vol.51 no.1 march 2017 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 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 Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psychology, Trieste, Italy Vassil Hadjidekov, Medical University, Department of Diagnostic Imaging, Sofia, Bulgaria 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 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 Marko Hočevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Miklós Kásler, National Institute of Oncology, Budapest, Hungary 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 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(1): A. 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: • 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 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 • 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(1): B. contents contents review Primary pulmonary choriocarcinoma Ziga Snoj, Igor Kocijancic, Erik Skof nuclear medicine PET/CT imaging in polymyalgia rheumatica: praepubic 18F-FDG uptake correlates with pectineus and adductor longus muscles enthesitis and with tenosynovitis Zdenek Rehak, Andrea Sprlakova-Pukova, Zbynek Bortlicek, Zdenek Fojtik, Tomas Kazda, Marek Joukal, Renata Koukalova, Jiri Vasina, Jana Eremiasova, Petr Nemec radiology 15 Echocardiography and cardiac biomarkers in patients with non-small cell lung cancer treated with platinum-based chemotherapy Daniel Omersa, Tanja Cufer, Robert Marcun, Mitja Lainscak 23 Contribution of diffusion weighted MRI to diagnosis and staging in gastric tumors and comparison with multi-detector computed tomography Harun Arslan, Mehmet Fatih Özbay, Iskan Çalli, Erkan Dogan, Sebahattin Çelik, Abdussamet Batur,Aydin Bora, Alpaslan Yavuz, Mehmet Deniz Bulut, Mesut Özgökçe, Mehmet Çetin Kotan experimental oncology 30 Electrotransfer of plasmid DNA radiosensitizes B16F10 tumors through activation of immune response Monika Savarin, Urska Kamensek, Maja Cemazar, Richard Heller, Gregor Sersa 40 Comparison between cryoablation and irreversible electroporation of rabbit livers at a location close to the gallbladder Jianying Zeng, Zilin Qin, Liang Zhou, Gang Fang, Jibing Chen, Jialiang Li, Lizhi Niu, Bing Liang, Kecheng Xu clinical oncology 47 Cancer burden in Slovenia with the time trends analysis Vesna Zadnik, Maja Primic Zakelj, Katarina Lokar, Katja Jarm, Urska Ivanus, Tina Zagar Radiol Oncol 2017; 51(1): C. contents 56 Leiomyosarcoma of the renal vein: analysis of outcome and prognostic factors in the world case series of 67 patients Marko Novak, Andraz Perhavec, Katherine E. Maturen, Snezana Pavlovic Djokic, Simona Jereb, Darja Erzen 65 Prognostic significance of uPA/PAI-1 level, HER2 status, and traditional histologic factors for survival in node-negative breast cancer patients Nina Fokter Dovnik, Iztok Takac 74 Safety and efficacy of doxorubicin-eluting superabsorbent polymer microspheres for the treatment of liver metastases from neuroendocrine tumours: preliminary results Lawrence Bonne, Chris Verslype, Annouschka Laenen, Sandra Cornelissen, Christophe M. Deroose, Hans Prenen, Vincent Vandecaveye, Eric Van Cutsem, Geert Maleux 81 Long-term outcomes of high dose treatment and autologous stem cell transplantation in follicular and mantle cell lymphomas – a single centre experience Lucka Boltezar, Karlo Pintaric, Jože Pretnar, Maja Pohar Perme, Barbara Jezersek Novakovic 88 Incidence of positive peritoneal cytology in patients with endometrial carcinoma after hysteroscopy vs. dilatation and curettage Andraz Dovnik, Bojana Crnobrnja, Branka Zegura, Iztok Takac, Maja Pakiz 94 Implant-prosthetic rehabilitation after radiation treatment in head and neck cancer patients: a case-series report of outcome Jasna Cotic, Jure Jamsek, Milan Kuhar, Natasa Ihan Hren, Andrej Kansky, Mutlu Özcan, Peter Jevnikar radiophysics 101 Determination of dosimetric parameters for shielded 153Gd source in prostate cancer brachytherapy Mahdi Ghorbani, Benyamin Khajetash, Najmeh Ghatei, Mohammad Mehrpouyan, Ali S. Meigooni, Ramin Shahraini 112 Optimal planning strategy among various arc arrangements for prostate stereotactic body radiotherapy with volumetric modulated arc therapy technique Sang Won Kang,Jin Beom Chung, Jae Sung Kim, In Ah Kim, Keun Yong Eom, Changhoon Song, Jeong Woo Lee, Jin Young Kim, Tae Suk Suh I slovenian abstracts Radiol Oncol 2017; 51(1): D. tant goal, since patients captured in early stages of the disease have higher survival rate.8 There is no standardized treatment for PPC. PPC grows rapidly and has high propensity to disseminate to other organs, such as bone, liver, brain, spleen and contralateral lung.4 Due to undifferentiated nature of malignancy, PPC has poor response to radia­tion treatment.9 The most appropriate regimen for chemotherapy seems to be BEP (bleomycin, meth­otrexate and cisplatin) or EMA-CO (etoposide, methotrexate, actinomycin D, cyclophospamide and vincristine).1,6,7,8,10 Despite the absence of rand­omized trials to prove its superiority, the combina­tion of EMA-CO has become the preferred regimen for initial treatment of high-risk gestational troph­oblastic disease in most countries.11 The aim of the article was to establish whether there are different clinical entities of PPC that de­served different diagnostic and therapeutic ap­proach. Furthermore, to illustrate the review we additionally present a patient referred to our insti­tution. In order to elucidate clinical characteristics and to determine optimal way of management of this rare neoplasm we analysed our patient togeth­er with other 54 reported cases. Patients and methods All published cases of PPC were collected with a PubMed search with the key word ‘primary pulmo­nary choriocarcinoma’ and the key word ‘solitary pulmonary choriocarcinoma’ that were published in English literature. The eligibility criteria were histological diagnosis of choriocarcinoma of the pulmonary tumour and thorough examination of reproductive organs to exclude potential primary choriocarcinoma in the gonads. Differences be­tween patients groups according to gender were es­timated using nonparametric Mann-Whitney test. Probabilities of survival were estimated using the Kaplan-Meier method and differences between pa­tient groups were evaluated with the log-rank test. Prognostic factors were analysed using the Cox pro­portional hazard model. Reported values are two-sided. Statistical significance was set at p < 0.05. The patient A 35 year old woman complaining of severe right sided chest pain was admitted to our hospital. The patient had three normal term deliveries in the past 15 years and three spontaneous abortions in the past year. The chest radiograph showed a round opacity in the right lungs. A computed to­mographic scan (CT) of the chest revealed the pres­ence of 6.4 x 5.4 x 5.9 cm pulmonary mass in the right lower lobe (Figure 1A). Transthoracic needle biopsy was performed and diagnosis of poorly dif­ferentiated giant cell carcinoma (GCC) was made. To exclude additional masses FDG-PET study was performed (Figure 1B). FDG accumulation in the pulmonary mass was low, maximum standardized uptake value (SUVmax) was 2.7, and only in the periphery of the mass. No other abnormal accumu­lation was detected in whole body including pelvic cavity. During hospitalization the patient’s condi­tion progresively worsened with depleting levels of hematocrit, hemoglobin and platelets. Urgent right sided bilobectomy with pericard excision was performed. Histopathologic workup of excised tumour showed poorly differentiated carcinoma with trophoblast differentiation, most consistent with choriocarcinoma. Excised nodes showed no tumour infiltration. Four weeks after surgery the patient was ad­mitted to our hospital to start chemotherapeutical treatment. In these four weeks the patient had two episodes of epileptical seizures. CT and MRI of the head showed 1 cm intracerebral metastasis with surrounding edema (Figure 1C). TABLE 1. Primary pulmonary choriocarcinoma: Summary of reported cases Gender, male/female 17/38 Median age all patients, years 34 Median age female, years 33 Median age male, years 60 Initial symptom Cough 22 Dyspnea 17 Hemoptysis 14 Chest pain 11 Asymptomatic 8 Location Right / Left lung 29/18 Right upper lobe 11 Right middle lobe 3 Right lower lobe 15 Left upper lobe 12 Left lower lobe 4 Bilateral 2 Tumor size, cm . 5 23 > 5, . 10 13 > 10 8 Treatment S 10 C 8 RT 3 S + C 24 S + C + RT 3 C = chemotherapy; RT = radiotherapy; S = surgery The patient began EMA-CO regimen chemo­therapy with high dosage methotrexate. At the start of the chemotherapy plasma hCG levels were 169396 IU/L, on eighth day fell to 20883 IU/L and after three months (4 cycles of chemotherapy) the plasma hCG levels fell within normal range. Patient underwent stereotactic radiotherapy of cerebral metastasis with dose 1 x 25 Gy after the fourth cycle and afterwards received two addition­al cycles of EMA-CO regimen with standard dose of metotrexate. To completely rule out the origin of the tumour in reproductive tract the vaginal total hysterectomy with bilateral salpingoectomy was performed after 6 cycles of chemotherapy and his­topathological examination was negative. According to FIGO clinical and prognostic stag­ing patient had stage IV disease with high-risk score.12 Brain MRI was performed 7 months after stereotactic radiotherapy and showed 3 mm re­sidual mass with no surrounding edema. Patient is on regular follow-up and is still on therapy with levetiracetam and dalteparin without evidence of relapse at 12 months following surgery. The patient was treated according to the Helsinki Declaration. She gave a written informed consent before treatment to use her clinical data for research. Results Patients We searched the literature with a PubMed search to establish characteristics of PPC using criteria de­scribed in Patients and methods. A review of the literature in English revealed 54 cases with previ­ous report of primary choriocarcinoma originating in the lung.1-10,13-15,17-44 The patient described in the present paper was analysed with other 54 reported cases. The profiles of the patients are summarized in Table 1; 