia- tion of 0.01% with the reference dose, as present- ed in Figure 5. This method also gave the highest mean DSC value of 0.824 ± 0.05 for the right pa- rotid gland and the lowest mean parotid dose vari- ation with 3.2% of the reference deformed dose, as demonstrated in Figure 6A. However, the SymHS method showed the best performance for left pa- rotid deformation, as demonstrated in Figure 3A, with the highest mean value of DSC = 0.824 ± 0.06, and also showed the lowest mean left parotid dose variation with 2.9% of the reference deformed dose, as illustrated in Figure 6B. For the spinal cord, the AsyHS method gave the best performance, as pre- sented in Figure 3, with the highest mean value of DSC = 0.806 ± 0.05, and also showed the lowest dose variation with 1.5% of the reference deformed dose, as presented in Figure 6C. Regarding the uncertainty of dose accumula- tion, the three DIR methods demonstrated satis- factory volume matching for accumulated dose application with DSC values more than 0.7 for all the methods. Moreover, the one-way ANOVA analysis demonstrated that there was no signifi- cant difference between the three DIR methods as regards ROI deformation and dose accumula- tion. However, when uncertainty was considered (difference between the maximum dose and the minimum dose) in the estimation of the accumu- lated dose for all the DIR methods, the average of the DSC value of all the targets by the three DIR methods was 0.782 ± 0.04. The mean uncertainty for estimating the target dose was 0.21 ± 0.11 Gy (range: 0.06–0.32 Gy). Regarding the uncertainty of the parotid dose, the averages of the DSC values by the three DIR methods were 0.805 ± 0.01 (right) and 0.814 ± 0.01 (left). This shows that the mean uncer- tainty values for estimating the parotid dose were 1.99 ± 0.76 Gy (range: 0.01–3.14 Gy) for the right parotid and 1.19 ± 0.24 Gy (range: 0.01–1.58 Gy) for the left parotid. For the spinal cord, the average of the DSC values was 0.791 ± 0.01 Gy, while the mean uncertainty value for the estimated dose was 0.41 A B FIGURE 7. Cumulative dose comparison, derived from helical tomotherapy planned adaptive software (HT) and DIRART software in median dose, D50, of (A) right parotid gland and (B) left parotid gland. Radiol Oncol 2017; 51(4): 438-446. Nobnop W et al. / Deformable image registration methods for dose accumulation446 ± 0.04 Gy (range: 0.04–0.57 Gy). The results in this study were lower that Rigaud et al.17 who showed the mean uncertainty (difference between the max- imum dose and the minimum dose, considering all the 10 DIR methods) to estimate the cumulated mean dose for the parotid gland (PG) was 4.03 Gy (SD = 2.27 Gy, range: 1.06–8.91 Gy). Further investigation would involve applying this methodology to other treatment areas to iden- tify patients who may benefit from adaptive treat- ment. DIR on megavoltage computed tomography imaging makes it possible to calculate daily and ac- cumulated doses. Significant dose variations were observed as a result of inter-fractional anatomic changes, which is information that would benefit adaptive treatment strategies. Acknowledgments The author offers many thanks to the staff of the Division of Therapeutic Radiology and Oncology, Faculty of Medicine, Chiang Mai University, for supporting this study. References 1. Hardcastle N, Tome W, Cannon D, Brouwer C, Wittendorp P, Dogan N, et al. A multi-institution evaluation of deformable image registration algorithms for automatic organ delineation in adaptive head and neck radiotherapy. Radiat Oncol J 2012; 7: 1-7. doi: 10.1186/1748-717X-7-90 2. Lee C, Langen KM, Lu W, Haimerl J, Schnarr E, Ruchala KJ, et al. Assessment of parotid gland dose changes during head and neck cancer radiotherapy using daily megavoltage computed tomography and deformable image registration. Int J Radiat Oncol Biol Phys 2008; 71: 1563-71. doi: 10.1016/j. ijrobp.2008.04.013 3. Lee C, Langen KM, Lu W, Haimerl J, Schnarr E, Ruchala KJ, et al. Evaluation of geometric changes of parotid glands during head and neck cancer radio- therapy using daily MVCT and automatic deformable registration. Radiother Oncol 2008; 89: 81-8. doi: 10.1016/j.radonc.2008.07.006 4. Castelli J, Simon A, Louvel G, Henry O, Chajon E, Nassef M, et al. Impact of head and neck cancer adaptive radiotherapy to spare the parotid glands and decrease the risk of xerostomia. Radiat Oncol J 2015; 10: 1-10. doi: 10.1186/s13014-014-0318-z 5. Kupelian PA, Langen KM, Zeidan OA, Meeks SL, Willoughby TR, Wagner TH, et al. Daily variations in delivered doses in patients treated with radio- therapy for localized prostate cancer. Int J Radiat Oncol Biol Phys 2006; 66: 876-82. doi: 10.1016/j.ijrobp.2006.06.011 6. Langen KM, Meeks SL, Poole DO, Wagner TH, Willoughby TR, Kupelian PA, et al. The use of megavoltage CT (MVCT) images for dose recomputations. Phys Med Biol 2005; 50: 4259-76. doi: 10.1088/0031-9155/50/18/002 7. Morin O, Chen J, Aubin M, Gillis A, Aubry JF, Bose S, et al. Dose calculation using megavoltage cone-beam CT. Int J Radiat Oncol Biol Phys 2007; 67: 1201-10. doi: 10.1016/j.ijrobp.2006.10.048 8. Yeo U, Supple J, Taylor M, Smith R, Kron T, Franich R. Performance of 12 DIR algorithms in low-contrast regions for mass and density conserving defor- mation. Med Phys 2013; 40: 1-12. doi: 10.1118/1.4819945 9. Nie K, Chuang C, Kirby N, Braunstein S, Pouliot J. Site-specific deformable imaging registration algorithm selection using patient-based simulated deformations. Med Phys 2013; 40: 041911. doi: 10.1118/1.4793723 10. Hardcastle N, Elmpt W, Cannon D, Ruysscher D, Bzdusek K, Tomé W. Accuracy of deformable image registration for contour propagation in adap- tive lung radiotherapy. Radiat Oncol J 2013; 8: 243-50. doi: 10.1186/1748- 717X-8-243 11. Yang D, Brame S, NaqaE. Technical Note: DIRART–A software suite for de- formable image registration and adaptive radiotherapy research. Med Phys 2011; 38: 71-7. doi: 10.1118/1.3521468 12. Lee N, Harris J, Garden AS, Straube W, Glisson B, Xia P, et al. Intensity- modulated radiation therapy with or without chemotherapy for naso- pharyngeal carcinoma: radiation therapy oncology group phase II trial 0225. J Clin Oncol 2009; 27: 3684-90. doi: 10.1200/JCO.2008.19.9109 13. Kristy KB. Image processing in radiation therapy. New York: Taylor & Francis Group; 2013. 14. Zimring D, Talos F, Bhagwat G, Haker J, Black P, Zou K. Statistical valida- tion of brain tumor shape approximation via spherical harmonics for image-guided neurosurgery. Acad Radiol 2005; 12: 459-66. doi: 10.1016/j. acra.2004.11.032 15. Varadhan R, Karangelis G, KrishnanK, Hui S. A framework for deformable image registration validation in radiotherapy clinical applications. J Appl Clin Med Phys 2013; 14: 4066. doi: 10.1120/jacmp.v14i1.4066 16. Luiza B, Mischa SH, Eliana M, Vasquez O, Ben JM. A symmetric nonrigid registration method to handle large organ deformations in cervical cancer patients. Med Phys 2010; 37: 3760. doi: 10.1118/1.3443436 17. Rigaud B, Antoine S, Castelli J, Gobeli M, Arango JO, Cazoulat G, et al. Evaluation of deformable image registration methods for dose moni- toring in head and neck radiotherapy. Biomed Res Int 2015; 1-16. doi: 10.1155/2015/726268 Radiol Oncol 2017; 51(4): 447-454. doi: 10.1515/raon-2017-0024 447 research article Long term results of radiotherapy in vulvar cancer patients in Slovenia between 1997–2004 Helena Barbara Zobec Logar Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2017; 51(4): 447-454. Received 2 March 2017 Accepted 24 April 2017 Correspondence to: Helena Barbara Zobec Logar, M.D., M.Sc., Institute of Oncology Ljubljana, Zaloška 2, 1000 Ljubljana, Slovenia. Phone: +386 1 5879 505; Fax: +386 1 5879 857; E-mail: hlogar@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of this retrospective single institution study was to analyse long term results of vulvar cancer treatment with conventional 2D radiotherapy in Slovenia between years 1997–2004. Patients and methods. Fifty-six patients, median age 74.4 years +/- 9.7 years, mainly stage T2 or T3, were included in the study. All patients were treated with radiotherapy, which was combined with surgery (group A), used as the pri- mary treatment (group B) or at the time of relapse (group C). Chemotherapy was added in some patients. Histology, grade, lymph node status, details of surgery, radiation dose to the primary tumour, inguinofemoral and pelvic area as well as local control (LC) and survival were evaluated. Results. Overall survival (OS), disease specific survival (DSS) and LC rates at 10-years for all patients were as follows: 22.7%, 34.5% and 41.1%, respectively. The best 10-years results of the treatment were achieved in the primary operated patients treated with adjuvant radiotherapy +/-chemotherapy (OS 31.9%, DSS 40.6% and LC 47.6%). Positive lymph nodes had a strong influence on LC. In case of positive nodes LC decreased by 60% (p = 0.03) and survival decreased by 50% (p = 0.2). There was a trend to a better LC with higher doses ≥ 54.0 Gy (p = 0.05). Conclusions. The best treatment option for patients with advanced vulvar cancer is combined treatment with sur- gery and radiotherapy +/- chemotherapy, if feasible. Radiotherapy with the dose of ≥ 54.0 Gy should be considered to achieve better LC if positive adverse factors are present. Key words: vulvar cancer; radiotherapy, surgery; local control; survival Introduction Vulvar cancer is a rare gynaecological cancer and it accounts for approximately 3–5% of all gynae- cological malignancies. The incidence in Slovenia between years 1997–2004 was 33 to 57 (average 40) patients per year, the highest incidence occurred in women over 60 years of age.1 Surgery is the most important modality in the treatment of early vul- var cancer, while (chemo)radiotherapy +/- surgery is the most important modality in the treatment of advanced vulvar cancer. Radiotherapy can be used preoperatively, postoperatively, or as the only treatment in locally advanced vulvar cancer with bulky unresectable primary tumour or groin disease. In operated patients positive margins and positive lymph nodes are the most important in- dications for adjuvant radiotherapy. Other prog- nostic factors such as large primary tumour lesion, deep tumour invasion and in recent years also lymphovascular space involvement (LVI) are the factors that are used to determine adjuvant radio- therapy as well.2,3 The majority of the patients with vulvar cancer are over 70 years of age, with many additional comorbidities, which also favour radio- therapy as the best treatment option. Recent data are showing improvement of the treatment by add- ing adjuvant chemotherapy.4-7 In the past different cytotoxic agents were utilized, but nowadays the preferred is weekly cisplatin, an option which was extrapolated from treatment of advanced cervical squamous cell carcinoma (SCC). Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients448 Surgical treatment Classical surgical approach is radical vulvectomy. More conservative surgery which reduced mor- bidity without compromising prognosis is used in early stage vulvar SCC. Wide local excision of the tumour with sentinel lymph node biopsy (SNB) with or without inguinofemoral lymphadenecto- my is an attractive alternative.8-12 The most suitable candidates for SNB are patients with tumour diam- eter < 4 cm and with no palpable groin nodes.9,13,14 Patients with positive sentinel-node metastases are candidates for additional treatment.8,9 The lymph node status and particularly inguinofemoral lymph node status is the most important prognos- tic factor for survival, while the tumour free mar- gin is the most important prognostic factor for local recurrence.3, 15-17 Postoperative radiotherapy Radiotherapy in postoperative adjuvant treatment is used to additionally treat the primary site, groins and/or pelvis. Radiotherapy to the primary site is indicated in case of positive or close margin (≤ 8 mm) and large primary tumour (> 4 cm).2,3,18 OS of patients with close/positive margins who received adjuvant radiotherapy was similar to those with negative margins.18 Radiotherapy to the groins is indicated in case of two or more microscopically positive groin nodes, in case of one or more mac- roscopically positive node, or in case of extracap- sular extension (ECE). When only one microscopi- cally positive groin node is identified, adjuvant radiotherapy can be omitted, although recent rec- ommendations show improvement in disease free survival (DFS) when adding radiotherapy also in a single microscopic node positive disease.5, 19-22 Lymph node involvement is the strongest negative predictive factor for survival. The 3-years PFS and OS for node positive disease was 35.2% and 56.2% compared to 75.2% and 90.2% for node negative disease.5,11,23 Despite adjuvant radiotherapy in node positive disease and clinical benefit in PFS, OS remains poor (57.7% with adjuvant radiother- apy vs 51.4% without radiotherapy at 3-years, p = 0.17).5 Locally advanced vulvar cancer Radiotherapy is the treatment option for bulky pri- mary site and/or extensive groin disease. The ad- dition of chemotherapy to radiotherapy improves survival in several SCC.6,7,24,25 As high as 60% of patients with locally advanced disease can present with nodal metastases, the reason that urge for more aggressive treatment which combines chem- otherapy and radiotherapy in locally advanced disease. After primary chemoradiotherapy surgery can be performed in selected cases. There are only a few studies, mainly single institution or phase II studies that report results of such approach.4,6,25-28 In this retrospective single institution study the long term results of vulvar cancer treatment in Slovenia between 1997 and 2004 with radiotherapy alone or combined with surgery or chemotherapy were analysed. Patients and methods Patients and tumours 69 patients with vulvar cancer treated with ra- diotherapy from January 1997 till December 2004 were retrospectively analysed. Thirteen patients were excluded from the study, because the pallia- tive dose was used to treat the cancer. At the end 56 patients with histologically confirmed vulvar can- cer treated with curative intent with radiotherapy, +/- surgery, +/- chemotherapy were enrolled and retrospectively analysed. Mean patient age was 74.4 +/- 9.7 years. All pa- tients were staged according to the FIGO 1988 stag- ing system (13). FIGO stage distribution, available in 94.6% of the patients, was as follows: stage I 5 (8.9%), stage II 4 (7.1%), stage III 33 (58.9%) and stage IV 11 (19.6%) and not specified in 3 patients. Histological type of the tumour was SCC in all patients included in the study. Other histological types ranging from malignant melanoma (2 pa- tients), adenocarcinoma of the Bartholin’s gland (1 patient) and Paget’s disease (1 patient) were rare and were excluded from further analysis. The di- agnostic work-up before the treatment consisted of clinical examination with biopsy and chest radiog- raphy in operated patients, while imaging studies such as abdominal and groin ultrasound or chest/ abdominal computer tomography (CT) were only rarely performed. Patients, tumour and treatment characteristics are listed in Table 1. The study was approved by the Protocol Review Board at the Institute of Oncology Ljubljana (ERID- KESOPKR-14). The investigation followed recom- mendations of the Helsinki Declaration (1964, with later amendments) and of the European Council Convention on Protection of Human Rights in Bio- Medicine (Oviedo 1997). Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients 449 TABLE 1. Patients, tumor and treatment characteristcs Characteristics All Primary operated Primary irradiated Relapse group group A group B C Number of patients (%) 56 (100.0%) 31 (55.4%) 11 (19.6%) 14 (25%) Mean age (years) (+/-SD) 74.4 (+/-9.7) 74.9 ( +/- 9.8) 67.9 (+/-10.8) 87.2 (+/- 6.1) Median follow-up time (months) 22.5 (2-203) 27 (8-203) 9 (2-72) 44 (5-134) FIGO stage I 5 (8.9%) 0 0 5 (35.7%) II 4 (7.1%) 0 0 4 (28.6%) III 33 (58.9%) 23 (74.2%) 6 (54.5%) 4 (28.6%) IV 11 (19.6%) 7 (22.6%) 4 (36.4%) 0 not specified 3 (5.4%) 1 (3.2%) 1 (9.1%) 1 (7.1%) Histopathologic grade G1 21 (37.5%) 11 (35.5%) 3 (27.3%) 7 (50.0%) G2 25 (44.6%) 15 (48.4) 7 (63.6%) 3 (21.4%) G3 5 (8.9%) 3 (9.7%) 1 (9.1%) 1 (7.1%) not specified 5 (8.9%) 2 (6.5%) 0 3 (21.4%) T stage T1 5 (8.9%) 2 (6.5%) 0 3 (21.4%) T2 26 (46.4%) 18 (58.1%) 1 (9.1%) 7 (50.0%) T3 16 (28.6%) 9 (29.0%) 7 (63.6%) 0 T4 3 (5.4%) 1 (3.2%) 2 (18.2%) 0 not specified 6 (10.7%) 1 (3.2%) 1 (9.1%) 4 (28.6%) Diameter of primary tumor ≤ 2 cm 10 (17.9%) 7 (22.6%) 0 3 (21.4%) > 2 and ≤ 4 cm 23 (41.1%) 16 (51.6%) 3 (27.3%) 4 (28.6%) > 4 cm 12 (21.5%) 5 (16.2%) 6 (54.5%) 1 (7.1%) not specified 11 (19.6%) 3 (9.7%) 2 (18.2%) 6 (42.9%) Tumor invasion lower urethra 9 (16.1%) 3 (9.7%) 6 (54.5%) 0 vagina 15 (26.8%) 8 (25.8%) 7 (63.6%) 0 anus 1 (1.8%) 0 1 (9.1%) 0 bladder wall 0 0 1 (9.1%) 0 rectal wall 1 (1.8%) 0 1 (9.1%) 0 pelvic bone 2 (7.1%) 1 (3.2%) 1 (9.1%) 0 Tumor location clitoris 6 (10.7%) 4 (12.9%) 0 2 (14.3%) labium major 10 (17.9%) 5 (16.1%) 1 (9.1%) 4 (28.6%) labium minor 8 (14.3%) 4 (12.9%) 0 4 (28.6%) comissura/more than one location 28 (50.0%) 16 (51.6%) 10 (90.9%) 2 (14.3%) not specified 4 (7.1%) 2 (6.5%) 0 2 (14.3%) Nodal status N0 17 (30.3%) 4 (12.9%) 5 (45.5%) 8 (57.1%) N1 25 (44.6%) 19 (61.3%) 2 (18.2%) 4 (28.6%) N2 10 (17.9%) 7 (22.6%) 3 (27.3%) 0 not specified 4 (7.1%) 1 (3.2%) 1 (9.1%) 2 (14.3%) Nodal metastases micrometastases 9 (16.1%) 7 (22.6%) not specified 2 (14.3%) macrometastases 25 (44.6%) 18 (58.1%) 5 (45.5%) 2 (14.3%) Exstracapsular tumor spread positive 10 (17.9%) 8 (25.8%) not specified 2 (14.3%) Residual tumor after surgery 20 (35.7%) 16 (51.6%) not specified 4 (28.6%) primary tumor site 15 (26.8%) 13 (41.9%) not specified 2 (14.3%) nodal site 7 (12.5%) 4 (12.9%) not specified 3 (21.4%) Mean dose to the tumor (Gy) (+/-SD) 47.4 (+/-7.9) 47.1 (+/-8.2) 46.7 (+/-8.7) 46.0 (+/-5.2) Mean dose to the inguinofemoral area (Gy) (+/-SD) ipsilateral 47.5 (+/-8.8) 50.7 (+/-7.9) 46.9 (+/-8.4) 40.1 (+/-6.7) contralateral 49.7 (+/-9.7) 53.4 (+/-7.8) 44.9 (+/-9.8) 37.4 (+/-6.9) Mean dose to the pelvic area (Gy) (+/-SD) ipsilateral 46.2 (+/-6.2) 47.0 (+/-6.8) 45.0 (+/-4.9) 43.5 (+/-6.2) contralateral 44.6 (+/-10.5) 44.7 (+/-12.5) 45.0 (+/-4.9) 39.1 (+/-4.9) Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients450 Treatment In this retrospective study radiotherapy was used as the part of the treatment in all patients and it was most frequently combined with surgery in 31 patients (55.4%). Chemoradiotherapy was used in 11 patients (17.9%). At that time the most common- ly used cytotoxic agent in adjuvant setting was ble- omycin or cisplatin. Radiotherapy was also used at the time of relapse in 14 patients (25%): local re- lapse at the site of primary tumour was detected in 6 patients, and nodal relapse in 10 patients. The majority of relapses developed within two years after the primary treatment. In order to evaluate LC and survival among dif- ferent treatment modalities, three treatment groups were formed (Table 1). 