17 men and 38 women were included with a median age of 34 years (range 0.3 to 77). At the time of discovery 47 patients (85%) produced symptoms, including persistent cough (40%), dysp­nea (31%), hemoptysis (25%) and chest pain (20%). At presentation 8 tumours were asymptomatic. A few of the women have presented with hormonal problems such as amenorrhea or vaginal bleeding. In men, signs of feminisation such as gynecomastia have been observed but are uncommon. Correlation between gender and clinicopathological factors We analysed the relationship between gender and other clinicopathological features in patients with PPC (Table 2). Men were older than women (p = 0.000). Men had the history of smoking more often than women (p = 0.000). No statistically significant difference between genders was observed for loca­tion, size, presence of metastases, history of haem­optysis or treatment. Survival Only 47 cases (16 men and 31 women) reported treatment outcome. The median survival time was All patients, n = 47 Women, n = 31 Men, n = 16 All patients, n = 47 Women with positivegestationalhistory, n = 14 Women with negativegestationalhistory, n = 17 Men, n = 16 FIGURE 2. Kaplan-Meier survival curves. (A) Survival for all reported cases with curves showing survival for female patients was longer than for male patients (p = 0.004). (B) Survival of all reported cases with survival of men and curves showing survival of female patients with the history of gestational event was longer than for female patients without the history of gestational event (p = 0.040). 8 months. The review of these 47 cases showed 1-, 2-, and 5- year survival rates of 61%, 57% and 49%, respectively. Important difference (p = 0.004) in survival time between genders was observed with women showing 1-, 2-, and 5- year survival rates of 77%, 77% and 64% while men showed 1-, 2-, and 5- year survival rates of 31%, 21% and 21%, respec­tively (Figure 2A). In Table 3 univariate analysis is shown. Younger patients (< 40 years) survived longer than older pa­ tients (. 40 years), (p = 0.009). Patients with smaller tumours (< 5cm) survived longer than patients with larger tumours (. 5cm), (p = 0.000). Survival of patients without metastases at presentation was longer than for patients with metastases (p = 0.000). Patients without the history of smoking survived longer than patients with the history of smoking TABLE 2. Differences between female and male patients primary pulmonary choriocarcinoma Age < 40 30 4 . 40 9 13 0.000 Size, cm < 5 17 7 . 5 12 8 0.162 Metastasis Yes 15 10 No 24 7 0.161 Smoking Yes 2 9 No 37 8 0.000 Hemoptysis Yes 13 5 No 25 11 0.780 Treatment S Yes 24 9 No 4 4 0.221 C Yes 21 10 No 7 3 0.895 S+C Yes 17 7 No 11 6 0.682 C = chemotherapy; S = surgey (p = 0.001). The women were further divided into two groups according to the history of gestational events (such as abortion, pregnancy or hydatiform mole) within the time period of less than 7 years prior the admission. Women with the history of gestational event (n = 14) had better survival out­come than women without the history of gesta­tional event (n = 17), the difference was statistically significant (p = 0.040), (Figure 2B). Patients treated with combination of surgery plus chemotherapy survived longer than those treated with optimal supportive care or either chemotherapy or surgery alone (p = 0.001). Patients treated with chemothera­py only survived less than the patients treated with surgery only or combination of chemotherapy and surgery (p = 0.016). Patients treated with combina­tion of surgery and chemotherapy survived longer than patients without combination of surgery and chemotherapy (p = 0.010). Treatment with surgery demonstrated no significant prognostic influence. As shown in Table 4, multivariate analysis of prognostic factors using the Cox proportional haz­ards model showed that the treatment combin­ing surgery with chemotherapy had independent prognostic significance (p = 0.007). Furthermore, TABLE 3. Possible prognostic factors Female 31 - 77.3 77.3 Gender Male 16 4 31.3 20.8 0.004 < 40 29 - 75.9 58.4 Age . 40 18 4 33.3 33.3 0.009 < 5 20 - 80.0 80.0 Size (cm) . 5 16 3 25.0 0.0 0.000 Yes 21 4 42.9 0.95 Metastasis No 26 - 84.6 84.6 0.000 Yes 11 4 0.0 0.0 Smoking No 36 - 69.4 69.4 0.001 Female – Positive 14 - 69.6 69.6 gestational history Negative 16 - 56.3 56.3 0.040 S Yes 9 - 55.6 55.6 No 32 - 71.0 64.5 0.458 C Yes 7 3 28.6 28.6 No 34 - 75.8 69.9 0.016 S+C Yes 24 - 83.3 56.3 No 17 4 41.2 41.2 0.010 C = chemotherapy; MST = median survival time; S = surgery the size of the lesion showed an independent prog­nostic significance (p = 0.008). Discussion In the present work we made a systematic review of the relevant literature. We found out that there are different clinical entities of PPC based on gen­der and reproductive history that show different survival. Furthermore, we showed it is important to capture and treat patients in the early stages of the disease. In addition we demonstrated that sur­gical treatment combined with chemotherapy is the best treatment regarding survival. Gender difference PPC occurs in women in younger ages than in men (33 vs. 60 median age), an observation already described by Umemori et al.8 Therefore, it can be derived that PPC in women is more likely to oc­cur in the reproductive period. Furthermore, there are more reported cases with women than men. Statistically significant difference in survival time between genders was observed with women show­ing better outcome. We further divided women in two groups due to the history of the gestational event within the time period of less than 7 years prior the admission. We chose the cut off of 7 years conservatively on the basis of three published cas­es with genetical examination.6,7 In the paper by Maesta et al. they present two patients with gesta­tional event within 4 years prior to PPC diagnosis and in both cases they proved it to be of gestational origin.6 In the paper by Vegh et al. the patient had the last gestational event 7 years prior to admission and with fluorescence in situ hybridization they excluded gestational origin, no paternal chromo­some was found in tissue examinated.7 Based on this assumption we considered that women with the history of gestational event less than 7 years had better survival outcome than women without such history of gestational event. Looking at aforementioned gender differen­cies, PPC seems to be of different etiology in men. Furthermore, we showed that men have a history of smoking more often than women. To support TABLE 4. Multivariate analysis of prognostic factors, Cox proportional hazards model Yes 0.147 0.036 – 0.598 0.007 No 1 Smoking Yes 1.827 0.418–7.982 0.423 No 1 Gender Male 1.266 0.248–6.466 0.776 Female 1 Tumour size, cm . 5 6.622 1.622–27.031 0.008 < 5 1 Age, years . 40 0.879 0.164–4.703 0.880 < 40 1 C = chemotherapy; HR = hazard ratio; S = surgery the theory of dedifferentiation four cases of PPC have been reported with co-existent pulmonary carcinoma.5,10,13,14 All were male patients, in three cases PPC was sinchronous and found at autopsy, in one case the PPC was metachronous arising 6 years after diagnosis of squamous carcinoma. Problematically primary pulmonary carcinomas can produce hCG. Ikura et al. reported more intense expression of hCG in PPC than in hCG producing GCC.14 Although this finding seemed to reflect the level of serum hCG the cut off point seemed to be ambigous.15 Because of the rarity and clinicopatho­logical similarity of PPC and hCG producing GCC the criteria for distinguishing them are unclear and the diagnosis is very difficult.14 Survival The survival data should be handled cautiously because of the possibility that mostly patients with good survival were reported in the literature and the rest were neglected. Nevertheless PPC is no­torious for having a poor prognosis, however, the present review shows 1-, 2-, and 5- year survival rates of 61%, 57% and 49%, which is reasonably good. Furthermore, if only female patients are ob­served the survival rates are even higher. Patients having smaller size tumour and being without me­tastases at diagnosis have higher survival rate, thus early diagnosis with optimal management is im­portant. The outcome is still worrying but in com­parison with the review article from the year 2004 by Umemori et al. where they reported 1-, 2-, and 5- year survival rates of 41%, 34%, 34% the outcome results are more promising. We noticed that the early reports of PPC had poorer outcome than the ones nowadays and that chemotherapeutical treat­ment was rarely used in the cases prior to year 1994 but if chemotherapy was used it was single modal­ity therapy. The evolvement of chemotherapeuti­cal treatment and better supportive therapy could influence the better outcomes seen in the present review, but we could not attribute the difference in survival rates to the specific factor due to the rarity and nonexistence of therapeutic guidelines. Treatment In order of finding out any relationship between treatment and survival the univariate and multivar­iate analysis of prognostic factors was carried out. Univariate analysis showed that treatment with chemotherapy only is not good choice of treatment however significant prognostic influence for com­bined treatment with surgery and chemotherapy was found. Furthermore, multivariate analysis of prognostic factors revealed that combined treat­ment with surgery and chemotherapy had inde­pendent prognostic significance. These findings are in concordance with already known facts that PPC is very aggressive disease, not just locally, but also by early spread to other organs.4 Therefore, com­bined modality treatment (surgery and chemother­apy) represents best possible way to improve sur­vival. Of course patients have to be fit enough for such aggressive treatment. No reports of extended survival were found in patients who underwent complete resection without chemotherapy. Conclusions PPC is an extreme rarity with variable clinical char­acteristics and outcome. It is important to capture and treat patients in the early stages of the disease. Women with the history of gestational event may show better survival, therefore genetic examina­tion could help us to predict patient’s prognosis. Surgery followed by adjuvant chemotherapy ap­pears to represent the best treatment for PPC. Acknowledgement We would like to thank M. Števanec for helping us with statistical analysis and Professor M. Snoj, M.D., Ph.D. for guidance with statistical interpre­tation. References 1. Serno J, Zeppernick F, Jäkel J, Schrading S, Maass N, Meinhold-Heerlein I, et al. Primary pulmonary choriocarcinoma: case report and review of the literature. Gynecol Obstet Invest 2012; 74: 171-6. 