31 patients were included in the primary operated group (group A), where the primary treatment modality was surgery with ra- diotherapy +/- chemotherapy as an adjuvant treat- ment. This was the largest group, reflecting that operation therapy +/- radiotherapy is the most im- portant treatment modality in the management of advanced vulvar cancer. Chemoradiotherapy was used only in 2/31 patients, respectively. In the pri- mary irradiated group (group B) 11 patients were included. Radiotherapy was used as the only treat- ment in 4/11 patients, combined with chemothera- py in 7/11 patients, used preoperatively in 1 or pre- and postoperatively in 1 patient, respectively. In the relapse group (group C) radiotherapy alone or combined with surgery was used at the time of re- lapse in 9/14 patients and 5/14 patients, respective- ly. Chemoradiotherapy was used in 2/14 patients. Surgical treatment Surgery is the cornerstone in the treatment of vul- var cancer. The most frequently used type of op- eration was radical vulvectomy in 29 and wide local excision in 16 patients. SNB was performed only in 6 patients. Vulvar cancer patients were diagnosed in different gynaecological centres in Slovenia and were mainly operated in the three main centres: University Medical Centre Ljubljana (33.9%), Institute of Oncology Ljubljana (37.5%) and University Medical Centre Maribor (5.4%). Unilateral and bilateral inguinofemoral lymph node dissection was performed in 26.8% and 33.9%, respectively. The mean number of removed lymph nodes was 7 (range 1–19). Among all operated pa- tients adjuvant radiotherapy was used in 65.1%, ra- diochemotherapy in 6.9% and radiotherapy at the time of relapse in 27.9%. Radiotherapy Radiotherapy is an important modality of the treat- ment in adjuvant setting, as a radical treatment in locally advanced cancer or as a treatment of choice at the time of relapse. External beam radiotherapy was delivered with 5–15 MV linear accelerator or Cobalt-60 machine. In addition, electrons were sometimes applied to boost inguinal areas or pri- mary tumour. Mostly supine position has been used, but “frogleg” position has been used as well, particularly in fat patients. Various two-dimen- sional (2D) techniques were used to radiate pri- mary tumour and/or inguinal nodes at that time. First technique used wide anteroposterior (AP) field that included the pelvic and inguinal area, with a narrow posteroanterior (PA) field covering pelvis only. The fields were weighted equally 1:1. The inguinal nodes were boosted to a certain depth determined by a clinician, with a separate anterior field. Bolus material on the primary tumour or in- guinal areas was used according to the physician’s decision. The physician’s determined depth of the inguinal nodes was usually 3 to 4 cm. The second technique used AP/PA field of the same dimension. An alternative technique consisted of a wide AP field and narrow PA field with a partial transmis- sion block at the central portion of the AP field. The dose at a specified depth to the inguinal areas was delivered through the AP field. The two opposite fields technique was applied in majority of the patients. Other techniques, such as four field (box) technique was used as well (16.2%). There were many variations in the prescribed dose to the inguinal nodes among patients due to dif- ferent inguinal depth chosen by a physician and different radiation techniques that were used. The daily dose was usually 1.8–2.0 Gy per fraction, a daily dose of 2.5–3.0 Gy per fraction was seldom used as well, with a total dose mainly in the range between 45–60 Gy. In order to enable comparison of the dose to the inguinal area, the depth dose was calculated for all patients at the same depth. The so called “inguinal depth” was determined on CT scans of the fifty women treated with pelvic radio- therapy at the time of collecting data for this study. The mean depth of the inguinal nodes was 5.5 cm (range 2–18 cm), which was more than arbitrary by a physician determined depth. Chemotherapy Today chemotherapy combined with radiotherapy is the fundamental strategy in the treatment of Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients 451 advanced vulvar cancer. At the time of this study it was not so widely used, altogether 11 patients (19.6%) got some kind of chemotherapy. Platinum- based chemotherapy is the treatment of choice in squamous cell carcinomas. In our study cisplatin was used alone or in combination with other cy- totoxic drugs such as bleomycin or 5-fluorouracil. Rarely, in case of renal dysfunction, carboplatin was used instead of cisplatin. Follow up First follow-up was usually performed 3 months after the treatment. Consecutive follow-ups were usually performed alternately by gynaecologist or radiation oncologist at 3 to 6 month intervals for the first 5 years and once per year later on. Statistical analysis The data were analysed using IBM SPSS statistical software package version 23.0 (SPSS Inc., Chicago, IL, USA), p values of < 0.05 were considered statisti- cally significant. Kaplan-Meier method was applied to calculate ac- tuarial survival and loco-regional control rates. The endpoint for OS was death from any cause, for DSS the disease related death and for LC any evidence of loco-regional recurrent disease. Patients without re- lapse were censored at the time of the last follow-up, visit or death. Surviving patients were censored at the time of the last follow-up. No effort was made to compare results between different treatment groups due to small number of patients in each group and consequently a low power of the test. The influence of positive lymph nodes, ECE, size of primary tumour and completeness of sur- gical resection on survival and LC were analysed. Arbitrary cut off points for positive lymph nodes and ECE (negative versus positive), size of primary tumour (up to 4 cm versus ≥ 4 cm) and complete- ness of surgical resection (complete R0 versus in- complete R1/R2) were used. The data regarding the side effects of the radio- therapy treatment or surgery were not systemati- cally recorded and were in general missing in the patient’s record, therefore the analysis of side ef- fects of the treatment was omitted in this study. Results At the time of the last follow up 21 patients (37.5%) proved to be disease free. Median follow-up for FIGURE 1. Local control for the group A (primary operated patients), B (primary irradiated patients), C (patients with relapse) and all patients. FIGURE 2. Overall survival for the group A, (primary operated patients), B (primary irradiated patients), C (patients with relapse) and all patients. Time (months) 2502001501005 00 Pr o b a b ili ty o f s ur vi va l 1 ,0 0,8 0,6 0,4 0,2 0,0 all patients-censored group C-censored group B-censored group A-censored all patients group C group B group A Time (months) 2502001501005 00 C um S ur vi va l 1 ,0 0,8 0,6 0,4 0,2 0,0 all patients-censored group C-censored group B-censored group A-censored all patients group C group B group A all patients was 22.5 months (range 2–203) and for surviving patients 101 months (range 72–157). For 4 surviving patients follow-up was lower than 10-years due to lost to follow-up, but according to the national Cancer Registry they were still alive at the close-out time of the study. The pattern of fail- ure at the last follow-up is represented in Table 2. Half of the patients with distant metastases had also locoregional recurrence. The skin was the most common site of metastatic disease, other pos- Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients452 sible sites were pelvic and paraaortic lymph nodes, lungs and bones. Overall survival (OS), disease specific survival (DSS) and local control (LC) for all vulvar cancer patients at 10-years were 22.7%, 34.5% and 41.1%, for primary operated group 31.9%, 40.6% and 47.6% respectively, in primary irradiated group there were no survivals at 10-years and for the relapse group survivals and LC were as follows 14.3%, 33.9% and 41.5% (Figure 1 and 2). Ten years LC for node negative disease was 57.1% for T1–2 and 55.6% for T3–4. For node positive disease T1– 4N1 and T1–4N2 it was 38.4% and 29.2%, respec- tively. The difference was not statistically signifi- cant (p = 0.5). OS, DSS and LC at 10-years for stage III were as follows 30.3%, 35.3% and 44%, and for stage IV 18.2%, 18.2% and 24.9%. Data for stage I and II are not reported, because only 2 patients were alive at 5 years and only 1 patient (FIGO stage II) was alive at 10 years (134 months). Factors that contribute to lower outcome in stage I and II were higher age (mean age 79.9 +/-6.5, p = 0.04) and treatment of the relapsed disease in all stage I and II patients (local relapse 3, inguinal 4, local and inguinal 2 patients). The mean equivalent dose in 2 Gy per daily frac- tion (EQD2) applied to the primary tumour was 47.4 Gy +/- 7.9 Gy. The mean equivalent dose to the inguinofemoral area was 47.5 +/- 8.8 Gy and 49.7 +/- 9.7 Gy for the ipsi- and contralateral side. The mean equivalent dose to the primary tumour and to the inguinal area for different treatment groups are listed in Table 1. Significantly higher dose was applied to the macroscopic positive lymph nodes as to the negative or microscopic lymph nodes (54.0 Gy against 40.0 Gy, p = 0.04). There was no statis- tically significant difference between the dose ap- plied to operated or non-operated patients. Despite positive adverse factors, such as positive lymph nodes, ECE or large primary tumour, the dose ap- plied to the primary site or groins was rather low. Only 16% of patients received the dose higher than 54.0 Gy (Figure 3). ECE had a negative impact on LC (p = 0.02; Figure 4). Positive lymph nodes had a strong in- fluence on LC (Figure 5). In case of positive nodes LC decreased by 60% (p = 0.03), and OS as well as DSS decreased by 50% (p = 0.2). The complete- ness of surgical resection had an impact on local and regional control in the first two years after the treatment, which did not prove to be statistically significant (p = 0.6). There was a trend to a better LC with higher doses over 54.0 Gy (p = 0.05). Vulvar and groin morbidity are important fac- tors which effect quality of life of the patient. It is compromised due to surgical treatment and radio- therapy. In this study acute and late side effects were not systematically recorded and these results were not evaluated. Discussion The best treatment option for early stage vulvar cancer is surgery, with adjuvant radiotherapy in- dicated in high risk disease with positive adverse factors such as large primary tumour size, close or positive surgical margins, positive lymph node/s or ECE. For patients, who are not candidates for primary surgery due to more extensive disease or comorbidities, radiotherapy combined with chem- otherapy if feasible, is the preferred treatment option. The distribution of the patients between different treatment groups reflects the treatment strategy which was used most frequently - sur- gery combined with adjuvant radiotherapy. SNB in patients with early stage disease and negative groin exam as an alternative to inguinofemoral lymphadenectomy, was performed only in 10.7%, reflecting early beginnings of using this method in Slovenia. Radiotherapy as the primary treatment TABLE 2. Pattern of failure at the time of last follow-up All Primary operated Primary irradiated Relapse group group A group B C Local failure 9 5 4 0 Inguinal failure 6 3 1 2 Distant failure 5 3 1 1 Local and inguinal failure 10 5 3 2 Local/inguinal and distant failure 5 2 1 2 Overall failure 35 18 10 7 No evidence of disease 21 13 1 7 negative vs positive lymph nodes 10 C o un t 1 0 8 6 4 2 0 ≥ 54 Gy < 54 Gy ECE yesno C ou nt 6 4 2 0 ≥ 54 Gy < 54 Gy tumor size ≥ 4 cm< 4 cm C o un t 1 2 1 0 8 6 4 2 0 ≥ 54 Gy < 54 Gy FIGURE 3. Number of patients with the dose applied to the positive lymph nodes, nodes with extracapsular lymph node extension (ECE) and large tumours. Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients 453 was used less frequently; concomitant chemo- therapy was used in 63.3% (7/11) of patients. The 2D treatment technique that was used frequently at that time, with dose prescription to the groins, usually at 3 cm depth, resulted in under-dosing of the inguinal nodes. Many studies, as well as ours, showed that the inguinal lymph nodes depth is in general more than 3 cm.1,29 With the use of 3D and other novel techniques such as intensity modulat- ed radiotherapy (IMRT) or volumetric modulated arc therapy (VMAT), the appropriate higher dose can be delivered to the groins with the dose reduc- tion to the uninvolved tissues.4 In the study there was no significant difference between the dose applied to the patients with different risk factors and the patients in different treatment groups. The radiation dose applied after surgery is different after complete tumour removal, it ranges between 45.0 to 50.0 Gy, than after incomplete tumour re- moval with remaining microscopic or macroscopic disease, where the applied dose should be higher (≥ 60.0 Gy); although recent report showed that for vulvar squamous cell carcinoma with positive margins an adequate dose lies between 54.0 to 59.9 Gy.30 The results from our study showed that rath- er low dose was applied to the primary irradiated patients group B and relapse group C, which was, besides the existing comorbidity factors, higher age and advanced tumours, the possible reason for lower LC and survival. The risk factors for the worse outcome that were recognized in other stud- ies were proved in this study as well, despite the small number of patients. Positive lymph nodes status proved to be one of the strongest predictor for lower disease control and survival, despite the use of adjuvant therapy.7,5,31 Despite the fact of a low quality data of the ret- rospective analysis with scarce diagnostic work-up performed before the treatment, the historical 2D radiotherapy technique used in majority of the pa- tients and lacking morbidity data of the treatment, this is up to now the first and the only study of SCC of the vulva treated with radiotherapy in Slovenia. These results can be used as the basis for further analysis and improvement of the treatment of vulvar cancer patients with modern radiotherapy techniques. Conclusions The best treatment option for patients with squa- mous cell vulvar cancer is surgery with adjuvant radiotherapy +/- chemotherapy in case of positive adverse factors. The dose over 54.0 Gy is associat- ed with better LC. More aggressive treatment with higher doses to the lymph node/s and adjuvant chemotherapy should be offered to the patients with positive lymph node/s. For the patients who are not candidates for primary surgery chemora- diotherapy is the treatment of choice. 2D radio- therapy technique with the dose prescription to the inguinal nodes at certain depth and consequently sometimes inadequate dose distribution to the inguinofemoral region can be bypassed by mod- ern radiotherapy techniques, such as IMRT and Time (months) 2502001501005 00 Pr o b a b ili ty o f s ur vi va l 1 ,0 0,8 0,6 0,4 0,2 0,0 positive-censored negative-censored positive negative extracapsular extension Time (months) 2502001501005 00 Pr o b a b ili ty o f s ur vi va l 1 ,0 0,8 0,6 0,4 0,2 0,0 one or more positive nodes-censored no positive nodes- censored 1 or more positive nodes no positive nodes number of positive lymph nodes FIGURE 5. The influence of positive nodes on local control (p = 0.03). FIGURE 4. The influence of extracapsular nodal extension on local control (p = 0.02). Radiol Oncol 2017; 51(4): 447-454. Zobec Logar HB / Long term results of radiotherapy in vulvar cancer patients454 VMAT. With the use of these techniques and more conservative surgery the side effects and cosmetic results of the treatment can be further improved. References 1. Zadnik V, Primic Zakelj M, Lokar K, Jarm K, Ivanus U, Zagar T. Cancer burden in Slovenia with the time trends analysis. Radiol Oncol 2017; 51(4): 447- 454.; 51: 47-55. doi: 10.1515/raon-2017-0008 2. Jolly S, Soni P, Gaffney DK, Biagioli M, Elshaikh MA, Jhingran A, et al. ACR Appropriateness Criteria(R) Adjuvant Therapy in Vulvar Cancer. Oncology 2015; 29: 867-75. 3. Heaps JM, Fu YS, Montz FJ, Hacker NF, Berek JS. Surgical-pathologic variables predictive of local recurrence in squamous cell carcinoma of the vulva. Gynecol Oncol 1990; 38: 309-14. 4. Beriwal S, Coon D, Heron DE, Kelley JL, Edwards RP, Sukumvanich P, et al. Preoperative intensity-modulated radiotherapy and chemotherapy for locally advanced vulvar carcinoma. Gynecol Oncol 2008; 109: 291-5. doi: 10.1016/j.ygyno.2007.10.026 5. Mahner S, Jueckstock J, Hilpert F, Neuser P, Harter P, de Gregorio N, et al. Adjuvant therapy in lymph node-positive vulvar cancer: the AGO-CaRE-1 study. J Natl Cancer Inst 2015; 107: 1-12. doi: 10.1093/jnci/dju426 6. Montana GS, Thomas GM, Moore DH, Saxer A, Mangan CE, Lentz SS, et al. Preoperative chemo-radiation for carcinoma of the vulva with N2/N3 nodes: a gynecologic oncology group study. Int J Radiat Oncol Biol Phys 2000; 48: 1007-13. doi: 10.1016/S0360-3016(00)00762-8 7. Gill BS, Bernard ME, Lin JF, Balasubramani GK, Rajagopalan MS, Sukumvanich P, et al. Impact of adjuvant chemotherapy with radiation for node-positive vulvar cancer: a National Cancer Data Base (NCDB) analysis. Gynecol Oncol 2015; 137: 365-72. doi: 10.1016/j.ygyno.2015.03.056 8. Te Grootenhuis NC, van der Zee AG, van Doorn HC, van der Velden J, Vergote I, Zanagnolo V, et al. Sentinel nodes in vulvar cancer: long-term follow-up of the GROningen INternational Study on Sentinel nodes in Vulvar cancer (GROINSS-V) I. Gynecol Oncol 2016; 140: 8-14. doi: 10.1016/j. ygyno.2015.09.077 9. Oonk MH, van Hemel BM, Hollema H, de Hullu JA, Ansink AC, Vergote I, et al. Size of sentinel-node metastasis and chances of non-sentinel-node in- volvement and survival in early stage vulvar cancer: results from GROINSS-V, a multicentre observational study. Lancet Oncol 2010; 11: 646-52. doi: 10.1016/S1470-2045(10)70104-2 10. Van Der Zee AGJ, Oonk MH, De Hullu JA, Ansink AC, Vergote I, Verheijen RH, et al. Sentinel node dissection is safe in the treatment of early-stage vulvar cancer. J Clin Oncol 2008; 26: 884-9. doi: 10.1200/JCO.2007.14.0566 11. de Hullu JA, van der Zee AG. Surgery and radiotherapy in vulvar cancer. Crit Rev Oncol Hematol 2006; 60: 38-58. doi: 10.1016/j.critrevonc.2006.02.008 12. Vakselj A, Bebar S. The role of sentinel lymph node detection in vulvar car- cinoma and the experiences at the Institute of Oncology Ljubljana. Radiol Oncol 2007; 41: 167-73. doi: 10.2478/v10019-007-0027-4 13. Benedet JL, Bender H, Jones H, Ngan H, Pecorelli S, Staging classifications and clinical practice guidelines of gynaecologic cancers. Int J Gynaecol Obstet 2000; 70: 207-312. doi: 10.1016/S0020-7292(00)90001-8 14. Levenback CF, Ali S, Coleman RL, Gold MA, Fowler JM, Judson PL, et al. Lymphatic mapping and sentinel lymph node biopsy in women with squa- mous cell carcinoma of the vulva: a Gynecologic Oncology Group study. J Clin Oncol 2012; 30: 3786-91. doi: 10.1200/JCO.2011.41.2528 15. Homesley HD, Bundy BN, Sedlis A, Adcock L. Radiation therapy versus pelvic node resection for carcinoma of the vulva with positive groin nodes. Obstet Gynecol 1986; 68: 733-40. 16. Homesley HD, Bundy BN, Sedlis A, Yordan E, Berek JS, Jahshan A, et al. Assessment of current International Federation of Gynecology and Obstetrics staging of vulvar carcinoma relative to prognostic factors for survival (a Gynecologic Oncology Group study). Am J Obstet Gynecol 1991; 164: 997-1003. 17. De Hullu JA, Hollema H, Lolkema S, Boezen M, Boonstra H, Burger MPM, et al. Vulvar carcinoma. The price of less radical surgery. Cancer 2002; 95: 2331-8. doi: 10.1002/cncr.10969 18. Ignatov T, Eggemann H, Burger E, Costa SD, Ignatov A. Adjuvant radiothera- py for vulvar cancer with close or positive surgical margins. J Cancer Res Clin Oncol 2016; 142: 489-95. doi: 10.1007/s00432-015-2060-9 19. Fons G, Groenen SMA, Oonk MHM, Ansink AC, van der Zee AGJ, Burger MPM, et al. Adjuvant radiotherapy in patients with vulvar cancer and one intra capsular lymph node metastasis is not beneficial. Gynecol Oncol 2009; 114: 343-5. doi: 10.1016/j.ygyno.2009.05.017 20. Viswanathan AN, Pinto AP, Schultz D, Berkowitz R, Crum CP. Relationship of margin status and radiation dose to recurrence in post-operative vulvar car- cinoma. Gynecol Oncol 2013; 130: 545-9. doi: 10.1016/j.ygyno.2013.05.036 21. Parthasarathy A, Cheung MK, Osann K, Husain A, Teng NN, Berek JS, et al. The benefit of adjuvant radiation therapy in single-node-positive squamous cell vulvar carcinoma. Gynecol Oncol 2006; 103: 1095-9. doi: 10.1016/j. ygyno.2006.06.030 22. Woelber L, Eulenburg C, Choschzick M, Kruell A, Petersen C, Gieseking F, et al. Prognostic role of lymph node metastases in vulvar cancer and implica- tions for adjuvant treatment. Int J Gynecol Cancer 2012; 22: 503-8. doi: 10.1097/IGC.0b013e31823eed4c 23. Farias-Eisner R, Cirisano FD, Grouse D, Leuchter RS, Karlan BY, Lagasse LD, et al. Conservative and individualized surgery for early squamous carcinoma of the vulva: the treatment of choice for Stage I and II (T1-2 N0-1 M0) Disease. Gynecol Oncol 1994; 53: 55-8. doi: 10.1006/gyno.1994.1087 24. Green JA, Kirwan JM, Tierney JF, Symonds P, Fresco L, Collingwood M, et al. Survival and recurrence after concomitant chemotherapy and radiotherapy for cancer of the uterine cervix: a systematic review and meta-analysis. Lancet 2001; 358: 781-6. doi: 10.1016/S0140-6736(01)05965-7 25. Eifel P. Prolonged continuous infusion cisplatin and 5-fluorouracil with radia- tion for locally advanced carcinoma of the vulva. Gynecol Oncol 1995; 59: 51-6. doi: 10.1006/gyno.1995.1267 26. Moore DH, Thomas GM, Montana GS, Saxer A, Gallup DG, Olt G. Preoperative chemoradiation for advanced vulvar cancer: a phase II study of the Gynecologic Oncology Group. Int J Radiat Oncol Biol Phys 1998; 42: 79-85. 27. Geisler JP, Manahan KJ, Buller RE. Neoadjuvant chemotherapy in vulvar cancer: avoiding primary exenteration. Gynecol Oncol 2006; 100: 53-7. doi: 10.1016/j.ygyno.2005.06.068 28. Gerszten K, Selvaraj RN, Kelley J, Faul C. Preoperative chemoradiation for lo- cally advanced carcinoma of the vulva. Gynecol Oncol 2005; 99: 640-4. doi: 10.1016/j.ygyno.2005.07.126 29. Koh WJ, ChiuM, Stelzer KJ, Greer BE, Mastras D, Comsia N, Russell KJ GT. Femoral vessel depth and the implications for groin node radiation. Int J Radiat Oncol Biol Phys 1993; 15: 969-74. 30. Chapman B V, Gill BS, Viswanathan AN, Balasubramani GK, Sukumvanich P, Beriwal S. Adjuvant radiation therapy for margin-positive vulvar squamous cell carcinoma: defining the ideal dose-response using the national cancer data base. Int J Radiat Oncol Biol Phys 2016; 97: 1-11. doi: 10.1016/j. ijrobp.2016.09.023 31. Chan JK, Sugiyama V, Pham H, Gu M, Rutgers J, Osann K, et al. Margin dis- tance and other clinico-pathologic prognostic factors in vulvar carcinoma: a multivariate analysis. Gynecol Oncol 2007; 104: 636-41. doi: 10.1016/j. ygyno.2006.10.004 Radiol Oncol 2017; 51(4): 455-462. doi: 10.1515/raon-2017-0040 455 research article Association between SLC19A1 gene polymorphism and high dose methotrexate toxicity in childhood acute lymphoblastic leukaemia and non Hodgkin malignant lymphoma: introducing a haplotype based approach Barbara Faganel Kotnik1, Janez Jazbec1, Petra Bohanec Grabar2, Cristina Rodriguez-Antona3, Vita Dolzan2 1 Department of Oncology and Haematology, University Children’s Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Pharmacogenetics Laboratory, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Hereditary Endocrine Cancer Group Human Cancer Genetics Programme, Spanish National Cancer Research Centre, Madrid, Spain Radiol Oncol 2017; 51(4): 455-462. Received 14 February 2017 Accepted 18 August 2017 Correspondence to: Prof. Vita Dolžan, M.D., Ph.D., Pharmacogenetics Laboratory, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2; SI-1000 Ljubljana, Slovenia. Phone: + 386 1 543 7670; E-mail: vita.dolzan@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. We investigated the clinical relevance of SLC19A1 genetic variability for high dose methotrexate (HD-MTX) related toxicities in children and adolescents with acute lymphoblastic leukaemia (ALL) and non Hodgkin malignant lymphoma (NHML). Patients and methods. Eighty-eight children and adolescents with ALL/NHML were investigated for the influence of SLC19A1 single nucleotide polymorphisms (SNPs) and haplotypes on HD-MTX induced toxicities. Results. Patients with rs2838958 TT genotype had higher probability for mucositis development as compared to car- riers of at least one rs2838958 C allele (OR 0.226 (0.071–0.725), p < 0.009). Haplotype TGTTCCG (H4) statistically signifi- cantly reduced the risk for the occurrence of adverse events during treatment with HD-MTX (OR 0.143 (0.023–0.852), p = 0.030). Conclusions. SLC19A1 SNP and haplotype analysis could provide additional information in a personalized HD-MTX therapy for children with ALL/NHML in order to achieve better treatment outcome. However further studies are needed to validate the results. Key words: acute lymphoblastic leukaemia; genetic polymorphism; haplotype; methotrexate Introduction Inter-individual variability in treatment responses and treatment related toxicities is an important is- sue in clinical practice. It can lead to therapeutic fail- ures or adverse drug events (ADE). Identification and characterization of genetic polymorphisms and haplotypes involved in drug metabolism, transport and mechanism of action would provide important information about individual inherited differences in drug metabolism and treatment re- sponse in order to optimize treatment outcome.1 The solute carrier 19A1 (SLC19A1), trivially re- ferred as reduced folate carrier (RFC), located in a Radiol Oncol 2017; 51(4): 455-462. Faganel Kotnik B et al. / SLC19A1 polymorphisms and methotrexate toxicity in childhood ALL 456 40719 bp region at chromosome 21q22.3, is coding for solute folate carrier (RFC1), a major transporter of folates and antifolates into the cell. The antifolate methotrexate (MTX) is a cytotoxic drug which is used in current treatment regimens for childhood acute lymphoblastic leukaemia (ALL)2, and non- Hodgkin malignant lymphoma (NHML).3,4 The most frequently studied SLC19A1 polymorphism is rs1051266 in the second exon of the SLC19A1, which results in amino acid substitution of argi- nine for histidine (H27R)5 in transmembrane do- main 1 (TMD1), a region implicated in substrate binding and/or translocation6,7, is expected to alter SLC transport properties. Other polymorphisms have been described to date, but the functional consequences of other single nucleotide polymor- phisms (SNPs) are not known yet.8 Several studies so far investigated the associa- tion of rs1051266 with high dose MTX (HD-MTX) treatment outcome and treatment related toxicities in ALL patients.9-13 Some of them reported a pro- tective role of rs1051266 AA genotype14,15, while in others either an increased risk of MTX-induced toxicities and fatal outcomes in rs1051266 A car- riers9,10, or even no association between SLC19A1 genotype and MTX-induced toxicities was report- ed.11,13,16,17 Recently, SNPs in microRNAs (miRNAs) that are involved in regulation of SLC19A1 were investigated. SNP in miR-595 which might affect SLC19A1 regulation and could affect MTX levels in patients with paediatric B-cell ALL was reported.18 To date all studies but one19 evaluated the role of individual SLC19A1 SNPs in the MTX toxicities and outcomes of ALL treatment. The discovery of HapMap (Humane gene database) made it possi- ble to analyse many SNPs at a time. Between the former and the latter a tagSNP approach has been introduced recently. A tagSNP is a representative SNP in a region of the genome with high linkage disequilibrium. Using the tagSNPs reduces num- ber of SNPs needed to be genotyped to capture the common variations across the genome.20 Relatively small numbers of tagSNPs will therefore mark each of the common haplotypes within the gene region.21 In the present study we investigated a possible association between SLC19A1 haplotypes, defined by common functional variants and tagSNPs, and MTX related toxicities in 88 children and adoles- cents with ALL/NHML. The aim was to provide additional insight into improved, cost-effective approach for a personalized HD-MTX therapy for children with ALL/NHML based on haplotype analysis in order to achieve better treatment out- come. Patients and methods Study design The study group included 88 children and ado- lescents (41 male, 47 female) with ALL/NHML diagnosed and treated in the period from 1990 to 2008 at the Department of oncology and haematol- ogy, University Children‘s Hospital in Ljubljana, Slovenia. All the patients were Central European Caucasians and below the age of 18 years at the time of diagnosis. Clinical and demographic data were obtained from the medical records and a question- naire by a single abstractor blinded to genotypes and are presented in Table 1. Each patient enrolled in the study received a high MTX dose (77.3% of patients received 5 g/m2 and 22.7% received 2 g/ m2 of MTX, according to treatment protocols) on 4 separate occasions and concomitant leucovorin rescue therapy to reduce MTX toxicity. Overall, 352 courses of HD-MTX applications were analysed. All patients and/or their parents or legal guard- ians gave their written informed consent to par- ticipate in the study. The study was approved by the Republic of Slovenia National Medical Ethics TABLE 1. Demographic and clinical characteristics of children with ALL/NHML and MTX treatment toxicity Characteristic ALL patients (N = 88) Gender (%) Male Female 41 (46.6) 47 (53.4) Median age at diagnosis, years (range) 4.58 (0.3–16.6) Median body surface area, m2 (range) Leukaemia subtype (%); B-cell T-cell Undetermined Median No. leukocytes at diagnosis, 109 cells/L (range) Median No. thrombocytes at diagnosis, 109 cells/L (range) Median haemoglobin conc., g/l (range) Median % of blasts at diagnosis (range) Median MTX dose, mg (range) 0.73 (0.4–1.9) 72 (81.8) 13 (14.7) 3 (3.4) 9.35 (1–1650) 72 (3–553) 84 (43–148) 23.5 (0–97) 3300 (540–9200) MTX response Present, N (%) Relapse 8 (9.2)** Exitus 4 (4.6)** All MTX-induced ADEs* 48 (54.5) Leukopenia 33 (37.5) Thrombocytopenia 16 (18.2) Mucositis 15 (17.0) Neurotoxicity 7 (8.0)** *data was collected after the first cycle of MTX, **data is missing for one patient Radiol Oncol 2017; 51(4): 455-462. Faganel Kotnik B et al. / SLC19A1 polymorphisms and methotrexate toxicity in childhood ALL 457 Committee and was carried out according to the Helsinki Declaration. Outcome measurement Adverse drug events (ADE) were recorded ret- rospectively from patients’ files throughout the course of the HD-MTX treatment. ADEs were clas- sified as (1) leukopenia, defined as white blood cells count less than 5*109 per litre, (2) thrombo- cytopenia, defined as thrombocyte count less than 140*109 per litre, (3) mucositis, determined and graded according to the WHO oral toxicity scale classification22 or based on physician’s descrip- tions in medical records, need for analgesia and/ or total parenteral nutrition and (4) neurotoxic- ity, determined and graded according to National Cancer Institute Common Toxicity Criteria Version 1 and included patients with seizures (regardless of grade) and patients with grade 3 or 4.23 The data on relapse and exitus were also recorded. All the reported ADEs were used for the evaluation of MTX toxicity. SLC19A1 SNP selection A total of seven SNPs (rs2838951, rs2838956, rs2838958, rs1051266, rs1131596, rs2838451 and rs17004785) in SLC19A1 spanning 40 kbp across the chromosomal 21q22.3 region were selected. SNP selection was based on known function from the lit- erature and/or National Center for Biotechnology Information SNP database (NCBI) and block-tag- ging ability. Among all, two SNPs were a func- tional variant (rs1051266 and rs1131596), while five (rs2838951, rs2838956, rs2838958, rs2838451, rs17004785) were tagging SNPs, selected based on International HapMap Project CEPH data (phase II, March 2008) with the following criteria: r2 > 0.8, and the frequency of minor allele greater than 5%. Tagging SNP selection was done using HaploView software version 4.1 (Supplementary Table 1).24 SLC19A1 genotyping In total 5 ml of peripheral blood was collected into tubes with sodium citrate and stored for short term at -20oC until DNA isolation. Genomic DNA was isolated from peripheral blood leukocytes using a Qiagen FlexiGene kit (Qiagen GmbH, Germany). Genotyping was performed using a fluorescence- based competitive allele-specific (KASPar) assay.25 Real time PCR was performed on the ABI 7900HT in a 4 µL reaction mix containing 0.055 µL of KASPar allele specific PCR assay, 2 µL of KASPar PCR Master Mix (2x concentration, containing KTaq polymerase and ROX), 2.2 mM MgCl2 and 10 ng of DNA. Statistical analysis SNP based analyses Binary logistic regression analysis with the addi- tion of independent variables, such as sex, age at time of diagnosis and body surface area (BSA) was done using SPSS for Windows version 16.0.1 soft- ware (Statistical Package for the Social Sciences, Chicago, IL). The risk for toxicity of MTX treatment was expressed as odds ratio (OR) with 95% confi- dence intervals (CI). The level of significance was set to 0.05. Deviation from Hardy-Weinberg equi- librium (HWE) was calculated for each SNP using Chi-square test. The level of deviation was set to 0.05. Power calculations were done using G*Power software version 3.1.0. Our study had an 80% sta- tistical power to detect medium effect sizes (0.331) with an alpha level of 0.05. The correction for false- positive findings was done using a method for detecting false-positive report probability (FPRP) described by Wacholder et al.26 The cut-off for noteworthy results was set as 0.5. The expected ORs were based on the previous re- ports of an association of SNPs in SLC19A1 with