2. Maruoka Y, Abe K, Baba S, Isoda T, Matsuo Y, Kubo Y, et al. A case of pulmo­nary choriocarcinoma metastasis with unusual FDG-PET and CT findings: correlation with pathology. Ann Nucl Med 2012; 26: 835-9. 3. Toda S, Inoue Y, Ishino T, Yonemitsu N, Terayama K, Miyabara S, et al. 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Arch Pathol Lab Med 1987; 111: 477-9. 29. Sullivan LG. Primary choriocarcinoma of the lung in a man. Arch Pathol Lab Med 1989; 113: 82-3. 30. Van Nostrand KM, Lucci JA, Liao SY, Di Saia PJ. Primary lung choriocarcinoma masquerading as a metastatic gestational neoplasm. Gynecol Oncol 1994; 53: 361-5. 31. Durieu I, Berger N, Loire R, Gamondes JP, Guillaud PH, Cordier JF. Contralateral haemorrhagic pulmonary metastases (“choriocarcinoma syn­drome”) after pneumonectomy for primary pulmonary choriocarcinoma. Thorax 1994; 49: 523-4. 32. Otsuka T, Ohshima Y, Sunaga Y, Nagashima K. Primary pulmonary choriocar­cinoma in a four month old boy complicated with precocious puberty. Acta Paediatr Jpn 1994; 36: 404-7. 33. Canver CC, Voytovich MC. Resection of an unsuspected primary pulmonary choriocarcinoma. Ann Thorac Surg 1996; 61: 1249-51. 34. Aparicio J, Oltra A, Martínez-Moragón E, Llorca C, Gómez-Aldaraví L, Pastor M. Extragonadal nongestational choriocarcinoma involving the lung: a report of three cases. Respiration 1996; 63: 251-3. 35. Aras EL, López PG, Lago J, Muguruza I. Primary lung choriocarcinoma. Clin Transl Oncol 2001; 3: 107-9. 36. Arslanian A, Pischedda F, Filosso PL, Di Marzio P, Oliaro A, Fraire F, et al. Primary choriocarcinoma of the lung. J Thorac Cardiovasc Surg 2003; 125: 193-6. 37. Vaideeswar P, Mehta J, Deshpande J. Primary pulmonary choriocarcinoma-a series of 7 cases. Indian J Pathol Microbiol 2004; 47: 494-6. 38. Shintaku M, Hwang MH, Amitani R. Primary choriocarcinoma of the lung manifesting as diffuse alveolar hemorrhage. Arch Pathol Lab Med 2006; 130: 540-3. 39. Tajiri S, Ozawa H, Komatsu M, Hayama N, Kondo Y, Ito M, et al. A case of cho­riocarcinoma of suspected lung origin manifesting pulmonary embolism. Nihon Kokyuki Gakkai Zasshi 2008; 46:1029-33. 40. Corpa Rodríguez ME, Fernández Lahera J, Guadalajara Labajo H, Vázquez Pelillo JC, Nistal Martín de Serrano M, García Sánchez-Giron J. Choriocarcinoma of the lung. Arch Bronconeumol 2009; 45: 153-5. 41. Seol HJ, Lee JH, Lee KY, Kim JH, Lee NW, Park HJ. Primary pulmonary cho­riocarcinoma presenting with a hemothorax. J Thorac Oncol 2009; 4: 653-5. 42. Hadgu A, Tindni A, Panda M. Primary pulmonary choriocarcinoma in a male. BMJ Case Rep 2010. doi: 10.1136/bcr.02.2010.2712. 43. Berthod G, Bouzourene H, Pachinger C, Peters S. Solitary choriocarcinoma in the lung. J Thorac Oncol 2010; 5: 574-5. 44. Ibi T, Hirai K, Bessho R, Kawamoto M, Koizumi K, Shimizu K. Choriocarcinoma of the lung: report of a case. Gen Thorac Cardiovasc Surg 2012; 60: 377-80. 45. Di Crescenzo V, Laperuta P, Napolitano F, Carlomagno C, Garzi A, Vitale M. An unusual case of primary choriocarcinoma of the lung. BMC Surg 2013; 13(Suppl 2): S33. research article PET/CT imaging in polymyalgia rheumatica: praepubic 18F-FDG uptake correlates with pectineus and adductor longus muscles enthesitis and with tenosynovitis Zdenek Rehak1,2,3, Andrea Sprlakova-Pukova4, Zbynek Bortlicek5, Zdenek Fojtik6, Tomas Kazda7, Marek Joukal8, Renata Koukalova1, Jiri Vasina1, Jana Eremiasova1, Petr Nemec9 1 Department of Nuclear Medicine and PET Center, Masaryk Memorial Cancer Institute, Brno, Czech Republic 2 Regional Center for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Brno, Czech Republic 3 Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute and Faculty of Medicine, Brno, Czech Republic 4 Department of Radiology, University Hospital Brno and Masaryk University, Brno, Czech Republic 5 Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk University, Brno, Czech Republic 6 Rheumatology Unit, Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic 7 Department of Radiation Oncology, Masaryk Memorial Cancer Institute and Faculty of Medicine, Masaryk University, Brno, Czech Republic 8 Department of Anatomy, Faculty of Medicine, Masaryk University, Brno, Czech Republic 9 Rheumatology Unit, 2nd Department of Internal Medicine, St. Anne’s University Hospital Brno and Masaryk University, Brno, Czech Republic Radiol Oncol 2017; 51(1): 8-14. Received 29 August 2016 Accepted 14 December 2016 Correspondence to: Zdenek Rehak, Department of Nuclear Medicine and PET Center, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic; Phone: +420 54313 1300; Fax: +420 54313 1350; E-mail: rehak@mou.cz Disclosure: No potential conflicts of interest were disclosed. Background. The role of 18F-fluorodeoxyglucose positron emission computed tomography (18F-FDG PET/CT) is increas­ing in the diagnosis of polymyalgia rheumatica (PMR), one of the most common inflammatory rheumatic diseases. In addition to other locations, increased 18F-FDG accumulation has been detected in the praepubic region in some patients. However, a deeper description and pathophysiological explanation of this increased praepubic accumula­tion has been lacking. The aim of the presented study is to confirm a decrease in praepubic 18F-FDG accumulation in response to therapy and to describe potential correlations to other 18F-FDG PET/CT scan characteristics during the course of disease. As a secondary objective, we describe the pathological aspects of the observed praepubic 18F-FDG uptake. Patients and methods. A retrospective review of patients with newly suspected PMR undergoing baseline and fol­low up 18F-FDG PET/CT between February 2010 and March 2016 is given. Those with a visually detected presence of praepubic 18F-FDG accumulation were further analysed. The uptake was assessed visually and also semi-quantitatively in the defined region of interest by calculation of target-to-liver ratios. Other regions typical for PMR were systemati­cally described as well (shoulders, hips, sternoclavicular joints, ischiogluteal bursae, spinous interspaces). Results. Twenty-three out of 89 screened patients (26%) presented with initial praepubic 18F-FDG PET/CT positivity, 15 of whom also underwent follow up 18F-FDG PET/CT examination. Five out of 15 patients presented with increased 18F-FDG accumulation in large arteries as a sign of giant cell arteritis. During follow up examination, decrease in 18F-FDG accumulation caused by therapeutic intervention was observed in all evaluated locations in all analysed patients and no new positivity was indicated, including periarticular, extraarticular tissues or target large vessels. Praepubical accumulation of 18F-FDG was diminished in all patients (15/15, 100%) after treatment with steroids. Conclusions. Increased praepubic 18F-FDG uptake in patients with PMR is relatively common and this region should be systematically evaluated during differential diagnosis of rheumatic and malignant disease. Praepubic inflamma­tion is probably related to enthesitis and tenosynovitis at the origin of pectineus and adductor longus muscles ventrally from the pubis. Key words: positron emission tomography, polymyalgia rheumatica, enthesitis, tenosynovitis, fluorodeoxyglucose Introduction Polymyalgia rheumatica (PMR) is the most com­mon inflammatory rheumatic disease in patients older than 50 years, with a higher incidence in women. PMR shares many pathogenetic and epide­miological features with giant cell arteritis (GCA)1, and 50% of patients with GCA also develop PMR symptomatology.2 The typical symptoms of PMR are bilateral aching of the shoulder girdle, neck and hip girdle, and morning stiffness lasting for 30 min­utes or more. These symptoms are probably relat­ed to inflammation of the subacromial, subdeltoid and trochanteric bursae, and the glenohumeral or hip joints.3,4 The diagnosis of PMR is made primar­ily on clinical grounds and is bolstered by labora­tory evidence of an acute phase reaction. There is no single diagnostic test for PMR, but several di­agnostic and classification criteria have been sug­gested by some groups.5-9 Each set of criteria has advantages and disadvantages. A PMR-associated ultrasound lesion(s) in the shoulders and/or hips is currently acknowledged as diagnostic criteria for the scoring algorithm in the differential diagnosis of PMR.10 However, additional imaging methods for assessing rheumatic diseases are warranted. Prolonged febrile illness with concomittant non-specific symptoms can be also a sign of PMR as well as GCA. Thus, patients may be referred during differential diagnostics of inflammatory or malignant disease to whole body positron emission tomography (PET) or a combination of PET with computed tomography (PET/CT) using 18F-fluorodeoxyglucose (18F-FDG).11-14 Both GCA and PMR have their own characteristic 18F-FDG PET/CT features, which may occur in a non-mutu­ally exclusive manner. PMR presents with increased 18F-FDG accumu­lation in periarticular areas of shoulder and hip girdle and of sternoclavicular joints.15-17 The other location with metabolically active inflammation in PMR patients are the extraarticular synovial struc­tures (bursae). It appears that 18F-FDG detection of extraarticular bursitis using PET/CT might be routinely achievable for PMR patients, with rea­sonable sensitivity (85.7%) and specificity (88.2%), by considering high 18F-FDG uptake in at least 2 of 3 locations (ischial tuberosity, greater trochanter, spinous processes).18 Contrasting at least in part with PMR, GCA imaging typically reveals increased avidity in the wall of whole aorta, including its branches (sub­clavian and brachial arteries, brachiocephalic