MTX-toxicity.9,10,11,14,15 Prior probability was set to high (π = 0.1) due to well documented association of SLC19A1 and MTX transport.27 Haplotype based analysis SLC19A1 haploblocks and linkage disequilibrium (LD) between the selected SNPs were visualized using HaploView software version 4.1.24 SLC19A1 haplotype construction was performed using Tagger pairwise method. Results Patient characteristics Of the 88 patients recruited, 48 (54.5%) patients suf- fered from MTX-induced adverse events. Specific MTX-induced ADEs after the first cycle of MTX and their frequencies are presented in Table 1. Association between individual SNPs and MTX toxicity The characteristics and the minor allele frequencies (MAF) of the investigated SNPs in SLC19A1 are Radiol Oncol 2017; 51(4): 455-462. Faganel Kotnik B et al. / SLC19A1 polymorphisms and methotrexate toxicity in childhood ALL 458 presented in Supplementary Table 1. In one patient genotyping failed for all SNPs, while genotyping for rs2838951 and rs17004785 failed in two patients. The observed frequencies of the studied SNPs in ALL/NHML patients did not deviate from HWE (data not shown). With regard to specific ADEs, univariate logistic regression analysis revealed only the association of rs2838958 with the occurrence of mucositis devel- opment (p = 0.009). We observed that patients with rs2838958 TT genotype had higher probability for mucositis development as compared to carriers of at least one polymorphic rs2838958 C allele (OR 0.226 (0.071–0.725), p < 0.009) (Table 2). We then investigated the possible association be- tween SLC19A1 polymorphisms and MTX-induced ADEs with the multivariate analysis using an ad- ditive model (per allele), but a statistically signifi- cant relationship could be established neither for overall nor for specific ADEs (Table 3). A margin- ally significant impact on the risk of mucositis was observed for rs2838951 (OR 2.160 (0.909–5.129), p = 0.081). We also performed haplotype analysis to ascer- tain whether the combination of polymorphisms that define a specific haplotype impacts the occur- rence of ADEs. As shown in Figure 1 the investi- gated polymorphisms were lying in the same hap- loblock. The frequencies of SLC19A1 haplotypes in pa- tients with ALL and NHML that were treated with HD-MTX are shown in Table 4. The Haplotype TGTTCCC (H2), which has wild type alleles pre- sent on all loci, was selected as the reference hap- lotype for the analysis. By frequency, it is the sec- ond most common haplotype in the patient test group with a frequency of 33.4%. The haplotype CACCCCG (H1) had polymorphic alleles present on the first four and on the last locus, with wild type alleles present on the fifth and sixth loci. The TABLE 2. The influence of rs2838958 genotype on mucositis development - univariate logistic regression analysis Degree of mucositis rs2838958 genotype OR (95% CI) P TT (%) TC + CC (%) 0 18 (66.7) 53 (89.8) reference 1 4 (14.8) 4 (6.8) 0.340 (0.077–1.500) 0.154 2 4 (14.8) 2 (3.4) 0.170 (0.029–1.007) 0.051 4 1 (3.7) 0 (0.0) - * 1–4 9 (33.3) 6 (10.2) 0.226 (0.071–0.725) 0.009 Mean value ± STD 0.59 0.14 0.007 * Calculation was not possible TABLE 3. The impact of SLC19A1 genotypes on the occurrence of any and specific MTX-induced adverse events using multivariate analysis – an additive model SNP ID Any adverse events Leukopenia Thrombocytopenia Mucositis Neurotoxicity OR (CI 95%) P OR (CI 95%) P OR (CI 95%) P OR (CI 95%) P OR (CI 95%) P rs1131596 0.719 (0.354–1.458) 0.360 0.609 (0.310–1.197) 0.150 1.170 (0.532–2.576) 0.696 1.292 (0.525–3.170) 0.579 0.542 (0.146–2.016) 0.361 rs1051266 1.392 (0.686–2.824) 0.360 1.643 (0.836–3.230) 0.150 0.855 (0.388–1.881) 0.696 0.775 (0.316–1.906) 0.579 0.542 (0.146–2.016) 0.361 rs2838958 1.221 (0.599–2.490) 0.583 1.684 (0.849–3.339) 0.136 0.556 (0.240–1.291) 0.172 0.476 (0.182–1.243) 0.130 0.453 (0.122–1.677) 0.236 rs2838956 1.411 (0.686–2.899) 0.349 1.742 (0.878–3.458) 0.113 0.689 (0.303–1.566) 0.374 0.705 (0.283–1.759) 0.454 0.596 (0.163–2.182) 0.434 rs17004785 0.849 (0.280–2.572) 0.772 1.393 (0.476–4.079) 0.545 0.854 (0.218–3.349) 0.821 0.672 (0.148–3.055) 0.606 1.226 (0.255–5.899) 0.800 rs12483377 1.475 (0.504–4.318) 0.478 1.825 (0.672–4.956) 0.238 1.688 (0.540–5.280) 0.368 0.930 (0.249–3.473) 0.914 1.061 (0.232–4.861) 0.939 rs2838951 1.261 (0.666–2.412) 0.483 0.740 (0.392–1.394) 0.351 1.652 (0.786–3.473) 0.185 2.160 (0.909–5.129) 0.081 1.693 (0.577–4.967) 0.337 The estimates for the odds ratio (OR) were adjusted to the following variables: age, sex, and treatment regimen. Radiol Oncol 2017; 51(4): 455-462. Faganel Kotnik B et al. / SLC19A1 polymorphisms and methotrexate toxicity in childhood ALL 459 haplotype TGTTGTC (H3) had wild type alleles present on the first four and on the last locus, with polymorphic alleles present on the fifth and sixth. The haplotype TGTTCCG (H4) had wild type al- leles present at all loci, except for the last one, while haplotype CATTCCC (H5) had polymorphic al- leles present on the first, second, fifth and sixth lo- cus, with wild type alleles on the remaining three. H1 was the most frequent haplotype in the patient test group with a frequency of 39.6%. Together, H1, H2, and H3 haplotypes accounted for 82.4% of all haplotypes. In patients with ALL and ML, the haplotypes H4 and H5 were more rarely represented, with the frequency of a single haplotype between 1 to 5%. Therefore haplotype H5 and the other very rare haplotypes have been grouped together as ‘rare’ for further statistical analysis. We examined whether there is a link between the SLC19A1 haplotypes and ADEs in patients treated with HD-MTX. We included the age and sex of the patient, and the treatment regimen as co-variables in the model. We found a statistical- ly significant association of haplotype H4 in the SLC19A1 gene and the treatment regimen on the occurrence of any possible MTX-induced ADEs. Patients who were treated with ALL-BFM 95 were at an increased risk for the occurrence of any MTX- induced ADEs (OR 2.050 (1.065–3.943), p = 0.031), while those with haplotype H4 had a reduced risk for the occurrence of any possible ADE after treat- ment with HD-MTX (OR 0.143 (0.023–0.852), p = 0.030) (Table 5). The impact of the SLC19A1 hap- lotypes on specific MTX-induced ADEs was not observed, probably due to the small number of in- dividual toxic events (Table 5). Discussion In the present study we have used haplotype based approach to investigate the association of genetic variability in SLC19A1 gene with the high-dose MTX-induced ADEs in paediatric ALL and NHML patients. SLC19A1 is one of the main influx trans- porters for folates and antifolates, however the re- ports on the impact on genetic variability on the MTX treatment outcome are conflicting. Protective role of rs1051266 AA genotype14,15, as well as in- creased risk for MTX-induced ADEs10 or no asso- ciation of rs1051266 was reported.11,13,16 Among the so far published studies on the role of SLC19A1 in the treatment of ALL with MTX all studies but one19 have only investigated the most common functional SNP rs1051266 (SLC19A1 G80A), which may alter the transport characteris- tics of SLC19A1.28 By using a haplotype based ap- proach we were able to investigate the impact of genetic variability of SNPs that are in linkage dise- quilibrium within the broader region within of the SLC19A1 on the occurrence of MTX-induced ADEs and disease outcome and effectiveness of treat- ment with HD-MTX in children and adolescents with ALL/NHML. Among the investigated SLC19A1 polymor- phisms univariate logistic regression analysis showed that the rs2838958 TT genotype increases the risk of developing mucositis. This association remained borderline significant in the multivari- ate analysis using the additive model. The other SLC19A1 polymorphisms were not associated with the occurrence of MTX-induced ADEs. In the haplotype based analysis we found that H4 haplotype (TGTTCCG), which, excluding the last locus (rs2838951), has wild type alleles present, FIGURE 1. Graphic presentation of haploblocks and selected SNPs in SLC19A1. TABLE 4. SLC19A1 haplotype frequencies in patients with ALL and NHML Haplotype* Frequency Standard error H1 = CACCCCG 0.396 0.037 H2 = TGTTCCC 0.334 0.036 H3 = TGTTGTC 0.094 0.022 H4 = TGTTCCG 0.053 0.017 H5 = CATTCCC 0.017 0.010 Other 0.106 * SNP rs1131596, rs1051266, rs2838958, rs2838956, rs17004785, rs12483377, rs2838951 from 5’ to 3’, respectively Radiol Oncol 2017; 51(4): 455-462. Faganel Kotnik B et al. / SLC19A1 polymorphisms and methotrexate toxicity in childhood ALL 460 statistically significantly reduces the risk for the occurrence of ADEs during treatment with HD- MTX. Since the frequency is low in our sample it is difficult to ascertain its clinical relevance given the number of patients and the small number of in- dividual toxic events. Haplotype based approaches have been rarely used when investigating HD-MTX induced ad- verse events in ALL and NHML patients. One study investigated the influence of MTHFR SNPs and haplotypes on treatment response29 and HD- MTX toxicity30,31, while the other studies focused on DNA repair and cell cycle processes on risk for leukaemia development using haplotype based ap- proach.32-36 Only study of Lopez-Lopez focused on MTX transporters. This study examined 384 SNPs in 12 transporter genes, including SLC19A1, and their relationship to MTX plasma levels and MTX- related toxicities.19 They found no association be- tween SLC19A1 haplotypes and MTX plasma lev- els. However they confound the results of Treviño and colleagues on association between SLCO1B1 rs11045879 polymorphism and toxicity and ob- served for the first time a significant association with MTX plasma levels and rs9516519 in ABCC4, rs3740065 and haplotype GCGGG in ABCC2.19 However, haplotype based approach has been used in analysing the influence of the polymor- phisms in the broader region of SLC19A1 on the occurrence of low dose MTX-induced ADEs in rheumatoid arthritis (RA) patients in order to fa- cilitate the design of personalized low dose MTX treatment.37,38 Bohanec Grabar et al. reported that individual SNP and haplotype analysis suggest that rs1051266 could be a functional variant alter- ing MTX toxicity; rs1051266 and rs1131956 were significantly associated with protection against discontinuation of treatment owing to MTX tox- icity. Rs2838956 was significantly associated with protection against skin ADEs, while two com- mon SLC19A1 haplotypes carrying rs1051266 and rs1131596 minor alleles had a protective effect to- wards MTX induced ADEs. Significant association was also found for rs1051266 and rs1131596 with infection.37 Lima et al. reported that SLC19A1 and genotypes and haplotypes may help to identify RA patients with increased risk of MTX-related gas- trointestinal toxicity since protective effect of car- riers of wild type allele of rs1051266 and rs2838956 regarding MTX induced gastrointestinal toxicity as well as association between GGAG haplotype for SLC19A1 rs7499, rs1051266, rs2838956 and rs3788200 with MTX gastrointestinal toxicity was established.38 The results of the haplotype based analysis indi- cate, that other polymorphisms that are in linkage disequilibrium with rs1051266 and rs1131596, may impact the functioning of SLC19A1 via, for exam- ple, alterations in SLC19A1 splicing.39,40 As well, other genes coding for proteins that are involved in the transport and metabolism of MTX can also impact pharmacokinetics and the occurrence of MTX-induced AE, such as SLCO1B1 SNPs that are associated with lower clearance of MTX and SLCO1A2 as well.41 Furthermore, SNP in miR-595 TABLE 5. The impact of gender, age, treatment regimen and SLC19A1 haplotypes on the occurrence of any and specific (thrombocytopenia, mucositis, and neurotoxicity) MTX-induced adverse events All Thrombocytopenia Mucositis Neurotoxicity OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P Gender 2.385 (0.908–6.259) 0.078 1.615 (5.134–5.076) 0.413 1.523 (0.423–5.472) 0.520 0.531 (0.093–3.024) 0.475 Age 1.028(0.931–1.134) 0.588 9.526 (8.422–1.078) 0.440 1.054 (0.935–1.190) 0.396 1.088 (0.931–1.272) 0.289 Treatment regimen 2.050 (1.065–3.943) 0.031* 9.839 (4.620–2.095) 0.962 1.676 (0.689–4.075) 0.255 1.282 (0.397–4.139) 0.678 H1 = CACCCCG 1.000 (0.472–2.117) 0.943 6.615 (2.769–1.579) 0.352 0.419 (0.150–1.167) 0.096 0.309 (0.070–1.364) 0.120 H3 = TGTTGTC 1.110 (0.335–3.618) 0.844 9.220 (2.254–3.750) 0.891 0.593 (0.122–2.877) 0.515 0.951 (0.175–5.150) 0.934 H4 = TGTTCCC 0.143 (0.023–0.852) 0.030* 3.617 (4.568–2.541) 0.379 0.005 (0.0–NC) 0.985 0.014 (0.0–NC) 0.992 Rare haplotypes 2.002 (0.580–6.775) 0.275 9.113 (2.607–3.171) 0.871 2.002 (0.580–6.775) 0.275 2.002 (0.580–6.775) 0.275 *p < 0.05; H2 was the reference haplotype Radiol Oncol 2017; 51(4): 455-462. Faganel Kotnik B et al. / SLC19A1 polymorphisms and methotrexate toxicity in childhood ALL 461 which might affect SLC19A1 regulation and could affect MTX levels in patients with paediatric B-cell ALL was recently reported.18 The results of our study show that SLC19A1 gen- otyping could be one of the factors that could con- tribute to safer and more effective treatment with HD-MTX, especially for identifying patient groups at risk of mucositis development in Slovenian pa- tients. As the occurrence of ADEs and the treatment outcome could be affected by other genetic factors involved in the transport and metabolism of MTX as well as by concomitant supportive therapy and the current pathophysiological condition of the pa- tient the results of our study should be confirmed in a larger number of patients. The main limitation of our study, which is prob- ably also one of the reasons for the inconsistent re- sults among studies published so far, is the limited sample size. Since ALL is a rare disease in children, the number of patients that could be as comparable as possible in the entire treatment process is lim- ited. We therefore also included five patients with NHML in the study, who were treated according to the same protocol as patients with ALL. The great advantage of our study is that all sub- jects come from a small and very homogeneous population42, that the patient group is clinically well defined and very homogeneous given the type and course of treatment, centralized treat- ment of patients with an established treatment and supportive regimen. The group was treated by the same medical team in a single centre according to standard criteria. For genotyping, we used a method that would be suitable for genetic testing in clinical practice, as it is easy to perform, fast, affordable, and enables simultaneous analysis of multiple samples and dif- ferent polymorphisms if they are run under the same conditions, and also require small amounts of DNA for analysis. Employing a haplotype based approach we tried to examine the impact of genetic variability in the entire region of the studied gene and also included the possible functional polymorphisms that are in linkage disequilibrium with the selected SNPs. With this approach an even stronger influence of genetic variability of SLC19A1 with MTX-induced ADEs could be shown as opposed to taking into account only individual SNPs. 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XRCC1 polymorphisms and hap- lotypes in Mexican patients with acute lymphoblastic leukemia. Genet Mol Res 2009; 8: 1451-8. doi: 10.4238/vol8-4gmr687 33. Chokkalingam AP, Bartley K, Wiemels JL, Metayer C, Barcellos LF, Hansen HM, et al. Haplotypes of DNA repair and cell cycle control genes, X-ray ex- posure, and risk of childhood acute lymphoblastic leukemia. Cancer Causes Control 2011; 22: 1721-30. doi: 10.1007/s10552-011-9848-y 34. Mosor M, Ziółkowska I, Januszkiewicz-Lewandowska D, Nowak J. Polymorphisms and haplotypes of the NBS1 gene in childhood acute leu- kaemia. Eur J Cancer 2008; 44: 2226-32. doi: 10.1016/j.ejca.2008.06.026 35. Semsei AF, Erdélyi DJ, Ungvári I, Kámory E, Csókay B, Andrikovics H, et al. Association of some rare haplotypes and genotype combinations in the MDR1 gene with childhood acute lymphoblastic leukaemia. Leuk Res 2008; 32: 1214-20. doi: 10.1016/j.leukres.2007.12.009 36. Pakakasama S, Sirirat T, Kanchanachumpol S, Udomsubpayakul U, Mahasirimongkol S, Kitpoka P, et al. Genetic polymorphisms and haplotypes of DNA repair genes in childhood acute lymphoblastic leukaemia. Pediatr Blood Cancer 2007; 48: 16-20. doi: 10.1002/pbc.20742 37. Bohanec Grabar P, Leandro-García LJ, Inglada-Pérez L, Logar D, Rodríguez- Antona C, Dolžan V. Genetic variation in the SLC19A1 gene and methotrex- ate toxicity in rheumatoid arthritis patients. Pharmacogenomics 2012; 13: 1583-94. doi: 10.2217/pgs.12.150 38. Lima A, Bernardes M, Azevedo R, Monteiro J, Sousa H, Medeiros R, Seabra V. SLC19A1, SLC46A1 and SLCO1B1 polymorphisms as predictors of metho- trexate-related toxicity in Portuguese rheumatoid arthritis patients. Toxicol Sci 2014; 142: 196-209. doi: 10.1093/toxsci/kfu162 39. Williams FM, Flintoff WF. Structural organization of the human reduced folate carrier gene: evidence for 5’ heterogeneity in lymphoblast mRNA. Somat Cell Mol Genet 1998; 24: 143-56. 40. 