trunk, common iliac arteries and femoral arter­ies).19-21 Importantly, from the clinical point of view, 18F-FDG uptake in pertinent locations de­creases in response to effective treatment in both PMR and GCA. Thus, 18F-FDG PET/CT evaluation may be used for monitoring therapy and for follow up.15,19,22,23 In our previous study, increased 18F-FDG accu­mulation was detected in the praepubic region in some patients.17 However, a deeper description and pathophysiological explanation of this increased praepubic accumulation is needed. The aim of the present study is to confirm a decrease of praepubic 18F-FDG accumulation in response to treatment and to describe potential correlations to other 18F-FDG PET/CT scan characteristics and to the course of disease, and thereby to support the validation of the praepubic avidity within the general 18F-FDG PET/ CT features of PMR. As a secondary objective, we provide a description of the pathological aspect of the observed praepubic 18F-FDG uptake. Patients and methods Patients with suspected new or relapsed PMR who underwent 18F-FDG PET/CT examination at Masaryk Memorial Cancer Institute in Brno be­tween February 2010 and March 2016 were retro­spectively screened for visually detected presence of praepubic 18F-FDG accumulation. Patients for whom follow up 18F-FDG PET/CT was performed during corticosteroid therapy within the clinical remission phase were eligible for further analysis. All patient had to meet ACR 2012 diagnostic crite­ria for PMR. Patients who were previously treated for known PMR (with at least 15 months from the termination of therapy) were eligible as well. All patients initially provided their signed informed consent with participation on further retrospec­tive studies and this analysis was approved by the institutional review board. Treatment consisted by prednisone, methylprednisolone or methotrexate in various dosages as listed in Table 1. 18F-FDG PET/CT examination was performed uti­lizing the hybrid scanner Biograph 64 HR+ Siemens Erlangen, Germany. CT scan was performed in low dose CT (25 mAs eff/120 kV) as well as diagnostic or contrast enhanced CT scan (160 mAs eff/12 kV) (intravenous Iomeron 400, BRACCO, Milan, Italy). All patients had standard preparation prior to ex­amination, including restriction of physical activity for 12 h, fasting for at least 6 h, capillary glycemia below 10 mmol/L (180 mg/dL) prior to 18F-FDG administration and peroral hydration with 500­ TABLE 1. Imaging and laboratory results of all patients. Reported baseline treatment was initiated several days after baseline PET/CT 1 R 11-18-2014 4.0 1.134 44 23 P 15 S 2.23, H 1.25, Scl 1.13, Isch 1.45 no positivity 3-20-2015 0.576 16 1.4 P 5 H 1.11 no positivity 2 N 2-23-2010 48.7 1.234 120 49 P 15 S 1.65, H 1.24, Scl 1.24, Isch 1.17, L 1.14 no positivity 3-15-2014 0.812 26 16.9 M 8 no positivity no positivity 3 N 9-1-2014 3.2 2.148 120 137 P 60 S 1.87, Scl 1.45, H 2.31, Isch 1.65, L 1.12, Th 1.14, C 1.24 no positivity 12-8-2014 0.992 35 16.7 P 10 S 1.36 no positivity 4 N 7-24-2010 6.1 1.891 120 56.8 P 40 S 1.78, Scl 1.54, H 1, Isch 2.21, L 1.08 no positivity 1-25-2011 0.345 30 5.7 P 2.5 no positivity no positivity 5 N 3-13-2014 12.0 1.772 60 28.5 P 20 S 2.02, H 1.47, Scl 1.24, Isch 2.02, L 1.23 V3/6 3-13-2015 0.987 7 1 P 0 no positivity V1/6 6 N 10-14-2013 16.9 1.298 54 45 P 20 S1.78, H1.88, Scl 1.95, L 1.25, Isch 1.16 no positivity 3-13-2015 0.537 6 5.3 P 2.5 no positivity no positivity 7 R 1-3-2014 13.1 1.302 77 78.3 M 16 S 1.78, H 1.87, Scl 1.35, Isch 1.65, L 1.12 V4/6 2-6-2015 0.403 5 1.9 M 2 no positivity no positivity 8 N 6-9-2012 33.1 1.835 50 76 P 30 S 2.21, H 2.16, Scl 1.8, Isch 1.78, L 1.2 V4/6 3-13-2015 0.54 14 3.1 P 0 H 1.23 V2/6 9 N 6-3-2015 3.1 1.502 60 41.5 P 15 S 2.36, H 2.24, Scl 2.03, Isch 2.04, C 1.11 no positivity 9-4-2015 0.76 16 13.4 P 10 no positivity no positivity 10 N 1-8-2014 21.7 1.209 80 78.3 P 60 S 1.84, H1.78, Scl 1.69, Isch 1.57, L 1.23 no positivity 10-30-2015 0.395 10 2.5 P 7.5 no positivity no positivity 11 R 3-4-2015 11-5-2015 8.1 1.546 0.811 62 30 16.5 3.3 M 48 + MTX 10/ week M 4 + MTX 10/ week S 2.66, H 2.74, Scl 2.0, Isch 1.72, C 1.47, L 1.48 H 1.14, C 1.08, S 1.11, Scl 1.07 V5/6 V3/6 12 N 2-6-2015 8.3 1.789 120 98.7 M 32 S 2.27, H 2.06, Scl 2.16, Isch 3.03, L 1.45 no positivity 10-16-2015 0.97 40 7.5 M 8 Scl 1.14, Isch 1.21 no positivity 13 N 1-23-2015 14 1.123 70 67.5 P 15 S 1.78, H 1.87, Scl 1.69, Isch 1.57, L 1.24 no positivity 3-24-2016 0.441 6 1.4 P 5 no positivity no positivity 9-16-2015 1.282 74 52 P 20 S 2.44 , H 3.04, Scl 2.07, L 1.21 no positivity 14 N 5.8 3-9-2016 0.41 24 3.2 P 7.5 no positivity no positivity 15 N 10-26-2015 3.2 1.892 80 118.8 P 30 S 2.93, H 2.97, Isch 3.22, C 1.44, L 1.24, Th 1.17, Scl 1.79 V4/6 2-1-2016 0.678 5 1.3 P20 no positivity no positivity B/C = baseline and control; C, L, Th = cervical, lumbal, thoracic interspinous space; FW = Fahraus-Westergren test; H = hip; M = methylprednisolone; Isch = ischiogluteal bursae; MTX = methotrexate; N = newly diagnosed; P = prednisone; R = relapse; S = shoulder; Scl = sternoclavicular joint; V = vascular uptake with number indicating presence in regions from 6 measured 1,000 mL of plain water. 18F-FDG (UJV Rez, Czech Republic) was administered in a dose range of 327­434 (median 366 MBq) in the baseline study and in a dose range of 301-400 (median 362 MBq) in the fol­low up examination. After an in vivo accumulation time of 55 to 75 minutes, whole body scanning from the proximal third of thighs to the skull base was performed in baseline as well as follow up study. All images were iteratively reconstructed and cor­rected for attenuation. 18F-FDG uptake was assessed visually and also semi-quantitatively in the defined region of inter­est (ROI) with calculation of target-to-liver ratios. Liver 18F-FDG uptake (SUVmax) was used as a reference base (measured within the ROI located in the centrum of the right liver lobe). Praepubic 18F-FDG accumulation was semiquantitavely as­sessed as SUVmax within elliptic ROI located through both preapubic regions while keeping in the safe distance from the bladder based on the investigator discretion. Praepubic-to-liver ratio (SUVmax) was subsequently calculated. Other regions typical for PMR were system­atically described with measurement of SUVmax: shoulders, hips and sternoclavicular joints and ex­traarticular sites – in ischial tuberosity and between spinous processes where ischiogluteal and inters­pinous bursae are often presented, respectively. A target-to-liver ratio higher than 1.0 was considered positive in all mentioned regions. For paired or­gans, the higher value (from the right or left site) was used for target-to-liver calculation. 18F-FDG uptake (SUVmax) within the typical sites for GCA was also systematically evaluated, namely in the walls of following arteries: thoracic aorta, abdomi­nal aorta, brachial and subclavian arteries, iliac and femoral arteries. An artery wall-to-liver ratio higher than 1.0 was also considered positive. The number of positive vascular regions out of six evaluated re­gions is reported. Again, for paired structures, the higher value (from the right or left site) was used for target-to-liver calculation. The same scan evalu­ation was performed for follow up scans. Results Patients characteristic From 89 screened patients, 23 (26%) presented with initial praepubic 18F-FDG PET/CT positivity. 15 patients, 10 women and 5 men with a median age of 70 years, range 53 to 78, underwent also fol­low up 18F-FDG PET/CT and met inclusion crite­ria for the presented analysis (Table 2). Twelve out TABLE 2. Patients‘ baseline characteristics Sex, n (%) females males Age at the disease onset median (min-max) Time to follow up exam median (min-max) 10 (66.7 %) 5 (33.3 %) 70 years (53-78) 8 months (3-49) TABLE 3. Changes in analysed laboratory parameters between baseline and follow up examination CRP 57 mg/l (17-137) 3 mg/l (1-17) 0.001 FW 74 mm/hod (44-120) 16 mm/hod (5-40) 0.001 TABLE 4. Nuclear medicine data at baseline and follow up examination 1.50 (1.12-2.15) 0.58 (0.35-0.99) 0.001 18F-FDG uptake FIGURE 1. Initial 18FDG-PET/CT examination of all 15 examined patients, showing transversal planes through maximal praepubic uptake. Visually detectable accumulation can be observed in all patients. of 15 patients were classified as newly diagnosed; prior to the initiation of corticosteroid therapy, the other three patients had been previously treated for known PMR (with at least 15 months from the termination of therapy). Laboratory values for FW and CRP were obtained for all patients, ± 14 days around 18F-FDG PET/CT (Table 3). Patient number 5 had accompanied giant cell arteritis confirmed histologically by temporal artery biopsy. Baseline 18F-FDG PET/CT characteristics 18F-FDG PET/CT characteristic including 18F-FDG dosage are summarized in Table 4. Increased 18F-FDG accumulation (positivity) was observed at baseline in all patients in the praepubic region, with praepubic-to-liver ratios higher than 1.0. This accumulation was obvious by visual evaluation only, as presented in Figure 1, and this finding was always accompanied by additional positivity in 18F-FDG PET/CT scans (Table 1). Other sites with increased accumulation were as follow: around the shoulder girdle in all 15 patients, around the hip girdle in 15 patients, and around sternoclavicular joints in 14 out of 15 patients. 