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Radiol Oncol 2017; 51(4): 463-468. doi: 10.1515/raon-2017-0041 463 research article Impact on radiation dose and volume V57 Gy of the brain on recurrence and survival of patients with glioblastoma multiformae Igor Stojkovski, Valentina Krstevska, Snezhana Smichkoska University Clinic of Radiotherapy and Oncology, Skopje, Macedonia Radiol Oncol 2017; 51(4): 463-468. Received 11 June 2017 Accepted 11 September 2017 Correspondence to: Igor Stojkovski, University Clinic of Radiotherapy and Oncology – Skopje, Majka Tereza (Vodnjanska) 17, 1000 Skopje, Macedonia. Phone: +389 7 639 7454; Fax: +389 2 311 1430; E-mail: istojkovski@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to analyze impact of irradiated brain volume V57 Gy (volume receiving 57 Gy and more) on time to progression and survival of patients with glioblastoma. Patients and methods. Dosimetric analysis of treatment plan data has been performed on 70 patients with glio- blastoma, treated with postoperative radiochemotherapy with temozolomide, followed by adjuvant temozolomide. Patients were treated with 2 different methods of definition of treatment volumes and prescription of radiation dose. First group of patients has been treated with one treatment volume receiving 60 Gy in 2 Gy daily fraction (31 patients) and second group of the patients has been treated with “cone-down” technique, which consisted of two phases of treatment: the first phase of 46 Gy in 2 Gy fraction followed by “cone-down” boost of 14 Gy in 2 Gy fraction (39 pa- tients). Quantification of V57 Gy and ratio brain volume/V57Gy has been done. Average values of both parameters have been taken as a threshold value and patients have been split into 2 groups for each parameter (values smaller/ lager than threshold value). Results. Mean value for V57 Gy was 593.39 cm3 (range 166.94 to 968.60 cm3), mean value of brain volume has was 1332.86 cm3 (range 1047.00 to 1671.90 cm3) and mean value of brain-to-V57Gy ratio was 2.46 (range 1.42 to 7.67). There was no significant difference between two groups for both V57 Gy and ratio between brain volume and V57 Gy. Conclusions. Irradiated volume with dose 57 Gy or more (V57 Gy) and ration between whole brain volume and 57 Gy had no impact on time to progression and survival of patients with glioblastoma. Key words: irradiation V57; glioblastoma Introduction Glioblastoma is most common and most aggres- sive brain tumor with incidence of 2-3 per 100 000 population according to GLOBOCAN.1 Incidence of glioblastoma accounts 12-15% of all intracra- nial tumors and approximately 50-60 of all astro- cytic tumors.2,3 Diagnosis, treatment and follow up of patients with glioblastoma and multidisci- plinary approach and best results are achieved in specialized centers, which can offer all treatment modalities when it is needed and which are more experienced with larger volume of cases.4 Mutual understanding and collaboration of team of profes- sionals is of paramount importance for obtaining best medical care and best clinical results (tumor control and survival). During last two decades, major advantages have been made in enhancing precision of radiation treatment and shaping of ra- diation dose to increase dose distribution in target and to decrease radiation dose in organs at risk. Three-dimensional conformal radiotherapy and its derivates, Intensity Modulated Radiation Therapy (IMRT) and Volumetric Arc Therapy (VMAT) are Radiol Oncol 2017; 51(4): 463-468. Stojkovski I et al. / Impact of irradiated brain volume V57 Gy464 now standard of treatment for patients with glio- blastoma.5-7 Standard postoperative treatment of patients with glioblastoma consist of postopera- tive radiotherapy with temozolomide followed by adjuvant temozolomide.8-11 Radiotherapy is corner-stone of multimodality approach and it is considered as treatment with highest benefit of all three treatment modalities. Despite the major ad- vantages in personalization and precision of the radiotherapy treatment, median survival of pa- tients with glioblastoma is still between 12 and 16 months from diagnosis.4 In general there are two major approaches in definition of gross tumor volume (GTV) in pa- tients with glioblastoma. In studies conducted by EORTC (European Organization for Research and Treatment of Cancer) only one contoured gross tumor volume is used which is defined as an en- hanced visible tumor on MR images prior the sur- gery expanded respectively to clinical target vol- ume (CTV) and planning target volume (PTV) ac- cording ESTRO-ACROP Guidelines.7 In contrast in studies conducted by RTOG (Radiation Therapy Oncology Group) definition of volumes in according to “cone-down” approach, which means that there are virtually two volumes defined on preoperative and/or postoperative MR, one initial (larger) volume and second “cone- down” volume or boost volume (smaller). With “cone-down” approach in some clinical situation it is possible do decrease radiation dose to the brain which could have impact on survivorship of pa- tients with glioblastoma.12,13 Volume of the tumor measured as initial tumor size or preoperative tumor size and residual dis- ease is generally considered as a prognostic factor for survival and recurrence in patients with glio- blastoma.14,15 There are various approaches and quantification of what is really visible tumor and consequently, what volume should be irradiated in order to minimize tumor recurrence, but reach- ing consensus between different research groups is still under debate. Definition of volume of the tumor, depends of imaging modality used, resolu- tion of imaging modality, processing algorithms and various other variables.15-18 After definition of gross tumor volume there is still debate, what is most appropriate clinical treat- ment volume (CTV). There are different approach- es, which are evolving together with advances of imaging. CTV as concept in glioblastoma is diffi- cult to define and different research groups have various definitions and no-one of these definitions is absolutely true or false.20-28 There are more or less well defined criteria for definition of CTV and PTV for patients with glio- blastoma specially treated in clinical trial setting, as AVAGlio and Centric trials, recently.28,29 Also it is well known fact that in daily clinical practice clinicians are adopting volume delineation according to their clinical setting and capabilities, and using delineation according to RTOG, EORTC or institutional standards.30 In our study we treated patients with two dif- ferent approaches on delineation on treatment volumes using one phase treatment EORTC “like” and RTOG “like” approach, but randomization was used for assignment of patients in the group as a part of the standard protocol for treatment devel- oped in the institution. Patients and methods This study was approved by the ethical commit- tee of Medical Faculty at University “Ss. Cyril and Methodius” in Skopje and University Clinic of Radiotherapy and Oncology in Skopje (Number: 03-2455/2) and was carried out according to the Declaration of Helsinki. Total of 70 patients, with glioblastoma mul- tiforme has been included in this study. Patient accrual has been performed in the period from January 2013 to December 2015. All patients have been previously surgically treated with maximal safe resection of the primary tumor and defini- tive histological diagnosis has been established as a glioblastoma multiforme according to the last World Health Organization classification.3 After referral to radiotherapy treatment, pa- tients have been scheduled for computed tomog- raphy (CT) simulation in treatment position. CT scan has encompassed cranial region according to TABLE 1. Patient characteristics Group 1 (1 v) 31 patients Group 2 (cone-down) 39 patients Age Range 29–71, median age 56 Range 27–75, median age 56 Sex Male - 14 Female - 17 Male - 21 Female - 18 Location of the primary lesion 5 - frontal lobe 6 - temporal lobe 11 - parietal lobe 2 - occipital lobe 7 - overlapping 8 - frontal lobe 15 - temporal lobe 9 - parietal lobe 1 - occipital lobe 6 - overlapping Average time from surgery to RT (days) 36 (25–55) 39 (24–74) V57 Gy < 600 cm 3- 15 > 600 cm3 - 16 < 600 cm3 - 23 > 600 cm3 - 16 Radiol Oncol 2017; 51(4): 463-468. Stojkovski I et al. / Impact of irradiated brain volume V57 Gy 465 institutional protocol with slice thickness of 2 mm. For immobilization purpose thermoplastics masks and head rests have been used during simulation and treatment. After computed tomography simulation, image fusion with preoperative and/or postoperative MR scan has been done using automatic MR-CT fu- sion using non deformable algorithm with manual correction, only if necessary, leaded by decision of radiation oncologist. MR-CT fusion has been done using transversal MR images using T1 with con- trast and T2/FLAIR sequence. In the analysis has been included only patents who finished complete treatment, total of 70 patients from included 78 pa- tients. Eight patients did not finish treatment, and were excluded from analysis. Patients have been randomly assigned to one of the groups on the basis of referral to the depart- ment. Patients with odd hospital number have been assigned to the first group, and patients with even hospital number have been assigned to sec- ond group. In the first group of the patients (total of 31 patients), delineation was based on T2/FLAIR and T1 with contrast enhancements and only one GTV volume has been contoured. After delineation of GTV, CTV was expanded for 2 cm, taking in ac- count anatomic boundaries and omitting, if possi- ble, organs at risk. CTV was expanded to PTV with addition of margin of 5 mm, which is considered institutional standard. In the second group (total of 39 patients), “cone-down” approach has been used and delineation of target volume has been done in two phases and two GTV volumes have been de- lineated. First or initial volume GTV46, has been delineated based on MR images using T2/FLAIR abnormalities. Expansion of GTV to CTV has been done with margin of 2 cm, taking in account ana- tomic boundaries and avoidance of organs at risk, similar as in first group. Further expansion of CTV to PTV was with margin of 5 mm. Cone down vol- ume or boost volume has been delineated on con- trasts enhanced T1 MR Image set. CTV has been expanded for 2 cm, and PTV expanded further for 0.5 cm, as initial delineated volume. Dose prescription for patients in the first group was 60 Gy in 30 daily fractions of 2 Gy and in sec- ond group, prescribed dose for initial volume was 46 Gy in 23 fractions and for the “cone-down” volume additional 14 Gy in 7 fractions. Initial and “cone-down” volumes have been treated with 2 Gy daily fractions. Treatment schedules for both groups were 5 fractions on each consecutive day in 7 days week. Treatment planning has been done using Varian Eclipse planning system version 10.0.45.0 and the most appropriate treatment plan have been select- ed in order to achieve dose distribution in target(s) and in organs at risk in order to fulfill QUANTEC criteria.31-33 Together with radiotherapy all patients were treated according to “Stupp protocol” and received concurrent chemotherapy with temozolomide, fol- lowed by adjuvant temozolomide.35 After treatment, patients undergo follow up which consisted of physical examination every month which corresponded with adjuvant chemo- therapy cycle, MR every 3-4 months and other clin- ical examination if necessary. Follow up strategy was in line with ESMO clinical recommendations and modified according to specific clinical situa- tion.9 Two volumetric parameters were selected as rel- evant in order to predict exposure to brain as an or- gan at risk in our series of patients. First parameter is volume which received 57 Gy and more in cm3, “V57Gy”, and second parameter is calculated as a ratio between brain volume and “V57Gy”, calcu- lated as numeric coefficient, decimal number. “V57Gy” was calculated using TPS software and using build in algorithms for converting isodose level to structure. This function is standard in ma- jority of modern treatment planning systems avail- able in the market and it is available as a standard option in our institutional TPS. Patients has been separated in 2 groups for both parameters and threshold values were estimated for “V57Gy” equal to 600 cm3 and for ratio between brain volume and “V57Gy” equal to 2.4 presented as a decimal number. Based on the first parameter, patents were split on 2 groups. The first group of patients with “V57Gy” up to 600 cm3 consisted of 38 patients, and second group of patients with “V57Gy” more than 600 cm3 consisted of 32 patients. According to second parameter, ratio between brain volume and “V57Gy”, patients were also split into 2 groups: patients with ratio less than 2.4 numeric value (40 patients) and patients with ratio of more than 2.4 (30 patients). Results Median follow up of all 70 patients was 12 months (range from 4 to 33 months). Median time to pro- gression (recurrence) was 12 months and median Radiol Oncol 2017; 51(4): 463-468. Stojkovski I et al. / Impact of irradiated brain volume V57 Gy466 survival was calculated as 24 months using Kaplan Meir method.35 Survival analysis using Kaplan Meir method has been done on two parameters for both time to progression (recurrence) and overall survival. Comparison of survival has been calculated us- ing Matel-Cox and Gehan-Breslow-Wilcoxon (log- rank) tests.36,37 For volumetric parameter of “V57Gy”, compari- son of patients with “V57 Gy” of less than 600 cm3 (38 patients) and more than 600 cm3 (32 patients) has been done. Time to progression for group of patients with “V57Gy” ≤ 600 cm3 was 11.43 months and for group of patients with “V57Gy” > 600 cm3 was 13.29 and for overall survival, 14.64 and 13.29 months. (Figure 1, 2). There was not any significant difference for both time to progression (p = 0.2065) and overall survival (p = 0.9970). For second parameter, calculated as numeric value, ration between whole brain volume and “V57Gy” volume, comparison for time to progres- sion and overall survival for patients with numeri- cal value ≤ 2.4 (40 patients) and numeric value > 2.4 (30 patients) has been done. Median time to pro- gression for group of patients with value ≤ 2.4 was 11.43 months and for group of patients with nu- meric value > 2.4 was 12.18 months. Overall surviv- al was 11.68 and 14.64 months respectively. There was no significant difference in time to progression (p = 0.2881) and overall survival (p = 0.8572) be- tween this two groups (Figure 3, 4). Discussion Based on this data we concluded that these volu- metric parameters did not have any impact on time to progression and overall survival on patients with glioblastoma, treated with postoperative ra- diochemotherapy. In general, in malignant tumors, size of the tumor is considered as an independent prognostic factor which is described as a T stage according to UICC Classification38, but due to specific characteristics of brain tumors, TNM clas- sification for prognostic values is not applicable, but rather WHO classification which do not corre- spond to size of the tumor. According to EORTC and NCIC nomogram for predicting outcome in patients with newly diagnosed glioblastoma, there are several factors which are predicting survival. Following parameters are suggested as potential prognostic factors, which should be reported in all clinical studies: MGMT promoter methylation status, age, performance status, extent of resection, and Mini Mental State Examination (MMSE).39 Volumetric parameters calculated in our study did not have impact on local control and overall survival. In our study threshold value was esti- mated as an average value from our series of the patients. In the future studies we are planning to include more patients for evaluation of volumetric parameters and to create more strict constraints with higher gradient. In this case we had approxi- mation that there will be difference in both recur- rence and survival for patients with smaller irradi- ated volumes compared with very large irradiated volume, which should be proven in future studies. Radiation treatment of CNS tumors has been evolved in the past two decades with introduction of more precise imaging and treatment devices in radiation oncology followed by development of more precise treatment techniques. Despite the fact that modern treatment devices are able to deliver higher dose to specified tumor volume, using pos- sibility to conform beams in order to protect criti- cal organs there, are not positive studies to prove that escalation of radiation dose beyond 60 Gy with standard fractionation will have impact of the dis- ease control.40 There are some exceptions regard- ing dose and fractionation for patients with poor performance status. Recent studies showed that FIGURE 1. Time to progression for “V57Gy” volume. Threshold value was 600 cm3. FIGURE 2. Overall survival for “V57Gy” volume. Threshold value was 600 cm3. FIGURE 3. Time to progression for ration between brain volume and “V57Gy” volume. Threshold value was 2.4. FIGURE 4. Overall survival for ration between brain volume and “V57Gy” volume. Threshold value was 2.4. Radiol Oncol 2017; 51(4): 463-468. Stojkovski I et al. / Impact of irradiated brain volume V57 Gy 467 shortening duration of radiotherapy treatment with increasing daily fraction (40 Gy in 15 fractions or 25 Gy in 5 fractions) is with equivalent results re- garding survival and quality of life.42 In our study we showed that decreasing of treated volume with cone-down approach did not have any impact on marginal recurrence in glioblastoma patients treated with radiotherapy and concurrent and ad- juvant temozolomide. 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Cilengitide combined with standard treatment for patients with newly diagnosed glio- blastoma with methylated MGMT promoter (CENTRIC EORTC 26071-22072 study): a multicentre, randomised, open-label, phase 3 trial. Lancet Oncol 2014; 15: 1100-8. doi: 10.1016/S1470-2045(14)70379-1 30. Ghose A, Lim G, Husain S. Treatment for glioblastoma multiforme: current guidelines and Canadian practice. Curr Oncol 2010; 17: 52-8. doi: 10.3747/ co.v17i6.574 31. Lawrence YR, Li XA, el Naqa I, Hahn CA, Lawrence BM, Merchant TE, et al. QUANTEC: Radiation dse - volume effects in the brain. Int J Radiat Oncol 2010; 76: S77-85. doi: 10.1016/j.ijrobp.2009.02.091 32. Mayo C, Yorke E, Merchant TE. Radiation associated brainstem injury. Int J Radiat Oncol Biol Phys 2010; 76(3 Suppl): 36-41. doi: 10.1016/j. ijrobp.2009.08.078 33. Mayo C, Martel MK, Marks LB, Flickinger J, Nam J, Kirkpatrick J. Radiation dose-volume effects of optic nerves and chiasm. 