18F-FDG PET/CT pos­itivity was observed also in extraarticular synovial structures, in ischiogluteal bursa in 14 patients and between spinous processes of the vertebrae in 14 patients, most commonly within lumbal region in 13 patients. Five out of 15 patients presented with increased 18F-FDG accumulation in large arteries as a sign of large vessel vasculitis of GCA. In those with large vessel vasculitis, 18F-FDG PET/CT positivity was at least in 3 of 6 evaluated vascular regions. Increased values of FW and CRP were detected in all patients during initial examination and were correlated to newly diagnosed PMR or to its relapse (Table 3). Follow up 18F-FDG PET/CT characteristics Given the retrospective nature of this study, it was not possible to keep a strict interval between base­line and follow up 18F-FDG PET/CT scans. Follow up examinations were timed by clinical purposes rather than by experimental needs. Follow up 18F-FDG PET/CT (median time 8 months, range 3-49) revealed continuing positivity around the shoulder girdle in only 2/15 patients, around hip joints in 3/15, around the sternoclavicular joint in 2/15, in extraarticular synovial structures in ischio­gluteal bursae in 1/15 and in interspinous regions of cervical vertebrae in 1/15 patients. In three out of 15 patients, positivity in continuous large ves­sels was observed, maximally in 3 vessel regions out of 6 measured. In all evaluated locations in all analysed patients, a decrease in 18F-FDG accumu­lation (target-to-liver ratio) was observed, and no new positivity was indicated, including periarticu­lar, extraarticular tissues or target large vessels. Praepubic accumulation of 18F-FDG was dimin­ished in all patients after treatment with steroids. Praepubic-to-liver ratio was lower than 1.0 post therapy and this decrease in accumulation was clear by visual assessment (Figure 2). During follow up 18F-FDG PET/CT examination, all patients were undergoing steroid treatment and they reported subjective improvement in health condition, and disease remission was confirmed by attending rheumatologist. The laboratory signs of inflammation, FW, and CRP were decreased as well (Table 3). Discussion In the presented retrospective analysis of 18F-FDG PET/CT findings in patients with proven PMR, 25.8% patients presented with praepubic 18F-FDG uptake with fifteen patients from our cohort be­ing able to undergo follow up 18F-FDG PET/CT examination. We were not able to perform follow up 18F-FDG PET/CT scan in a prospectively defined phase of treatment; instead, the aim was to ob­tain 18F-FDG PET/CT study during disease remis­sion. During this follow up 18F-FDG PET/CT exam, praepubic accumulation of 18F-FDG was dimin­ished in all 15 patients after treatment with steroids. In our previous study, praepubic positivity was described only in 5/67 (8%) PMR patients repre­senting only a relatively rare sign of the disease.17 However, 32/67 (48%) patients were examined on an older PET scanner with the rest undergoing ex­amination in a newer hybrid PET/CT scanner with better image resolution. Lower detectability of targed lesions may have occured on 18F-FDG PET scanner alone and also underevaluation of accu­mulation quantification may result in false nega­tivity (praepubic-to-liver ratio < 1.0). Even if visual accumulation was clearly positive, no patient from our previous cohort met the criterion for positivity. Given the above findings, it is reasonable to specu­late that the percentage of positive preapubic ac­cumulation in patients with active PMR would be higher when examined on a hybrid 18F-FDG PET/ CT rather than a PET camera. The overall number of patients with initial praepubic 18F-FDG PET/CT positivity was 23/89 (25.8%). Thus, praepubic accu­mulation should not be considered a constant and frequent sign of 18F-FDG PET/CT results as it is in periarticular 18F-FDG accumulation in shoulders and hips. In cohorts from previous studies, accu­mulation in the shoulder groin girdle was observed in 33/35 (94%), in 12/14 (86%) and in 58/67 (87%) and in the hips in 31/35 (89%), in 12/14 (86%) and in 47/67 (70%) PMR patients, respectively.15-17 In a recently published report of 15 patients with PMR and of 9 patients with Elderly-Onset Rheumatoid Arthritis (EORA), praepubic 18F-FDG uptake was recommended as one of the signs to enable differentiation between these two condi­tions. 24 The incidence of praepubic 18F-FDG uptake (pectineus enthesitis) was relatively high with 9 patients from 15 evaluated which may be related to sample size bias. Patients with PMR developed significantly higher uptake compared to those with EORA.24 Patients in the present study represent a retrospectively described cohort of patients with praepubic 18F-FDG PET/CT positivity that includes post-treatment follow up examination. In light of these findings, it seems possible that increased praepubic accumulation may have been present in previously published case reports including those where axial slices indicating ischiogluteal bursi­tis were published.18,25-27 Sondag et al. observed 18F-FDG uptake in 11/50 (22%) patients; however, the cohort was a mixture of those with and with­out treatment with 22/50 (44%) with administra­tion of steroids.28 Mackie et al. published the first MRI findings of inflammation in the front of the symphysis in patients with PMR in 2015.29 A simi­lar observation was observed in our cohort (patient number 11); data not shown. It is difficult to exactly determine the patho­logical background of increased 18F-FDG uptake in the praepubic region in patients with PMR. The described accumulation seems to be relatively bor­dered and with spheric shape in some patients, while with blurry margins in others. MRI may be helpful in further evaluation of this region. Based on the MRI finding presented by Mackie et al. and Wakura et al., we assume another type of extraarticular inflammation is responsible for the observed PET findings.24,30 Namely, it appears to represent features of enthesitis and tenosynovitis of pectineus and adductor longus muscles. There is probably no bursa in the locations under the ten­dons of these muscles, which is also supported by findings on MRI, where no fluid collection with evidence of thickened wall suggestive for bursitis was observed. However, some reports have recent­ly suggested a combination of PMR and tenosyno­vitis in other locations, namely in the long head of biceps brachii 31 or in extensor tenosynovitis of the hand32 or in the vicinity of the enthesis of the rectus femoris.24 Our observations confirm that 18F-FDG PET/CT examination seems to be an advantageous one-step diagnostic modality for detecting different variants of PMR, for assessing extent and severity, and for excluding occult malignancy. The follow up exam may be useful in monitoring disease activity in­cluding the praepubic location. It is conceivable that the praepubic region will become part of tar­geted examination for other imaging strategies as is US or MRI and that clinical significance and cor­relations will be further discovered. Conclusions Increased praepubic tracer accumulation is becom­ing an integral part of 18F-FDG PET/CT evaluation of polymyalgia rheumatica, and is probably a cor­relate of enthesitis and tenosynovitis at the origin of pectineus and adductor longus muscles ventrally from the pubis. The findings described here were consistently presented in combination with other periarticular accumulations (shoulder and hip gir­dle and surrounded bursae, sternoclavicular joint) and other extraarticular bursae at some distance from joints such as the ischiogluteal bursae and interspinous bursae in spine. Some patients pre­sented with signs of large vessels vasculitis of GCA. In accordance with other 18F-FDG PET/CT positive locations, praepubic accumulation was decreased in relation to PMR treatment. Our findings support the clinical value of 18F-FDG PET/CT examination of patients with suspected or proved PMR. Acknowledgments and funding This work was supported in part by the Ministry of Health, Czech Republic – Conceptual Development of Research Organization (MMCI 00209805, FNBr, 65269705) and project MEYS-NPS I-LO1413. References 1. Nesjet G, Nesjet R. Giant cell arteritis and polymyalgia rheumatica. In: Ball GV, Bridges SL Jr., editors. Vasculitis (2nd ed.), Oxford University Press; 2008. p.307-16. 2. Pipitone N, Salvarani C. Update on polymyalgia rheumatica. Eur J Intern Med 2013; 24: 583–9. doi:10.1016/j.ejim.2013.03.003 3. Salvarani C, Cantini F, Olivieri I, Barozzi L, Machioni L, Niccoli L, et al. Proximal bursitis in active polymyalgia rheumatica. Ann Intern Med 1997; 127: 27-31. doi:10.7326/0003-4819-127-1-199707010-00005 4. Cantini F, Nicoli L, Nannini C, Padula A, Olivieri I, Boiardi L, et al. Inflammatory changes of the hip synovial structures in polymyalgia rheumatica. Clin Exp Rheumatol 2005; 23: 462-8. 5. Chuang TY, Hunder GG, Ilstrup DM, Kurland LT. Polymyalgia rheumatica: a 10-year epidemiologic and clinical study. Ann Intern Med 1982; 97: 672-80. doi:10.7326/0003-4819-97-5-672 6. Hamrin B. Polymyalgia arteritica. Acta Med Scand 1972; 533: 1-131. 7. Bird HA, Esselinckx W, Dixon AS, Mowat AG, Wood PH. An evaluation of criteria for polymyalgia rheumatica. Ann Rheum Dis 1979; 38: 434-9. doi:10.1136/ard.38.5.434 8. Jones JG, Hazleman BL. Prognosis and management of polymyalgia rheu­matica. Ann Rheum Dis 1981; 40: 1-5. doi:10.1136/ard.40.1.1 9. Healey LA. Long-term follow-up of polymyalgia rheumatica: evidence for synovitis. Semin Arthritis Rheum 1984; 13: 322-8. doi:10.1016/0049­0172(84)90012-X 10. Dasgupta B, Cimmino MA, Maradit-Kremers H, Schmidt WA, Schirmer M, Salvarani C, et al. 2012 provisional classification criteria for polymyalgia rheumatica: a European League Against Rheumatism/American College of Rheumatology collaborative initiative. Ann Rheum Dis 2012; 71: 484-92. doi: 10.1136/annrheumdis-2011-200329 11. Glaudemans AWJM, Signore A. FDG-PET/CT in infections: the imaging method of choice? Eur J Nucl Med Mol Imaging 2010; 37: 1986-91. doi: 10.1007/s00259-010-1587-x 12. Jaruskova M, Belohlavek O. Role of FDG-PET and PET/CT in the diagnosis of prolonged febrile states. Eur J Nucl Med Mol Imaging 2006; 33: 913–8. doi: 10.1007/s00259-006-0064-z 13. Kubota K, Nakamoto Y, Tamaki N, Kanegae K, Fukuda H, Kaneda T, et al. FDG­PET for the diagnosis of fever of unknown origin: a Japanese multi-center study. Ann Nucl Med 2011; 25: 355-64. doi: 10.1007/s12149-011-0470-6 14. Vaidyanathan S, Patel CN, Scarsbrook AF, Chowdhury FU. FDG PET/CT in infection and inflammation-current and emerging clinical applications. Clin radiol 2015; 70: 787-800. doi: 10.1016/j.crad.2015.03.010 15. Blockmans D, De Ceuninck L, Vanderschueren S, Knockaert D, Mortelmans L, Bobbaers H. Repetitive 18-fluorodeoxyglucose positron emission tomog­raphy in isolated polymyalgia rheumatica: a prospective study in 35 pa­tients. Rheumatology 2007; 46: 672-7. doi: 10.1093/rheumatology/kel376. 16. Yamashita H, Inoue M, Takahashi Y, Kano T, Mimori A. The natural history of asymptomatic positron emission tomography: positive giant cell arteritis after a case of self-limiting polymyalgia rheumatica. Mod Rheumatol 2012; 22: 942-6. doi: 10.1007/s10165-012-0689-7 17. Rehak Z, Vasina J, Nemec P, Fojtik Z, Koukalova R, Bortlicek Z, et al. Various forms of 18F-FDG PET and PET/CT findings in patients with polymyalgia rheumatica. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2015; 159: 629-36. doi: 10.5507/bp.2015.026 18. Yamashita H, Kubota K, Takahashi Y, Minaminoto R, Morooka M, Ito K, et al. Whole-body fluorodeoxyglucose positron emission tomography/computed tomography in patiens with active polymyalgia rheumatica: evidence for distinctive bursitis and large-vessel vasculitis. Mod Rheumatol 2012; 22: 705-11. doi: 10.1007/s10165-011-0581-x 19. Blockmans D, De Ceuninck L, Vanderschueren S, Knockaert D, Mortelmans L, Bobbaers H. Repetitive 18F-fluorodeoxyglucose positron emission to­mography in giant cell arteritis: a prospective study in 35 patients. Arthritis Rheum 2006; 55: 131-7. doi: 10.1002/art.21699 20. Lensen KDF, Comans EFI, Voskuyl AE, Van der Laken CJ, Brouwer E, Zwijnenburg AT, et al. Large-Vessel Vasculitis: Interobserver Agreement and Diagnostic Accuracy of 18 F-FDG-PET/CT. Biomed Res Int 2015; 2015: 914692. doi: 10.1155/2015/914692 21. Fuchs M, Briel M, Daikeler T, Walker UA, Rasch H, Berg S, et al. The impact of 18F-FDG PET on the management of patients with suspected large ves­sel vasculitis. Eur J Nucl Med Mol Imaging 2012; 39: 344-53. doi: 10.1007/ s00259-011-1967-x 22. Taniguchi Y, Nakayama S, Terada Y. Clinical implication of FDG-PET/CT in monitoring disease activity in large-vessel giant cell arteritis linked with secondary polymyalgia rheumatica. Case Reports in Internal Medicine 2014; 1: 6-9. doi: 10.5430/crim.v1n1p6 23. Glaudemans AWJM, de Vries EFJ, Galli F, Dierckx RAJO, Slart RHJA, Signore A. The use of 18F-FDG-PET/CT for Diagnosis and Treatment Monitoring of Inflammatory and Infectious Diseases. Clinical and Developmental Immunology 2013; 2013: 623036. doi: 10.1155/2013/623036 24. Wakura D, Kotani T, Takeuchi T, Komori T, Yoshida ,1 Makino S, et al. Differentiation between Polymyalgia Rheumatica (PMR) and Elderly-Onset Rheumatoid Arthritis Using 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography: Is Enthesitis a New Pathological Lesion in PMR? PLoS One 2016 ; 11: e0158509. doi: 10.1371/journal. pone.0158509 25. Toriihara A, Seto Y, Yoshida K, Umehara I, Nakagawa T, Tassei MD, et al. F-18 FDG PET/CT of Polymyalgia Rheumatica. Clin Nucl Med 2009; 34: 305-6. doi: 10.1097/RLU.0b013e31819e51fd 26. Kotani T, Komori T, Kanzaki Y, Takeuchi T, Wakura D, Iimori A, et al. FDG­PET/CT of polymyalgia rheumatica. Mod Rheumatol 2011; 21: 334-6. doi: 10.1007/s10165-010-0382-7 27. Park JS, Pyo JY, Park HJ, Lee HS, Kang Y, Kang MI, et al. Typical 18-FDG-PET/CT Findings of Polymyalgia Rheumatica: A Case Report. Journal of Rheumatic Diseases 2013; 20: 113-7. doi: 10.4078/jrd.2013.20.2.113 28. Sondag M, Guillot X, Verhoeven F, Blagosklonov O, Prati C, Boulahdour H et al. Utility of 18F-fluoro-dexoxyglucose positron emission tomography for the diagnosis of polymyalgia rheumatica: a controlled study. Rheumatology 2016; 55:1452-7. doi: 10.1093/rheumatology/kew202 29. Mackie SL, Pease CT, Fukuba E, Harris E, Emery P, Hodgson R, et al. Whole-body MRI of patients with polymyalgia rheumatica identifies a distinct subset with complete patient-reported response to glucocorticoids. Ann Rheum Dis 2015; 74: 2188-92. doi: 10.1136/annrheumdis-2015-207395 30. Mackie SL, Mc Gonagle DG. Response to:‘A relationship between extra-capsular involvement and response to steroid treatment in polymyalgia rheumatica: too soon to conclude?’by Yang et al. Ann Rheum Dis 2016; 75: e17. doi: 10.1136/annrheumdis-2015-208962 31. Ruta S, Rosa J, Navarta DA, Saucedo C, Catoggio LJ, Monaco RG, et al. Ultrasound assessment of new onset bilateral painful shoulder in patiens with polymyalgia rheumatica and rheumatoid arthritis. Clin Rheumatol 2012; 31: 1383–7. doi: 10.1007/s10067-012-2016-2 32. Cimmino MA, Parodi M, Zampogna G, Barbieri F, Garlaschi G. Polymyalgia rheumatica is associated with extensor tendon tenosynovitis but not with synovitis of the hands: a magnetic resonance imaging study. Rheumatology 2011; 50: 494–9. doi: 10.1093/rheumatology/keq367 research article Echocardiography and cardiac biomarkers in patients with non-small cell lung cancer treated with platinum-based chemotherapy Daniel Omersa1, Tanja Cufer2,3, Robert Marcun2, Mitja Lainscak3,4 1 National Institute of Public Health, Ljubljana, Slovenia 2 University Clinic Golnik, Golnik, Slovenia 3 Faculty of Medicine, Ljubljana, Slovenia 4 Departments of Cardiology and Research and Education, General Hospital Celje, Celje, Slovenia Radiol Oncol 2017; 51(1): 15-22. Received 9 February 2016 Accepted 8 May 2016 Correspondence to: Mitja Lainščak, M.D., General Hospital Celje, Department of Cardiology, Oblakova 5, SI-3000 Celje, Slovenia. Phone: +386 3 423 38 00; Fax: +386 3 423 37 54; E-mail: mitja.lainscak@guest.arnes.si Disclosure: No potential conflicts of interest were disclosed. Background. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and remains an impor­tant cause of cancer death worldwide. Platinum-based chemotherapy (PBC) for NSCLC can modify outcome while the risk of cardiotoxicity remains poorly researched. We aimed to evaluate the incidence and severity of cardiac injury during PBC in patients with NSCLC and to identify patients at risk. Methods. This was a single-centre, prospective, observational study of patients with early and advanced stage NSCLC referred for PBC. In addition to standard care, patients were examined and evaluated for cardiotoxicity be­fore the first dose (visit 1), at the last dose (visit 2) and 6 months after the last dose of PBC (visit 3). Cardiotoxicity (at visit 2 and 3) was defined as increase in the ultrasensitive troponin T, N-terminal pro-B type natriuretic peptide or decrease in left ventricular ejection fraction (LVEF). Results. Overall, 41 patients (mean age 61 ± 9; 54% men; 68% advanced lung cancer) were included. The median number of PBC cycles was 4. During the study period, there were no incidents of heart failure, and 3 deaths caused by tumour progression were recorded. The mean values of biomarkers and LVEF did not change significantly (p > 0.20). However, 10 (25%) had cardiotoxicity which was independently associated with a history of ischemic heart disease (p = 0.026). Conclusions. In NSCLC, cardiac assessment and lifestyle modifications may be pursued in patients with a history of cardiac disease and in patients with longer life expectancy. Key words: cardiotoxicity; platinum-based chemotherapy; lung cancer; cardiac biomarkers Introduction Currently, lung cancer is not only the leading cause of cancer-related deaths, but is also the most fre­quently diagnosed cancer worldwide.1 Non-small cell lung cancer (NSCLC) accounts for 80–85% of all lung cancers. According to recent guidelines, platinum-based doublet chemotherapy is used as first line therapy in NSCLC. In addition to plati­num agents (cisplatin or carboplatin), gemcitabine, pemetrexed, vinorelbine or a taxane is used.2-4 Cancer chemotherapeutic drugs, particularly anthracyclines, monoclonal antibodies and tar­geted drugs such as multiple tyrosine kinase in­hibitors can induce cardiac damage.5 In general, chemotherapy can cause cardiac damage by spe­cifically targeting receptors on cardiomyocytes or by inducing oxidative stress, as shown in trastu­zumab or anthracycline-induced cardiotoxicity, re­spectively.6,7 Screening of patients before treatment and monitoring cardiac function during therapy has traditionally relied on left ventricular ejection fraction (LVEF).6 However, because assessment of LVEF lacks the sensitivity needed for detecting early subclinical changes, newer and more sensi­tive metrics of cardiotoxicity are needed. The most commonly used biomarkers to detect subclinical cardiac toxicity are cardiac troponins and atrial or B-type natriuretic peptides. Cardiac troponins reflect cardiomyocyte injury or death, whereas natriuretic peptides are released due to cardiac stretch and strain during congestive heart failure.8,9 In addition to the risk of cardiotoxicity related to specific anti-cancer therapy, cancer patients are at increased risk of cardiovascular (CV) disease due to older age and shared lifestyle risk factors (e.g., smoking and obesity). With the introduction of new treatment modalities, the life expectancy of many cancer patients has improved substantially, and treatment-related comorbidities have become an important issue for cancer survivors. Cancer pa­tients with longer life expectancy (childhood can­cer, breast, prostate and colorectal carcinoma) are already more likely to die from CV disease than any other cause.10 In addition, for NSCLC, new de­velopments such as targeted therapies are showing potential for longer survival, and detection of early cardiac injury is becoming crucial for these patients. Data regarding cardiotoxicity following plati­num-based chemotherapy are scarce. There have been several studies reporting CV events and in­duced cardiotoxicity in animals following plati­num-based chemotherapy.11-17 Studies evaluating the long term effects of platinum-based chemo­therapy in testicular cancer have shown a higher rate of CV events such as decreased LVEF and higher mortality due to CV disease in patients ex­posed to platinum-based regimens.18-20 In addition, a large retrospective study showed that patients with NSCLC treated with chemotherapy and ra­diotherapy were more likely to develop heart fail­ure as well as cardiac dysfunction.21 To our knowl­edge, there has been only one prospective study of patients with NSCLC that evaluated cisplatin and anthracycline-based cardiotoxicity, and that study found no significant decrease in LVEF after treat­ment with cisplatin.22 Even though some degree of cardiac damage has been shown following treatment with platinum agents, it is still unclear whether platinum-based chemotherapy induces