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Sobin LH, Gospodarowicz MK, Wittekind C, editors. 7th Edition. Oxford: Wiley-Blackwell; 2011. 39. Gorlia T, van den Bent MJ, Hegi ME, Mirimanoff RO, Weller M, Cairncross JG, et al. Nomograms for predicting survival of patients with newly diagnosed glioblastoma: prognostic factor analysis of EORTC and NCIC trial 26981- 22981/CE.3. Lancet Oncol 2008; 9: 29-38. doi: http://dx.doi.org/10.1016/ S1470-2045(07)70384-4 40. Khan L, Soliman H, Sahgal A, Perry J, Xu W, Tsao MN. External beam ra- diation dose escalation for high grade glioma. Cochrane Database Syst Rev 2016; 8: CD011475. doi: 10.1002/14651858.CD011475.pub2 41. Roa W, Kepka L, Kumar N, Sinaika V, Matiello J, Lomidze D, et al. International Atomic Energy Agency randomized phase III study of radiation therapy in el- derly and/or frail patients with newly diagnosed glioblastoma multiforme. J Clin Oncol 2015; 33: 4145-50. doi: 10.1200/JCO.2015.62.6606 42. McDonald MW, Shu H-KG, Curran WJ, Crocker IR. Pattern of failure after lim- ited margin radiotherapy and temozolomide for glioblastoma. Int J Radiat Oncol 2011; 79: 130-6. doi: 10.1016/j.ijrobp.2009.10.048 43. Minniti G, Amelio D, Amichetti M, Salvati M, Muni R, Bozzao A, et al. Patterns of failure and comparison of different target volume delineations in patients with glioblastoma treated with conformal radiotherapy plus concomitant and adjuvant temozolomide. Radiother Oncol 2010; 97: 377- 81. doi: 10.1016/j.radonc.2010.08.020 44. Dobelbower MC, Burnett III OL, Nordal RA, Nabors LB, Markert JM, Hyatt MD, et al. Patterns of failure for glioblastoma multiforme following concur- rent radiation and temozolomide. J Med Imaging Radiat Oncol 2011; 55: 77-81. doi: 10.1111/j.1754-9485.2010.02232.x 45. Gebhardt BJ, Dobelbower MC, Ennis WH, Bag AK, Markert JM, Fiveash JB. Patterns of failure for glioblastoma multiforme following limited-margin radiation and concurrent temozolomide. Radiat Oncol 2014; 9: 130. doi: 10.1186/1748-717X-9-130 Radiol Oncol 2017; 51(4): 469-474. doi: 10.1515/raon-2017-0050 469 research article Quality assurance procedures based on dosimetric, gamma analysis as a fast reliable tool for commissioning brachytherapy treatment planning systems Grzegorz Zwierzchowski1,2, Grzegorz Bieleda1,2, Janusz Skowronek1,3 1 Poznan University of Medical Sciences, Faculty of Health Sciences, Poznana, Poland 2 Greater Poland Cancer Centre, Medical Physics Department, Poznan, Poland 3 Greater Poland Cancer Centre, Brachytherapy Department, Poznan, Poland Radiol Oncol 2017; 51(4): 469-474. Received 25 November 2016 Accepted 10 October 2017 Correspondence to: Dr. Grzegorz Zwierzchowski, Greater Poland Cancer Centre, Medical Physics Department / Brachytherapy Department, Garbary 15 Str, PL 61-855 Poznan, Poland. E-mail: grzegorz.zwierzchowski@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Fast and easily repeatable methods for commissioning procedures for brachytherapy (BT) treatment planning systems (TPS) are needed. Radiochromic film dosimetry with gamma analysis is widely used in external beam quality assurance (QA) procedures and planar film dosimetry is also increasingly used for verification of the dose dis- tribution in BT applications. Using the gamma analysis method for comparing calculated and measured dose data could be used for commissioning procedures of the newly developed TG-186 and MBDCA calculation algorithms. The aim of this study was dosimetric verification of the calculation algorithm used in TPS when the CT/MRI ring applicator is used. Materials and methods. Ring applicators with 26 and 30 mm diameters and a 60 mm intra-uterine tube with 60º angle were used for verification. Gafchromic® EBT films were used as dosimetric media. Dose grids, corresponding to each plane (dosimetric film location), were exported from the TPS as a raw data. Gafchromic® films were digitized after irradiation. gamma analysis of the data were performed using the OMNI Pro I’mRT® system, as recommended by the AAPM TG-119 rapport criterion for gamma analysis of 3%, 3 mm and a level of 95%. Results. For the 26 mm and 30 mm rings, the average gamma ranged, respectively, from 0.1 to 0.44 and from 0.1 to 0.27. In both cases, 99% of the measured points corresponded with the calculated data. Conclusions. This analysis showed excellent agreement between the dose distribution calculated with the TPS and the doses measured by Gafchromic films. This finding confirms the viability of using film dosimetry in BT. Key words: brachytherapy; quality assurance; film dosimetry; ring applicator Introduction Radiochromic film dosimetry with gamma analy- sis is widely used in quality assurance procedures for external beam radiotherapy (EBRT). The main advantage of this method is the ability to collect dosimetric data with very high planar resolution in contrast to other methods that are based on point dose measurements. Introducing fast and easily repeatable methods for commissioning procedures for brachytherapy (BT) treatment planning systems (TPS) is a com- plex undertaking in most cases.1,2 The relatively low energy range of BT sources induce very high spatial dose gradients in irradiated volumes; as a result, it is difficult to use point dose measurement in high dose volumes because small errors in de- tector positioning can induce large uncertainties in the measured values. Planar film dosimetry is increasingly used to verify dose distributions in BT applications. When the stepping source is used, the overall dose distri- Radiol Oncol 2017; 51(4): 469-474. Zwierzchowski G et al. / Film dosimetry procedures for quality assurances in brachytherapy470 bution in the medium is a product of the contribu- tion from each source position and the modulated step time, which are governed by optimization routines.3,4 An advantage of film dosimetry is that it offers the possibility of collecting the dose data in the planar area and using techniques of additive measurements.5,6 Self-developing dosimetry films have long been used to successfully verify dose distributions.7,8 Although this approach still needs to be custom- ized—in terms of its technical possibilities and BT TPS realization regime in particular facilities. Most TPS used for treatment plan preparation are still based on the TG-43 recommendations. These algorithms and optimization routines have been verified by many authors in homogenous conditions.9,10 The Sivert integral and modular dose calculation models allow calculations of dose rates under the assumption that the elemental source position is surrounded by a homogenous water en- vironment.11,12 The TG-186 recommendations use the MBDCA (Model Based Dose Calculation Algorithm). Because these recommendations deliver accurate tissue segmentation and take into account the ele- mental composition of the structure, their use con- tinues to make inroads because they provide better accuracy in BT dosimetry.13,14,15 Ring applicators are widely used in high dose rate (HDR) BT applications for patients with cervix cancer. This model is convenient to use due to its fixed geometry and availability in versions com- patible with computed tomography (CT)/magnetic resonance imaging (MRI) and many variations in- cluding additional interstitial needles. The most common observed problems with setup are associ- ated with source positioning uncertainties during treatment compared to the dwell position place- ments used to calculate the dose distribution. This problem occurs when source path models from li- braries are used and can also occur when the path is modeled manually.16 To our knowledge, no previous studies have at- tempted to perform dosimetric verification of the calculation algorithm used in TPS when the CT/ MRI ring applicator is used. For this reason, we conducted the present study, in which we also sought to develop treatment planning commis- sioning procedures for current and further use based on the planar film dosimetry. Materials and methods For the preparation of the treatment plans Oncentra Brachy® 4.3 were used as this is the main treatment planning software used in authors department. This version of the system was equipped with TG43 based calculation algorithms only. Two treatment plans were prepared for evalu- ation purposes. Standard CT/MRI ring applicator sets were used. The setup was based on 26 and 30 mm diameter rings and a 60 mm intra-uterine tube with 60º bent angle. Reconstruction of the ap- plication geometry was based on the applicator library module in Oncentra Brachy® 4.3. The refer- ence dose of 3 Gy was prescribed to the standard Manchester A points using a HDR iridium source. Gafchromic® EBT films were used as the do- simetric media. Irradiation setup was based on a PMMA phantom. The phantom was build using large PMMA blocks with prepared cavity for intrautherine probe. Blocks of PMMA with different thickness were used to prepare repeatable setup and for as- suring dosimetric media flatness and proper (par- allel and perpendicular) positioning relative to the applicator. The dose to water in PMMA (calibra- tion) and dose to water in water (measurements and TG43-based dose calculations) are not equiv- alent. However, Dw,w and Dw,PMMA differ only by 0.8% for distances of 25 mm from an 192Ir source. For this reason, relative character of the performed measurements and that PMMA is commonly avail- able in radiotherapy facilities this material was chosen and used. FIGURE 1. Geometry of the measurements, tandem ring applicator and Gafchromic® film located in planes (A), (B) and (C), respectively. Radiol Oncol 2017; 51(4): 469-474. Zwierzchowski G et al. / Film dosimetry procedures for quality assurances in brachytherapy 471 Dosimetric films (Gafchromic® EBT) were then placed in three planes. The first plane (plane A) was located at the surface of the ring part of the applica- tor with a round hole for the intra-uterine probe. Plane B was located on the surface of the probe in parallel orientation to the plane where Manchester A points were located. The third film (plane C) was placed on the surface of the probe perpendicular to the previous (plane B) localization. Geometry of the measurements is presented on the Figure 1. After preparation of the treatment plan dose grids corresponding to each plane (dosimetric film localization), these were exported from the TPS as a raw data. The smallest available dose grid resolu- tion was 1 mm x 1 mm, although under this con- dition only a small part of the data collected from film analysis could be used for comparisons. In the plane A area that included the hole data were re- moved from the analysis to avoid obvious differ- ences in the no-film area. Additionally, the dose limit in the export module of the TPS was set to 400% of the reference dose and any dose values ex- ceeding this limit were exported as 400% value in the dose grid. After 72 hours, the irradiated Gafchromic® films were digitized with a flat table scanner (Epson® Perfection V750 Pro), all with the same orienta- tion. Bitmap representation of the digitized films was then converted to the dose data and then local gamma analysis of the data were performed using the OMNI Pro I’mRT ® package with 3% and 3 mm criteria. Calibration data for the films were collected by separately irradiating 14 sheets (20 mm x 30 mm) of Gafchromic® EBT (Lot #: 47207-031, ISP) films with doses ranging from 0.25 Gy to 8.0 Gy us- ing an HDR Ir-192 (192-Ir-mHDR-v2) source. To assure homogenous dose distribution, the films were placed between two blocks of 25 mm thick PMMA and two catheters were placed above and below the films at a distance of 25 mm. The doses were prescribed to the dose points in the centre of the film. After 72 hours, the films were digi- tized with a flat table scanner (Epson® Perfection V750 Pro) with light source on the one side and the detector on the other side of the film, all with the same orientation. Mean values from the most homogenous central part of the film (10 mm x 5 mm) were calculated using the VeriSoft® package. In region of interest of 10 mm x 5 mm the dose variation was estimated below 5%. Then the cali- bration curve was prepared and used recalculate the optical density (analog to digital conversion value; ADC) to the doses.17 Results Co-registration of data imported from TPS and dose distribution from scanned film were guided by cen- tre of the intrauterine probe. Dose was normalized to 400% as maximum measured dose by OmniPro® software for each analyzed film. The films were scanned at 250 DPI (Dots Per Inch) resolution, al- ways with the same orientation. Treatment plan- ning system export files contains dose data with 1 mm resolution therefore the data from film have to be downsampled by analyzing software. The dose inside the hole in the films - prepared for insertion intrauterine probe was manually changed to 0 Gy, in both - film data and TPS exported ones. We assumed that dose distributions (planned and measured) were consistent if the count of pixel values from 0.00 to 1.00 (blue and green represen- tation) in the gamma analysis was over 95% and the average pixel value was lower than 1.00. Pixel values higher than 1.00 (yellow and red representa- tion) show the regions of inconsistencies (Figures 3–8). The gamma analysis showed that all meas- ured dose distributions were consistent with the planned distributions. Table 1 presents average and maximum gammas and percentage of the ana- lyzed dose points that met the 3% and 3 mm cri- teria. It’s difficult to clearly state the uncertainties dur- ing realizing this study (based on gamma analysis), the main source of possible error is the mechani- FIGURE 2. Digitized dosimetric films as a visual input data for two applicators setup and for all three analyzed dose planes. (I) Applicator ring diameter 26 mm, Gafchromic® film located in planes (A), (B) and (C), respectively. (II) Applicator ring diameter 30 mm: Gafchromic® film located in planes (A), (B) and (C), respectively. Radiol Oncol 2017; 51(4): 469-474. Zwierzchowski G et al. / Film dosimetry procedures for quality assurances in brachytherapy472 FIGURE 7. Data analysis for the 30 mm ring in plane B. (I) - dose distribution obtained from film, (II) - dose distribution obtained from TPS, (III) - isodoses obtained from film (dots) and TPS (lines), (IV) - gamma factor map. FIGURE 8. Data analysis for the 30 mm ring in plane C. (I) - dose distribution obtained from film, (II) - dose distribution obtained from TPS, (III) - isodoses obtained from film (dots) and TPS (lines), (IV) - gamma factor map. FIGURE 3. Data analysis for the 26 mm ring in plane A. (I)- dose distribution obtained from film, (II) - dose distribution obtained from TPS, (III) - isodoses obtained from film (dots) and TPS (lines), (IV) - gamma factor map. FIGURE 4. Data analysis for the 26 mm ring in plane B. (I) - dose distribution obtained from film, (II) - dose distribution obtained from TPS, (III) - isodoses obtained from film (dots) and TPS (lines), (IV) - gamma factor map. FIGURE 5. Data analysis for the 26 mm ring in plane C. (I) - dose distribution obtained from film, (II) - dose distribution obtained from TPS, (III) - isodoses obtained from film (dots) and TPS (lines), (IV) - gamma factor map. FIGURE 6. Data analysis for the 30 mm ring in plane A. (I) - dose distribution obtained from film, (II) - dose distribution obtained from TPS, (III) - isodoses obtained from film (dots) and TPS (lines), (IV) - gamma factor map. cal positioning of the films in phantom. Another problem could be find in the spatial aligning of the two types of analyzed data. Authors decided to manually register two data series using intrauter- ine probe position assuming that the film was flat and properly placed. Authors estimated possible positioning errors at less than 1 mm level. During the calibration - homogenous irradiation of film detector with a point source in one position isn’t achievable, therefore a special arrangement Radiol Oncol 2017; 51(4): 469-474. Zwierzchowski G et al. / Film dosimetry procedures for quality assurances in brachytherapy 473 of irradiation based on Khushdeep Singh’s18 work with two catheters was used, which allowed to de- posit not less than 95% with standard deviation of 0,82% of reference dose on a defined film area. Discussion In the study, we have attempted to use the typi- cal BT setup with tandem ring applicator as a test platform to develop a commissioning procedure to verify the MBDCA algorithm recommended in TG- 186. The main finding was that self-developing flat film dosimetry is a fast and reliable commission- ing method in TG-43 conditions that could easily be adopted to almost any clinical setup in which point dose dosimetry is difficult to use and cannot provide valuable information. The data packages obtained from digitizing the Gafchromic® films were prepared for comparison with the planar dose distribution data exported from the TPS. The OMNI Pro I’mRT® software typ- ically used for IMRT was used to perform gamma analysis of the data. In this approach, a commer- cially available method, typically used for external beam radiotherapy (EBRT), was used to compare the calculated and measured dose distribution. Overall dose distributions from EBDRT and BT is significantly different in terms