cardiotoxicity in patients with NSCLC. Hence, our aims were to evaluate cardiac function with echocardiography and bio­markers in patients with NSCLC treated with plat­inum-based chemotherapy and to identify those at risk for cardiotoxicity. Patients and methods Study design and patients This was a single-centre, prospective, observa­tional study. All patients with NSCLC who were referred for platinum-based chemotherapy in our clinic between June 2012 and September 2013 were screened to include those with early and advanced stage NSCLC treated with platinum-based dou­blets as adjuvant or metastatic therapy. Patients with adjuvant radiotherapy or concomitant chem­oradiotherapy were not included in the study. Platinum doublets with vinorelbine, gemcitabine or pemetrexed were given according to guide­lines and established clinical practice.2,3 Cisplatin 80 mg/m2 and carboplatin AUC 5 were used and modified according to the standard practice toxic­ity guidelines. Patients with a history, symptoms or signs of heart disease, or renal disease received carboplatin, while others received cisplatin. In ad­dition to standard examinations, patients had eval­uation of cardiac function by echocardiography and biomarkers before the first (visit 1), last cycle of chemotherapy (visit 2) and 6 months after com­pletion of platinum-based chemotherapy (visit 3). Between visit 2 and visit 3, none of the patients was re-challenged with platinum-based chemotherapy. Physicians involved in the care of patients partici­pating in the study were not aware of the results of biomarkers and echocardiography assessment, un­less findings were pathological and would lead to a change in a patient’s management. This study was conducted according to the Declaration of Helsinki and was approved by National Ethics Committee (Approval Nr. 37/07/09). All patients have received verbal and written information before written in­formed consent was obtained. Biomarkers and echocardiography Levels of biomarkers - ultrasensitive troponin T (usTnT) and N-terminal pro-B type natriuretic peptide (NT-proBNP) - were assessed at each visit. Whole blood samples were drawn by venepunc­ture and analysed using Roche’s Elecsys® diag­nostic analysis (Basel, Switzerland). A standard echocardiographic examination was performed by two experienced physicians (ML and RM). Cardiac chambers, valve abnormalities, systolic and dias­tolic function were evaluated. Left ventricular (LV) end-diastolic and end-systolic volumes were cal­culated using the Teicholtz or Simpson’s biplane method.23 The Teicholtz formula and Simpson’s biplane method were used in patients with a struc­turally normal LV and a structurally abnormal LV or diminished systolic function, respectively. Systolic function was evaluated by calculating left ventricular ejection fraction (LVEF) from LV end-diastolic and end-systolic volumes. Diastolic func­tion was assessed using measurements of flow velocity at the opening of the mitral valve, septal tissue Doppler of the mitral annulus and volume of the left atrium. The ratio between E wave veloc­ity at the mitral valve (E) and septal velocity of the mitral annulus (e’) was calculated (E/e’).24 Cut points and endpoint definitions Elevated usTnT and NT-proBNP levels at visits 2 and 3 were defined as a > 30% increase from the vis­ it 1 value. LVEF reduction was defined as a . 10% or . 5% decrease of LVEF to value . 55% in asymp­tomatic and symptomatic patients, respectively, as suggested in trastuzumab trials.25 Diastolic dysfunction was defined as E/e’ . 15 or E/e’ < 15 and . 8 with an enlarged left atrium, as recom­mended by the European Society of Cardiology.24 Cardiotoxicity for an individual patient was de­fined as having increased usTnT or NT-proBNP or decreased LVEF at either visit 2 or 3. Primary endpoint was cardiotoxicity, second­ary endpoints were symptomatic heart failure, in­creased usTnT, NT-proBNP, LVEF reduction and diastolic dysfunction at visit 2 and 3, as described above. Statistical analysis Statistical analysis was performed using R 3.0.2 (R Development Core Team, Vienna, Austria) and the R package CVST (Krueger and Braun). Results are presented as the mean ± standard deviation for numeric variables and number (%) for nomi­nal variables, except for the chemotherapy cycles, which are presented as median value and range. The paired t-test and Cochran’s Q test were used to assess the changes at visit 2 and 3 from those at visit 1 for numeric variables and for binary vari­ables, respectively. In univariate logistic regression models, we included cardiotoxicity as the depend­ent variable and sex, age, history of ischemic heart disease, history of arterial hypertension, metastatic lung cancer, number of chemotherapy cycles and usTnT, NT-proBNP and LVEF values at visit 1 as independent variables. In multivariate logistic re­gression models, we included cardiotoxicity as the dependent variable and history of ischemic heart disease, sex, age and LVEF at visit 1 as independent FIGURE 1. Study flowchart. variables. For these analyses, results are presented as odds ratio (OR) with corresponding 95% confi­dence intervals (CI). A p value of less than 0.05 was considered to be statistically significant. Results Overall, 41 patients were included in the study between June 2012 and September 2013. 3 of the patients died during the study, all of them due to the cancer progression. At visit 2 and visit 3, 6 and 19 patients had an incomplete cardiac evaluation, respectively, mostly due to capacity related diffi­culties and patients’ refusal at visit 2, and disease progression at visit 3. In all 3 visits, biomarkers were assessed in 7 patients, echocardiography was performed in 12 patients and both were assessed in 4 patients (Figure 1). All 40 patients who survived until visit 2 had information regarding primary endpoint. Of 41 patients, 13 patients had early stage NSCLC and received adjuvant chemotherapy and 28 patients were given chemotherapy for meta­static disease (Table 2). In addition to cisplatin or carboplatin, all patients received one additional TABLE 1. Patients’ characteristics Age – yr 61 ± 9 Male sex – no. (%) 22 (54) Arterial hypertension – no. (%) 17 (41) Ischemic heart disease – no. (%) 4 (10) Cardiovascular disease – no. (%) 5 (12) Glomerular filtration rate - ml/min/1.73 m2 102 ± 28 Advanced disease – no. (%) 28 (68) Where not mentioned, results are shown as mean values ± standard deviation. For calculating glomerular filtration rate, a modification of diet in renal disease study (MDRD) method was used. TABLE 2. Chemotherapy treatment characteristics Chemotherapy cycles – median (range) 4 (2-6) Chemotherapy – no. (%) Cisplatin 28 (68) Carboplatin 8 (20) Both, in sequence 5 (12) and Vinorelbine 11 (27) Pemetrexed 22 (54) Gemcitabine 8 (19) FIGURE 2. Number of patients at visit 2 or 3 with elevated usTnT, NT-proBNP and decreased LVEF form visit 1 values. Normal and abnormal was defined as > 30% increase in usTnT and NT-proBNP, and > 10% in symptomatic patients or > 5% in asymptomatic patients decrease to value lower than 50% in LVEF from visit 1 value. LVEF = left ventricular ejection fraction; n = terminal pro BNP; NT = proBNP; usTnT = ultrasensitive troponin T chemotherapeutic drug (vinorelbine, pemetrexed or gemcitabine). At visit 1 or during the course of chemotherapy, none of the patients had overt symptoms or signs suggestive of heart failure or any other heart dis­ease. CV disease was present in 5 patients (4 ischae­mic heart disease and 1 peripheral artery disease). Of those with ischaemic heart disease 1 had older myocardial infarction while 3 had chronic ischae­mic heart disease or angina pectoris. Differences in biomarkers and echocardiographic variables at each visit are shown in Table 3 and Figure 2. The values of usTnT and NT-proBNP gradually insig­nificantly decreased during the study, while the mean values of LVEF were not different between visits. ANOVA analysis included only patients with all measurements for each variable, therefore 7 and 12 patients were included in the analysis of biomarkers and LVEF, respectively. An increase of usTnT and NT-proBNP was observed in 4 and 7 patients at visit 2 or 3, respectively. None of the patients had decreased LVEF at visit 2, while 1 pa­tient had decreased LVEF at visit 3. Patient who had decreased LVEF did not have increased values of NT-proBNP or usTnT, and one patient who had elevated NT-proBNP to more than 1800 pg/ml did not have decreased LVEF. While in some of the pa­tients, the value of biomarkers increased, others ex­perienced a decrease in values (Figure 3). Primary endpoint was observed in 25% (10) out of 40 patients who survived until visit 2: 9 had in­creased values of biomarkers, and 1 had decreased LVEF. Univariate logistic regression models are shown in Table 4 where associations of different variables with corresponding ORs and 95% CI are given for each variable. Of all the variables tested in univariate logistic regression, history of ischemic heart disease was statistically significantly linked to cardiotoxicity (OR 12.86, 95% CI 1.16–142.88; p = 0.04). After adjusting for age, sex and LVEF value at visit 1, history of ischemic heart disease remained predictive of cardiotoxicity (OR 35.22, 95% CI 7.08– 175.32, p = 0.023) (Table 5). Discussion Our results show that the treatment with platinum-based therapy did not induce clinically evident car­diotoxicity such as overt heart failure or other car­diac disease. Although no statistically significant or clinically important increase in the mean value for usTnT, NT-proBNP or decrease in LVEF or di­astolic dysfunction was observed among the entire TABLE 3. Biomarkers and echocardiography data usTnT – pg/ml 11.4 ± 5.4 10.9 ± 5.1 19 0.93 8.3 ±3.2 8 1 >30% increase in usTnT from Visit 1 – no. (%) 3 (15.8) 19 1 2 (25) 8 1 NT-proBNP – pg/ml 266 ± 250 258 ± 378 17 0.55 226 ± 430 8 0.60 >30% increase in NT-proBNP from Visit 1 – no. (%) 6 (35.3) 17 1 2 (25) 8 1 LVEF - % 68 ± 8 67 ± 8 22 0.66 69 ± 9 16 0.63 >30% increase in LVEF from Visit 1 – no. (%) 0 (0) 22 1 1 (6) 16 1 Diastolic dysfunction – no. (%) 9 (27.3) 6 (27.3) 21 1 4 (23.5) 17 1 Results are shown as