of the dose gra- dients.19 For brachytherapy dose distributions we observe much larger dose ranges in small volumes. As a result, the use of point dose detectors to make measurements is highly difficult due to positioning errors. In contrast, film dosimetry seems to be offer a fast and reliable method for commissioning cal- culation algorithms, and planning and treatment delivery procedure.2,20,21 The common rule for the gamma criterion when BT verification using gafrchromic films is per- formed has not been established. For purposes of this study, we adopted the AAPM TG-119 rapport criterion for gamma analysis: 3%, 3 mm and a level of 95%. The results of the analysis were acceptable for two applicator size on all three planes at the 95% level and above. This results confirms the correct- ness of this measuring method and is a positive reference for further analysis in more complex cas- es with more complicated density and geometry setup. Another important issue is the use of shields and commissioning of the calculation result for shielded applicators, where high density material is placed in the close proximity of the source.22 In most TPS in current use, the elemental com- position of the structures is not taken into account during the calculation (atomic number - Z) and dose distribution is based on geometric models of the shields. The TG-186 recommendations with MBDCA introduce the need to develop reliable verification methods that are more convenient and accurate, which can be performed in a more repeat- able manner than point dose methods.23,24 Better accuracy in BT dosimetry appears to be a common need when the benefits from accurate tissue segmentation and the structure’s elemental composition are considered as an important step up.13,14,25 It bears mentioning that for the analysis per- formed in the present study, we only used a small amount of the data from the digitized dosimetric films. The planar resolution of the films is very high and limited only by the chemical structure of the dosimetric media itself and physical resolution of the scanner. On the other hand, the maximum resolution of the exported dose grid was only 1 mm. But the most important factor for QA in BT is the possibility to export data exactly from the plane where the film was located during the measure- ments. This allows for the design of very effective phantoms with convenient and repeatable geom- etry.2,26 The film dosimetry used to verify the dose dis- tribution and also for the direct reconstruction of the source path should be considered the method of choice for commissioning these newly-designed applicators. Precise reconstruction of the real source positions allows the dose distributions cal- culated by the planning system to be checked and leads to more conformal treatment planning.27 It should be noted that introducing effective, repeat- able and easy-to-use QA procedures for BT is es- TABLE 1. Results of Gamma analysis for the 26 mm ring and 30 mm ring applicator Ring, 26 mm Gamma average Gamma max Gamma (0.00 – 1.00) plane A 0.22 1.42 99.04% plane B 0.10 1.29 99.31% plane C 0.44 1.75 98.88% Ring, 30 mm Gamma average Gamma max Gamma (0.00 – 1.00) plane A 0.25 1.96 98.11% plane B 0.27 2.00 97.94% plane C 0.10 1.22 99.54% Radiol Oncol 2017; 51(4): 469-474. Zwierzchowski G et al. / Film dosimetry procedures for quality assurances in brachytherapy474 sential. Numerous factors, including relatively high doses, dose gradients, limited number of frac- tions, and advanced optimization algorithms with extensive dwell time modulation, make it nearly impossible to apply in-vivo dosimetry, thus leaving virtually no room to implement plan corrections during treatment. In these conditions, developing convenient “offline” methods for commissioning calculation routines, applicators and also for more frequently used QA procedures is very important and merits more study. Conclusions The analysis performed in this study showed ex- cellent agreement between the dose distributions calculated using TPS and the doses measured by Gafchromic films. This confirms the viability of us- ing film dosimetry in brachytherapy. The proposed commissioning procedure for further use with the MBDCA algorithm was established for use at the authors’ facility, and it seems likely that the same procedure could be replicated at other centers. References 1. Malicki J. 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Zwierzchowski G, Bielęda G, Skowronek J, Mazur M. Film based verification of calculation algorithms used for brachytherapy planning-getting ready for upcoming challenges of MBDCA. J Contemp Brachytherapy 2016; 8: 326-35. doi: 10.5114/jcb.2016.61828 18. Singh K. Investigation of the energy response of EBT-2 gafchromic film model. Montreal: Department of Medical Physics, McGill University; 2011. 19. Manikandan A, Biplab S, David PA, Holla R, Vivek TR, Sujatha N. Relative dosimetrical verification in high dose rate brachytherapy using two-di- mensional detector array IMatriXX. J Med Phys 2011; 36: 171-5. doi: 10.4103/0971-6203.83491 20. Vijande J, Ballester F, Ouhib Z, Granero D, Pujades-Claumarchirant MC, Perez-Calatayud J. Dosimetry comparison between TG-43 and Monte Carlo calculations using the Freiburg flap for skin high-dose-rate brachytherapy. Brachytherapy 2012; 11: 528-35. doi: 10.1016/j.brachy.2011.11.005 21. Rivard MJ, Venselaar JL, Beaulieu L. The evolution of brachytherapy treat- ment planning. Med Phys 2009; 36: 2136-53. doi: 10.1118/1.3125136 22. Hira M, Podgorsak MB, Jaggernauth W, Malhotra HK. Measurement of dose perturbation around shielded ovoids in high-dose-rate brachytherapy. Brachytherapy 2011; 10: 232-41. doi: 10.1016/j.brachy.2010.08.008 23. Uniyal SC, Sharma SD, Naithani UC. A dosimetry method in the transverse plane of HDR Ir-192 brachytherapy source using gafchromic EBT2 film. Phys Med 2012; 28: 129-33. doi: 10.1016/j.ejmp.2011.03.005 24. Landry G, Reniers B, Murrer L, Lutgens L, Gurp EB, Pignol JP, et al. Sensitivity of low energy brachytherapy Monte Carlo dose calculations to uncertain- ties in human tissue composition. Med Phys 2010; 37: 5188-98. doi: 10.1118/1.3477161 25. Kirisits C, Rivard MJ, Baltas D, Ballester F, De Brabandere M, van der Laarse R, et al. Review of clinical brachytherapy uncertainties: analysis guidelines of GEC-ESTRO and the AAPM. Radiother Oncol 2014; 110: 199-212. doi: 10.1016/j.radonc.2013.11.002 26. Niatsetski Y, Fekkes S, Vreeken H. Source path measurements for ring ap- plicators. [Abstract]. Radiother Oncol 2011; 99: 278. Poster No. 695 27. Berger D, Dimopoulos J, Georg P, Georg D, Pötter R, Kirisits C. Uncertainties in assessment of the vaginal dose for intracavitary brachytherapy of cervical cancer using a tandem-ring applicator. Int J Radiat Oncol Biol Phys 2007; 67: 1451-9. doi: 10.1016/j.ijrobp.2006.11.021 Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. I Radiol Oncol 2017; 51(4): 369-377. doi: 10.1515/raon-2017-0044 Karcinogeneza inducirana z nizkimi dozami sevanja Piotrowski I, Kulcenty K, Maria Suchorska W, Skrobała A, Skórska M, Kruszyna-Mochalska M, Kowalik A, Jackowiak W, Malicki J Izhodišča. Učinki visokodoznega sevanja na človeške celice in tkiva so sorazmerno dobro znani, ni pa še enotnega mne- nja o učinkih nizkih in ultra nizkih doz sevanja. Znano je, da ionizirajoče sevanje povzroča karcinogenezo z indukcijo genskih mutacij in kromosomskih aberacij. Tako lahko z radioterapijo onkoloških bolnikov povzročimo nastanek sekundarnih rakov. Obsevalne aparature - pri intenzitetno modulirani radioterapiji - izpostavijo velika področja zdravih tkiv nizkim dozam seva- nja. To bi lahko vplivalo na povečano incidence rakov pri uspešno zdravljenjih bolnikih. Raziskave kažejo, da je tveganje za karcinogenezo večje pri nizkih dozah sevanja, glede na izračune linearnega modela. Tudi epidemiološki podatki delavcev v nuklernih reaktorjih in preživelih po atomski bombi potrjujejo, da lahko nizke doze sevanja prispevajo k tveganju za nastanek raka. To tveganje morda korelira s starostjo ob izpostavitvi. Zaključki. Razumevanje molekulrnih mehanizmov odgovorov na sevanje z nizko dozo je ključno za pravilno oceno tveganj in dobrobiti teh sevanj. Upoštevati bi jih bilo potrebno tudi pri planiranju radioterapije in izpostavljenjsti zaposlenih, ki delajo v območju sevanja. Radiol Oncol 2017; 51(4): 378-385. doi: 10.1515/raon-2017-0045 Klinični pomen 18F-DFG pozitronske emisijske tomografije/računalniške tomografije pri sledenju kolorektalnega raka. Iskanje načinov za povečanje specifičnosti pri odkrivanju ponovitev Ince S, Okuyucu K, Hancerliogulları O, Alagoz E, San H, Arslan N Izhodišča. Kolorektalni rak se v 2 letih po resekciji primarnega tumorja ponovi pri skoraj 40 % bolnikov. Slikanje z 18F-FDG pozitronsko emisijsko tomografijo/računalniško tomografijo (PET/CT) je novejša preiskava, ki jo pogosto uporabljamo za oceno metastatskega širjenja v obdobju sledenja. Naš namen je bil raziskati diagnostični pomen podatkov 18F-FDG-PET/CT pri kolo- rektalnem raku. Ocenjevali smo vrednosti maksimalnega standardiziranega privzema izotopa (SUVmax), vrednosti celokupne glikolize v leziji (TLG) in razlike v SUVmax pri dveh ponovitvah slikanja v določenem časovnem razmiku. Bolniki in metode. Pri 53 bolnikih s kolorektalnim rakom smo analizirali na kontrolnem ali ponovnem 18F-FDG-PET/CT vrednosti SUVmax posameznih lezij. Vse lezije s povišanimi vrednostmi SUVmax so bile potrjene s kolonoskopijo ali histološko. Rezultate PET/CT smo primerjala s konvencionalnimi slikovnimi načini (CT, MRI) in tumorskimi označevalci (karbohidratni anti- gen 19-9 [Ca 19-9], karcinoembrionalni antigen [CEA]). Rezultati. V skupini benignih lezij je bil povprečni SUVmax 6,9 ± 5,6 in v skupini malignih lezij 12,7 ± 6,1. Povprečna vrednost TLG v skupini benignih in malignih lezij je bila 401 oz. 148. Rezultati preiskave 18F-FDG-PET/CT so bili pravilno pozitivni pri 48 % bolnikov z normalnimi vrednostmi Ca19-9 ali CEA in pravilno negativni pri 10 % primerov s povišanim Ca 19-9 ali CEA. CT ali MRI sta prepoznala sumljivo maligno lezijo pri 32 % bolnikih in rezultati 18F-FDG-PET/CT so bili pravilno negativni pri 35 % primerov. Najpomembnejšo in jasno statistično razliko v vrednosti TLG smo našli med skupinama z benignimi boleznimi in recidivnimi lezijami. Zaključki. Čeprav je SUVmax močan metabolni pokazatelj (p = 0,008), se kaže TLG kot najboljši napovedovalec recidiva pri kolorektalnem raku (p = 0,001). Oba zvišujeta specifičnost preiskave z 18F-FDG-PET/CT. Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. II Radiol Oncol 2017; 51(4): 386-392. doi: 10.1515/raon-2017-0049 Pomen ultrazvoka s kontrastnim sredstvom v zaznavi in biopsiji lezij dojke zaznavnih le s preiskavo MR? Nykänen A, Arponen O, Sutela A, Vanninen R, Sudah M Izhodišča. Namen raziskave je bil oceniti uporabnost ultrazvoka s kontrastnim sredstvom (UZKS) in UZKS vodenih posegov v diagnostiki lezij, ki so vidne na MR ampak nezaznavne s klasično ultrazvočno (UZ) preiskavo. Bolniki in metode. Z retrospektivno raziskavo smo zajeli 10 žensk, ki so imele 10 z UZ nezaznavne lezije in smo jih zdravili v obdobju od julija 2014 do aprila 2017. Vse lezije so bile ponovno ocenjene z UZKS, kjer smo aplicirali 2,4 ml kontrasta SonoVue®. Ko smo lezijo spoznali za pozitivno z UZKS, smo uporabili enako količino kontrastnega sredstva še za UZKS voden poseg. Rezultati. Z MR smo odkrili 8 lezij, ki so privzemale kontrastno sredstvo s povprečno velikostjo 9 mm (razpon 5–16 mm) in 2 leziji, ki nista privzemali kontrastnega sredstva velikosti 10 ter 20 mm. Z usmerjeno UZ preiskavo nismo odkrili skladnih lezij. Z UZKS smo odkrili 5 od 10 lezij (50 %). Z UZKS vodenim posegom smo vzeli biopsijo 4 lezij in 1 lezija je bila označena s sponko ter kasneje kirurško odstranjena. Štiri lezije so bile histološko potrjeno maligne. Pet lezij ni bilo zaznavnih z UZKS; pri 3 smo opravili MR vodeno biopsijo; 1 lezija ni bila dostopna za MR vodeno biopsijo in smo jo sledili; pri 1 leziji smo opravili UZ vodeno biopsijo z mesta kjer so bile vidne arhitekturne nepravilnosti, čeprav je morfološko nismo z gotovostjo uspeli identificirati. Tri lezije, UZKS nezaznavne, so bile histološko potrjeno maligne. Zaključki. Na podlagi preliminarnih rezultatov je UZKS možno orodje pri zaznavi lezij, ki so vidne le z MR in lahko odraža fi- nančno in časovno učinkovito prakso. UZKS je bolj priročno nadomestilo MR vodeni biopsiji in bi ga lahko vključili v diagnostični algoritem, ki ocenjuje lezije vidne le z MR. Radiol Oncol 2017; 51(4): 393-400.; doi: 10.1515/raon-2017-0030 Izhodiščni privzem Lipiodola po transarterijski kemoembolizaciji pri hepatocelularnih rakih. Določitev pražnih vrednosti, ki napovedujejo tumorski odgovor Matsui Y, Horikawa M, Noudeh YJ, Kaufman JA, Kolbeck KJ, Farsad K Izhodišča. Namen raziskave je bil oceniti povezavo med izhodiščnimi vrednostmi privzema Lipiodola in zgodnjo ponovitvijo hepatocelularnega raka (HCC) po transarterijski kemoembolizaciji (TACE) ter določiti pražne vrednosti izhodiščnega privze- ma, ki napoveduje odgovor tumorja. Bolniki in metode. Retrospektivno smo pregledali bazo podatkov o HCC, pri katerih smo opravili TACE z Lipiodolom. S ke- moterapevtsko lipiodolsko emulzijo smo zdravili 46 tumorjev pri 30 bolnikih, nismo pa naredili dodatno embolizacija z majhnimi delci. Izhodiščni privzem lipiodola smo merili kot srednje vrednosti v Hounsfieldovih enotah (HU) s pomočjo računalniške tomo- grafije (CT) 1 teden po TACE. Izračunali smo hitrost izpiranja kot razmerje razlik med izhodiščnimi vrednostmi HU in vrednosmi HU po določenem času sledenja (HU/mesec). Coxov regresijski model sorazmernega tveganja smo uporabili za ugotavljanje po- vezanosti med izhodiščnim privzemom Lipiodola in drugimi spremenljivkami tumorskega odgovora. Uporabili smo analizo ROC (receiver operating characteristics) za opredelitev izhodiščnega praga privzema Lipiodola, ki napoveduje odgovor tumorjev. Rezultati. V obdobju spremljanja (povprečno 5,6 mesecev) se je ponovilo 19 (41,3 %) tumorjev (povprečni čas do po- novitve je bil 3,6 mesecev). V multivariatnem modelu sta bila nizki izhodiščni privzem Lipiodola (P = 0.001) in večja hitrost izplavljanja (P < 0.0001) statistično značilna napovedna dejavnika zgodnje ponovitve tumorja. Analiza ROC je pokazala, da so izhodiščne pražne vrednosti privzema Lipiodola 270,2 HU značilno povezane z odgovorom tumorjev (95 % občutljivost, 93 % specifičnost). Zaključki. Izhodiščni privzem Lipiodola in hitrost izplavljanja v obdobju sledenja sta neodvisna napovedna dejavnika zgo- dnje ponovitve tumorja. Pražne vrednosti izhodiščnega privzema Lipiodola več kot 270 HU so imele največjo občutljivost in specifičnost za napoved odgovora tumorja na zdravljenje. Naše ugotovitve bi bile lahko koristne pri odločanju o nadaljnjih strategijah zdravljenja po TACE z Lipiodolom. Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. III Radiol Oncol 2017; 51(4): 401-406.; doi: 10.1515/raon-2017-0053 Hondromalacija patele, ruptura medialnega meniskusa in medialni periartikularni burzitis pri bolnikih z osteoartrozo Resorlu M, Doner D, Karatag O, Akgun Toprak C Izhodišča. V raziskavi smo ocenjevali prisotnost burzitisa v medialnem kompartmentu kolena (burze tetiv pes anserinus, semimembranoznega tibialnega kolateralnega ligamenta in medialnega kolateralnega ligamenta) pri osteoartrozi, hondro- malaciji patele in raztrganini medialnega meniskusa. Bolniki in metode. Dva radiologa sta retrospektivno pregledala 100 magnetnoresonančnih preiskav kolena pri bolnikih napotenih zaradi bolečine v kolenu. Prvi radiolog je ocenjeval osteoartrozo, hondromalacijo patele in rupturo medialnega meniskusa. Drugi radiolog je brez podatkov najdb prvega ocenjeval prisotnost burzitisa. Rezultati. Znake blage osteoartroze (stopnje I in II) je imelo 55 bolnikov, znake težje osteoartroze (stopnje III in IV) pa 45. Ohranjen hrustanec patele je imelo 25 bolnikov, medtem ko je imelo znake blage hondromalacije patele (stopnje I in II) 29 bolnikov in znake izrazite hondromalacije patele (stopnje III in IV) 46 bolnikov. Rupturo medialnega meniskusa je imelo 51 bolni- kov. Med težjo osteoartrozo in hondromalacijo patele je obstajala pozitivna korelacija (p < 0.001 in p = 0.018). Pomembno ko- relacijo smo ugotovili tudi pri ugotovitvi medialnega burzitisa in rupture medialnega meniskusa (p = 0.023). Pozitivno korelacijo smo videli tudi med medialnim burzitisom in stopnjo osteoartroze, ne pa med burzitisom in hondromalacijo patele (p = 0.023 in p = 0.479). Burzitis lateralnega kolateralnega ligamenta smo našli pri dveh bolnikih, iliotibialni burzitis pa pri petih bolnikih. Zaključki. Bolniki s težjo osteoartrozo so imeli večjo prevelanco rupture medialnega meniskusa ter burzitisa v medialnem kompartmentu. Radiol Oncol 2017; 51(4): 407-414. doi: 10.1515/raon-2017-0048 Fitoterapevtika oridonin in ponicidin imata in vitro aditivni učinek z obsevanjem na celicah raka trebušne slinavke Liermann J, Naumann P, Fortunato F, E. Schmid TE, Weber KJ, Debus J, Combs SE Izhodišča. Kemoradioterapija lokalno napredovalega raka trebušne slinavke lahko pripomore k njegovi operabilnosti. V raziskavi smo preučevali radisenzibilizirajoči učinek oridonina in ponicidina na celice raka trebušne slinavke. Obe učinkovini sta bili izolirani iz Isodon rubescens, rastline znane iz tradicionalne kitajske medicine, in imata dokazano protitumorsko delovanje. Materiali in metode. Celice raka trebušne slinavke AsPC-1, BxPC-3, Panc-1 in MIA PaCa-2 smo izpostavili oridoninu ali ponicidinu in jih nato obsevali z 2 Gy ali 6 Gy. Merili smo njihovo klonogenost, vpliv na razporeditev v celičnem ciklusu, na- stajanje žarišč γH2AX kot indikatorja dvojnih DNA prelomov ter izražanje proteinov Ku70 in XRCC4, ki so udeleženi v popravilu dvojnih DNA prelomov. Rezultati. Oridonin in ponicidin sta imela citotoksičen učinek na celične linije trebušneg slinavke in sta povzročala dvojne prelome DNA. V kombinaciji z obsevanjem smo zasledili aditivni učinek in podaljšano fazo zaostanka v G2/M. Nismo zaznali sprememb v izražanju proteinov, ki so udeleženi v popravilu dvojnih prelomov DNA. Zaključki. Kombinirano zdravljenje celic z oridoninom ali ponicidinom in obsevanjem je imelo aditivni učinek z zaustavitvijo celic v G2/m fazi celičnega ciklusa. Obe učinkovini povzročita dvojne prelome DNA in onemogočata večje popravilo dvojnih prelomov. Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. IV Radiol Oncol 2017; 51(4): 415-421. doi: 10.1515/raon-2017-0051 Usmerjena elektroporacija podganjih jeter s pomočjo slane infuzije v portalno veno. Preliminarni rezultati različne konduktivnosti Pañella C, Castellví Q, Moll X, Quesada R, Villanueva A, Iglesias M, Naranjo D, Sánchez-Velázquez P, Andaluz A, Grande G, Ivorra A, Burdío F Izhodšča. Jetrni tumorji in metastaze pogosto niso primerni za kirurško ali ablativno zdravljenje. Namen raziskave je bil pre- učiti izvedljivost in varnost povečanja konduktivnosti jeter z infuzijo slane raztopine v portalno veno. Predpostavljali smo, da s tem lahko zdravimo več tumorskih nodulov hkrati z ireverzibilno elektroporacijo. Materiali in metode. Uporabili smo podgane Sprague Dawley (Skupina A, n = 10) in atimične podgane s tumorji v jetrih (Skupina B, n = 8). Slano raztopino smo injicirali v portalno veno, hkrati pa za neutralizacijo hipernatremije dejoniziran serum in furesimid intraarterijsko. Merili smo spremembe konduktivnosti jeter in tumorjev v jetrih. Čas, ko je bila jetrna konduktivnost večja od tumorske konduktivnosti, smo imenovali terapevtsko okno. Živali smo spremljali še en mesec po terapiji, nato smo jih humano usmrtili in izvedli histološko analizo vzorcev. Rezultati. Večina živali, 82,4 % obravnavanih je preživela postopek. Srednja vrednost največje jetrne konduktivnosti se je povečala za 2,7 in 3,5 krat od bazalne vrednosti v skupini A in skupini B. Tudi jetrna konduktivnost v primerjavi s tumorsko kon- duktivnostjo se je povečala za 1,4 krat, kar je terapevtsko okno za selektivno aplikacijo ireverzibilne elektroporacije. Zaključki. Infuzija slane raztopine ob kompenzacije hipernatrijemije je varna metoda za povečanje jetrne konduktivnosti in za usmerjeno delovanje ireverzibilne elektroporacije na jetrne tumorje. Radiol Oncol 2017; 51(4): 422-430. doi: 10.1515/raon-2017-0043 Minimalno invaziven postopek elektrokemoterapije za zdravljenje nosnih tumorjev psov z enoigelno elektrodo Maglietti F, Tellado M, Olaiz N, Michinski S, Marshall G Izhodišča. Tumorje nosne votline odkrijemo pozno, ko že infiltrirajo okoljska tkiva in je zato potrebno agresivno zdravljenje. V raziskavi smo za zdravljenje takih tumorjev uporabili elektrokemoterapijo. Materiali in metode. V primeru zdravljenja globoko ležečih tumorjev je glavna omejitev elektrokemoterapije pokritost celotnega tumorja s primernim električnim poljem. Zato smo razvili enoigelno elektrodo (SiNE), ki ob minimalni invazivnosti lahko dobro pokrije tumor z električnim poljem in je enostavna za uporabo. S tako elektrodo smo zdravili 11 psov s spontanimi tumorji ter primerjali učinkovitost z 10 psmi, ki so bili zdravljeni s kirurgijo in adjuvantno kemoterapijo. Rezultati. Elektrokemoterapije je bil uspešna, pri 27 % tumorjev smo ugotovili popolni odgovorov, pri 64 % delni odgovor in pri 9 % stabilno bolezen. S tem smo dosegli 91 % objektivnih odgovorov in 16,86 mesecev celokupnega preživetja (4–32 mesecev; srednja vrednost 16,5 mesecev), kar pomeni značilno podaljšanje preživetja (p = 0,0008) v primerjavi s kontrolno skupino zdravljenih psov. Stranski učinek je bilo vnetje nosne votline, ki se je pomirilo po zdravljenju s kortikosteroidi. Eno leto po zdravljenju je bilo živih 60 % psov zdravljenih z elektrokemoterapijo in 10 % psov v kontrolni skupini, po 32 mesecih pa 30 % po elektrokemoterapiji in 10 % v kontrolni skupini. Zaključki. Elektrokemoterapija s SiNE elektrodo je varna in učinkovita pri zdravljenju nosnih tumorjev psov. Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. V Radiol Oncol 2017; 51(4): 431-437. doi: 10.1515/raon-2017-0042 Metformin in vitro v glioblastomih poveča občutljivost za obsevanje z zaustavitvijo celičnega cikla v fazi G2/M in s povečanim izražanjem adenozin-5′-monofosfat-aktivirajoče proteinske kinaze Adeberg S, Bernhardt D, Harrabi SB, Nicolay NH, Hörner-Rieber J, König L, Repka M, Mohr A, Abdollahi A, Weber KJ, Debus J, Rieken S Izhodišča. Predpostavljamo, da ima metabolizem pomembno vlogo pri nadzoru rakavih celic. Nedavno smo pokazali iz- boljšano preživetje brez napredovanja bolezni pri bolnikih z glioblastomom, ki so med kemoradioterapijo prejemali metformin kot antidiabetično zdravilo. Čeprav metformin uporabljamo v klinični praksi, je njegov vpliv pri glioblastom še razmeroma slabo raziskan, še posebej v kombinaciji z drugimi načini zdravljenja, kot sta obsevanje in temozolomid. Materiali in metode. V raziskavi smo proučevali vpliv metformina v kombinaciji z obsevanjem in temozolomidom na preživetje celic (klonogeno preživetje) celični ciklus (rutinska citometrijska analiza FACScan) in nivojem fosforilirane adenozin- 5′-monofosfat-aktivirajoče proteinske kinaze (AMPK) (Phopho-AMPKalpha1 - ELISA) v glioblastomskih celičnih linijah LN18 in LN229. Rezultati. Metformin in temozolomid sta izboljšala učinkovitost fotonskega obsevanja v celicah glioblastoma. Toksičnost ce- lic je bila izrazitejša pri celicah LN18 z ne-metiliranim promotorjem za O6-metilgvanin DNK metiltransferazo (MGMT). Zaustavitev celičnega ciklusa v fazi G2/M z metforminom in kombiniranim zdravljenjem smo opazili v času do 72 ur. Ti učinki po obsevanju, metforminu in temozolomidu so bili povezani s povišanimi vrednostmi aktivirane AMPK v celični liniji LN229, ne pa tudi v celični liniji LN18. Zaključki. Radiosenzitivni učinki metformina na glioblastomske celice, zdravljene z obsevanjem in temozolomidom in vitro, se kaže z zaustavitvijo celičnega ciklusa v fazi G2/M in spremembami pAMPK. Radiol Oncol 2017; 51(4): 438-446.; doi: 10.1515/raon-2017-0033 Ocena metod preoblikovane registracije slik za akumulacijo doze pri bolnikih z rakom nosnega žrela med radioterapijo Nobnop W, Chitapanarux I, Neamin H, Wanwilairat S, Lorvidhaya V, Sanghangthum T Izhodišča. Preoblikovano registracijo slik (PRS) uporabljamo za prilagoditev struktur glede na anatomske spremembe pri opazovanju dozimetričnega učinka. V raziskavi smo uporabili slike megavoltnega računalniškega tomografa (MVCT) za iz- delavo kumulativnih doz pri bolnikih z rakom nosnega žrela z različnimi metodami PRS. Analizirali smo izvedbo različnih metod PRS in vpliv akumulacije doze. Bolniki in metode. V raziskavo smo vključili pet bolnikov z rakom nosnega žrela, ki smo jih zdravili s helično tomoterapijo. Za oceno akumulacije doze s štirimi metodami PRS smo uporabili tedenske slike MVCT, ki smo jih posneli ob 1., 6., 11., 16., 21., 26. in 31. frakciji obsevanja. Analizirali smo deviacije kumulativne doze od izhodiščnega obsevalnega načrta in preučene korelacije teh variacij z anatomskimi spremembami in metodami PRS. Rezultati. Tarčna doza ob koncu zdravljenja je bila nekoliko različna glede na izhodiščni načrt. Z napredovanjem zdra- vljenja so razlike v dozah za posamezne organe naraščale in sicer do 6,8 % (razpon: 2,2−10,9 %) za desno parotidno žlezo, do 15,2 % (razpon: -1,7−36,3 %) za levo parotidno žlezo in do 6,4 % (razpon: -1.6−13.2 %) za hrbtenjačo. Povprečne vrednosti nenatančnosti za oceno akumuliranih doz za vse metode PRS so bile 0,21 ± 0,11 Gy (tarčna doza), 1,99 ± 0,76 Gy (desna parotidna žleza), 1,19 ± 0,24 Gy (leva parotidna žleza) in 0,41 ± 0,04 Gy (hrbtenjača). Zaključki. Natančnost metod PRS vpliva na oceno akumuliranja doze. Vpliva tako na tarčno dozo kot na dozo, ki jo prej- mejo organi. Metode PRS vključujejo ustrezno tehniko ocene doze za opazovanje kot rezultat anatomskih sprememb med frakcijami obsevanja. Primerne so za uporabo pri adaptivnih terapevtskih strategijah. Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. VI Radiol Oncol 2017; 51(4): 447-454. doi: 10.1515/raon-2017-0024 Dolgoročni rezultati zdravljenja z obsevanjem pri bolnicah z rakom zunanjega spolovila v Sloveniji v letih 1997–2004 Zobec Logar HB Izhodišča. Namen raziskave je bila retrospektivna analiza bolnic z rakom zunanjega spolovila zdravljenih z dvodimenzional- nim (2D) obsevanjem na Onkološkem inštitutu Ljubljana v letih 1997–2004. Bolnice in metode. V raziskavo smo vključili 56 bolnic, srednje starosti 74,4 +/- 9,7 let, večinoma stadija T2 in T3. Vse bolnice smo zdravili z obsevanjem, bodisi v kombinaciji z operacijo (skupina A), z obsevanjem kot primarnim zdravljenjem (skupina B) ali z obsevanjem ob ponovitvi bolezni (skupina C). Nekatere bolnice so prejele tudi kemoterapijo. Analizirali smo histološki tip tumorja, stopnjo diferenciacije, število in velikost pozitivnih bezgavk, širjenje bolezni preko bezgavčne kapsule, vrsto operativ- nega posega, dozo obsevanja na področje primarnega tumorja, dimeljskih in medeničnih bezgavk, lokalno kontrolo bolezni in preživetje bolnic. Rezultati. 10-letno celokupno preživetje je bilo 22,7 %, za bolezen specifično preživetje 34,5 % in lokalna kontrola bolezni 41,1 %. Najboljše 10-letne rezultate zdravljenja smo dosegli v skupini primarno operiranih bolnic, zdravljenih z dopolnilnim obse- vanjem z ali brez kemoterapije (celokupno preživetje 31,9 %, za bolezen specifično preživetje 40,6 %, lokalna kontrola bolezni 47,6 %). Prisotnost pozitivnih bezgavk je imela velik vpliv na lokalno kontrolo bolezni. V primeru pozitivnih bezgavk se je lokalna kontrola zmanjšala za 60 % (p = 0,03), preživetje pa za 50 % (p = 0,2). Pri dozi, ki je bila enaka ali višja od 54 Gy, se je pokazala težnja k boljši lokalni kontroli bolezni (p = 0.05). Zaključki. Najboljši način zdravljenja lokalno napredovalega raka zunanjega spolovila nudi kombinacija operacije in ob- sevanja z ali brez kemoterapije. V primeru prisotnosti negativnih dejavnikov tveganja je zaradi boljše lokalne kontrole smiselno obsevanje z dozo ≥ 54 Gy. Radiol Oncol 2017; 51(4): 455-462. doi: 10.1515/raon-2017-0040 Povezava med polimorfizmi gena SLC19A1 in toksičnostjo visokih odmerkov metotreksata pri otrocih z akutno limfoblastno levkemijo in ne-Hodgkinovim malignim limfomom. Uvedba obravnave, ki temelji na haplotipih Faganel Kotnik B, Jazbec J, Bohanec Grabar P, Rodriguez-Antona C, Dolžan V Izhodišča. Raziskovali smo klinično pomembnost vpliva genetske variabilnosti SLC19A1 na z metotreksatom v visokem od- merku (HD-MTX) povzročene toksičnosti pri otrocih in adolescentih, ki smo jih zdravili zaradi akutne limfoblastne levkemije (ALL) in ne-Hodgkinovega malignega limfoma (NHML). Bolniki in metode. Pri 88 otrocih in mladostnikih z ALL/NHML smo raziskovali vpliv polimorfizmov posameznega nukleotida (SNP) in haplotipov SLC19A1 na toksičnost, povzročeno s HD-MTX. Rezultati. Bolniki z genotipom TT rs2838958 so imeli večjo verjetnost za razvoj mukozitisa v primerjavi z nosilci najmanj enega C alela rs2838958 (Razmerje obetov [OR] 0,226 [0,071–0,725], p < 0,009). Haplotip TGTTCCG (H4) je statistično značilno zmanjšal tveganje za pojav neželenih učinkov med zdravljenjem s HD-MTX (OR 0,143 [0,02274–0,852], p = 0,0303). Zaključki. Analiza SLC19A1 SNP in haplotipov bi lahko prinesla dodatne informacije za bolniku prilagojeno zdravljenje s HD-MTX pri otrocih z ALL/NHML. To bi lahko omogočilo boljši izid zdravljenja. Za validacijo rezultatov so potrebne nadaljnje raziskave. Slovenian abstracts Radiol Oncol 2017; 51(4): I-VII. VII Radiol Oncol 2017; 51(4): 463-468. doi: 10.1515/raon-2017-0041 Vpliv obsevalne doze in možganskega volumna V57 Gy na napredovanje bolezni in preživetje pri bolnikih z multiformnim glioblastomom Stojkovski I, Krstevska V, Smichkoska S Izhodišča. Namen raziskave je bil analizirati vpliv obsevanega volumna možgan V57 Gy (volumna, ki je prejel 57 Gy ali več) na čas do napredovanja bolezni in na preživetje pri bolnikih z glioblastomov. Bolniki in metode. Naredili smo dozimetrično analizo obsevalnih načrtov pri 70 bolnikov z glioblastomom, ki so prejeli pooperativno radiokemoterapijo s temozolamidom in nato še z adjuvantno terapijo prav tako s temozolamidom. Bolnike smo zdravili z dvema različnima načinoma, ki smo ju uporabili pri opredelitvi obsevanega volumna in pri predpisu obsevalne doze. Prvo skupino bolnikov smo obsevali z enim volumnom, ki je prejel 60 Gy v dnevnih frakcijah po 2 Gy (31 bolnikov). Drugo sku- pino bolnikov pa smo obsevali s stožčasto tehniko cone-downe, sestavljeno iz dveh faz. Prvi fazi s 46 Gy v frakcijah po 2 Gy je sledil dodatek cone-down s 14 Gy v frakcijah po 2 Gy (39 bolnikov). Določili smo volumen V57 Gy in razmerje med volumnom možgan in V57 Gy. Povprečne vrednosti obeh parametrov smo obravnavali kot pražne vrednosti. Tako smo bolnike razdelili v dve skupini glede na vsak parameter (manjše oz. večje vrednosti od pražne). Rezultati. Povprečna vrednost volumna V57 Gy je bila 593,39 cm3 (razpon 166,94 do 968,60 cm3), povprečna vrednost volumna možgan 1332,86 cm3 (razpon 1047 do 1671,90 cm3) in povprečna vrednost razmerja možgani/V57 Gy 2,46 (razpon 1,42 do 7,67). Med obama skupinama ni bilo pomembne razlike glede V57 Gy in razmerja med volumnov možgan in V57 Gy. Zaključki. Volumen, ki smo ga obsevali z dozo več kot 57 Gy (V57 Gy) in razmerje med volumnom celih možgan in V57 Gy nista imela vpliva na čas do napredovanja bolezni in na preživetje pri bolnikih z glioblastomom. Radiol Oncol 2017; 51(4): 469-474. doi: 10.1515/raon-2017-0050 Zagotavljanje kakovosti pri uporabi analize gama za radiokromsko filmsko dozimetrijo Zwierzchowski G, Bieleda G, Skowronek J Izhodišča. Za komisioniranje načrtovalnih sistemov v brahiterapiji potrebujemo hitre in enostavno ponovljive postopke. V teleradioterapiji je uporaba analize gama pri radiokromski filmski dozimetriji že dobro uveljavljena metoda pri postopkih za zagotavljanje kakovosti. Obenem to metodo vedno bolj uporabljamo pri preverjanju dvodimenzionalne dozne porazdelitve v brahiterapiji. Analizo gama lahko uporabljamo kot metodo za primerjavo med izračunanimi in izmerjenimi dozami v skladu s poročilom TG-186 in za računske algoritme MBDCA. Namen raziskave je bil dozimetrično preverjanje računskih algoritmov v načrtovalnih sistemih za brahiterapijo, ko uporabljamo krožne aplikatorje CT/MRI. Materiali in metode. V raziskavi smo uporabljali krožna aplikatorja s polmeroma 26 in 30 mm ter 60 mm maternično son- do. Za same meritve doze pa smo uporabljali filme Gafchromic® EBT. Dozne porazdelitve smo izvozili iz načrtovalnih sistemov za vsako ravnino, v kateri smo uporabili filme, ki smo jih po obsevanju digitalizirali. Za analizo gama smo uporabili kriterij 3 % / 3 mm pri stopnji 95 %, kar je v skladu s priporočili iz AAPM TG-119. Podatke pa smo obdelali s sistemom OMNI Pro I’mRT®. Rezultati. Povprečne vrednosti gama so bile od 0,1 do 0,44 za 26 mm krožni aplikator in od 0,1 do 0,27 za 30 mm krožni aplikator. V obeh primerih je bila stopnja ujemanja z izračunanimi vrednostmi 99 %. Zaključki. Predstavljena analiza je pokazala odlično ujemanje med doznimi porazdelitvami izračunanimi z načrtovalnimi sistemi in doznimi porazdelitvami, ki smo jih izmerili z gafkromskimi filmi. Naše ugotovitve potrjujejo primernost uporabe filmske dozimetrije v brahiterapiji. Fundacija "Docent dr. J. Cholewa" je neprofitno, neinstitucionalno in nestrankarsko združenje posameznikov, ustanov in organizacij, ki želijo materialno spodbujati in poglabljati raziskovalno dejavnost v onkologiji. Dunajska 106 1000 Ljubljana IBAN: SI56 0203 3001 7879 431 Activity of “Dr. J. Cholewa” Foundation for Cancer Research and Education – a report for the final quarter of 2017 Dr. Josip Cholewa Foundation for cancer research and education continues with its planned activities in the final quarter of 2017. Its primary focus remains the provision of grants and scholarships and oth- er forms of financial assistance for basic, clinical and public health research in the field of oncology. In parallel, it also makes efforts to provide financial and other support for the organisation of congresses, symposia and other forms of meetings to spread the knowledge about prevention and treatment of can- cer, and finally about rehabilitation for cancer patients. In Foundation's strategy the spread of knowl- edge should not be restricted only to the professionals that treat cancer patients, but also to the patients themselves and to the general public. The Foundation continues to provide support for »Radiology and Oncology«, a quarterly scientific magazine with a respectable impact factor that publishes research and review articles about all aspects of cancer. The magazine is edited and published in Ljubljana, Slovenia. »Radiology and Oncology« is an open access journal available to everyone free of charge. Its long tradition represents a guarantee for the continuity of international exchange of ideas and research results in the field of oncology for all in Slovenia that are interested and involved in helping people affected by many different aspects of can- cer. The Foundation will continue with its activities in the future, especially since the problems associated with cancer affect more and more people in Slovenia and elsewhere. Ever more successful treatment results in longer survival in many patients with previously incurable cancer conditions, thus adding many new dimensions in life of cancer survivors and their families. Viljem Kovač, M.D., Ph.D. Borut Štabuc, M.D., Ph.D. Tomaž Benulič, M.D. Andrej Plesničar, M.D., M.Sc. 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