mean values ± standard deviation and differences were analysed with Paired t-test for numeric and Cochran’s Q test for binomial variables. LVEF = left ventricular ejection fraction; N = of patients with complete evaluation at visit 1 and 2 or visit 1 and 3, NT= proBNP, n-terminal pro BNP; usTnT = ultrasensitive troponin T group, 25% of patients met the definition of cardio­toxicity. This phenomenon was largely driven by the known history of CV disease, even when ad­justed to clinically relevant confounders. Although platinum-based chemotherapy, as re­ported in animal models and case reports, could be associated with cardiotoxicity in patients with NSCLC, data from observational studies are lack­ ing.15-17 To our best knowledge, only one pro­spective study that investigated platinum-based cardiotoxicity in patients with NSCLC has been published.22 The authors of that study measured LVEF with a multiple gated acquisition (MUGA) scan before and 3 months after treatment with cisplatin-gemcitabine (31 patients) or epirubicin­gemcitabine (38 patients). Study results showed a small and marginally significant decrease in the LVEF of 2% in the cisplatin-gemcitabine arm and a larger significant decrease in the LVEF of 7% in the epirubicin-gemcitabine arm. A larger decrease in the anthracycline arm was expected, which con­firms that anthracycline chemotherapy is more cardiotoxic than platinum-based chemotherapy. Our results show a similar decrease in LVEF af­ter platinum-based chemotherapy measured with echocardiography, which is less accurate for LVEF assessment but generally available, less expensive and can identify several other cardiac diseases compared to MUGA scanning. Due to similar re­sults in both studies, echocardiography is possibly as efficient in detecting decreased LVEF as a sign of platinum-based cardiotoxicity as MUGA and rep­resents a valid method that is more feasible for use in clinical practice. Cisplatin and other platinum-based chemo­therapy regimens are also used in other types of cancers, such as ovarian, testicular, prostatic and FIGURE 3. The mean with 95% CI and for each individual patient values of usTnT, NT­proBNP and LVEF at visit 1 and visit 2 or 3, whichever value was higher for usTnT and NT-proBNP and lower for LVEF. CI = confidence interval; LVEF = left ventricular ejection fraction; n = terminal pro BNP; NT = proBNP; usTnT = ultrasensitive troponin T bladder. Some studies have measured cardiotoxic­ity of cisplatin in patients treated for testicular can­cer.18-20,26 Based on longer survival rates observed in patients with testicular cancer, there were several cohort studies that assessed long-term cardiotoxic­ity of more than 6 years.19,20,26 The main findings of these studies were increased diastolic dysfunction and slightly decreased LVEF. Although the median age of patients diagnosed and treated for testicu­lar cancer was approximately 35 years, several patients treated with cisplatin chemotherapy had cardiac events in the following years. Although the mean LVEF did not change after treatment, patients treated with cisplatin had a significantly higher risk for cardiovascular mortality and an in­creased rate of CV events compared to the general population. Similarly, in our study with a much shorter follow-up period, several patients expe­rienced an important increase in biomarkers or TABLE 4. P values for univariate logistic regression models for cardiotoxicity Age in years 1.044 (0.955–1.142) 0.34 Male sex 0.824 (0.198–3.432) 0.79 Metastatic disease 0.765 (0.182–3.210) 0.71 History of ischemic heart disease 12.857 (1.157–142.880) 0.04 History of arterial hypertension 1.583 (0.377–6.649) 0.53 Glomerular filtration rate at Visit 1 1.000 (0.972–1.029) 1.00 No. of chemotherapy cycles 0.931 (0.474–1.827) 0.83 usTnT in pg/ml at Visit 1 0.961 (0.823–1.122) 0.62 NT-proBNP in pg/ml at Visit 1 1.000 (0.997–1.003) 0.93 LVEF in % at Visit 1 1.356 (0.000–37889.6) 0.95 LVEF = left ventricular ejection fraction; n = terminal pro BNP; NT = proBNP; usTnT = ultrasensitive troponin T TABLE 5. Multivariate regression model for cardiotoxicity Age in years 1.044 (0.955 – 1.142) 0.52 Male sex 0.824 (0.198 – 3.432) 0.21 History of ischemic heart disease 12.857 (1.157 – 142.880) 0.026 LVEF in % at Visit 1 1.356 (0.000 – 37889.6) 0.92 OR = odds ratio; LVEF = left ventricular ejection fraction decrease in LVEF. As none of these patients devel­oped any symptoms or signs suggestive of heart failure or cardiac disease, the importance of this finding is uncertain. While elevations in troponin and BNP can indicate early preclinical cardiotox­icity, a decrease in LV systolic function measured by LVEF shows more extensive cardiac damage.27 We can assume that patients who had elevations in biomarkers or lowered LVEF would have an in­creased risk for developing cardiac disease in the future.28 For a minority of our patients with early lung cancer, a continuous cardiac surveillance is already planned. As we have evidence-based ther­apy for heart failure, screening (at least in high risk patients) should be part of routine clinical practice to detect or even prevent cardiac damage and pos­sible cardiac events. The findings of logistic regression model analy­sis suggest that patients who had a positive his­tory of ischemic heart disease were more likely to have increased values of biomarkers or a decrease in LVEF after treatment and could be considered as those at risk and eligible for screening. Another group that would likely benefit from screening is the patients with a history of heart failure or left ventricular systolic dysfunction. With no such pa­tients at visit 1, we were not able to test this hy­pothesis; given the risks and the availability of spe­cific cardiac treatment29,30, clinicians would hardly exclude these patients from a closer follow-up dur­ing chemotherapy. We are aware of several limitations to this study. First, the sample size and several missing values for biomarkers and echocardiography could in­duce bias and limit the interpretation of the re­sults. Patients were frequently assessed only us­ing biomarkers or echocardiography, often only in visit 2 or visit 3, therefore complete assessment of both biomarkers and echocardiography is miss­ing in several patients. Combining the elevated biomarker and decreased LVEF at either visit 2 or 3 allowed complete assessment of cardiotoxicity. Even though usTnT, NT-proBNP and LVEF are ef­fected by different mechanisms, in asymptomatic patients, they are related to the development of preclinical cardiotoxicity. To further evaluate the extent of incomplete data a post hoc study power was calculated. For the same standard deviation and number of paired data as in our sample, we estimate that we had > 80% power to detect a > 3 pg/ml, > 250 pg/ml and > 7% increase/decrease in usTnT, NT-proBNP and LVEF, respectively. Second, blood for biomarkers was not taken imme­diately after the chemotherapy cycle but on day 1 of the following cycle. Therefore, any changes in biomarker values could not reflect the acute effect of the platinum-based chemotherapy cycles, as el­evations in NT-proBNP and usTnT persist for 1-2 weeks, but the interval between platinum doublets used in our patients was 3 weeks.31 Last, in our 6-month follow-up, we were unable to evaluate long term cardiotoxicity. Although patients with advanced stage NSCLC most likely would not de­velop long-term cardiotoxicity, patients with early stage NSCLC with longer survival could. To an­swer this question, future studies exploring LVEF and biomarker changes and possible development of overt heart failure in those with preclinical car­diotoxicity in early stage NSCLC patients treated with platinum-based chemotherapy are necessary. Despite its limitations, we think that this study is important, because it is the second study to evaluate cardiotoxicity in NSCLC patients treated with platinum-based chemotherapy. We were able to detect changes in cardiac biomarkers and to identify patients who may be at risk for devel­oping cardiotoxicity. Long-term platinum-based cardiotoxicity has already been observed in other cancers19,20,26, and with the invention of new anti­cancer therapies leading to a longer life expectancy for patients with NSCLC, screening for early car­diac injury might become an important part of care for patients with NSCLC, particularly those with early disease. With other concomitant or sequen­tial treatments, such as radiotherapy and targeted therapies, even small cardiotoxic effects of individ­ual type of therapy could contribute to late cardio­toxicity and higher cardiac mortalities as shown in other types of cancers.10 Detection of early cardiac injury may facilitate early therapeutic measures and healthy lifestyle modifications in patients with NSCLC who have a good prognosis, thus leading to better survival rates in those patients. Conclusions and clinical implications Our study shows that platinum-based therapy in patients with NSCLC did not induce clinically overt acute or subacute cardiotoxicity. Despite the fact that mean values of biomarkers and LVEF did not change significantly after treatment, 25% of patients experienced changes defined as cardio­toxic. We have shown that patients with a history of ischemic heart disease were more likely to ex­perience an increase in laboratory biomarkers or a decrease in LVEF. Therefore, we suggest assess­ing LVEF and laboratory biomarkers to screen for cardiotoxicity in patients with NSCLC treated with platinum-based chemotherapy who have a history of ischemic heart disease, heart failure or asympto­matic left ventricular dysfunction. Further research should focus on patients with a history of cardiac disease, patients with longer life expectancy - es­pecially in early stage NSCLC - and any novel bio­markers that may emerge in the future. Acknowledgements We would like to thank Ivanka Kržišnik for her dedicated and meticulous collection of data. The study was publicly funded by the Slovenian Research Agency (ARRS) grant number J3-2394 (B). References 1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. GLOBOCAN 2012 v1.0, Cancer incidence and mortality worldwide: IARC CancerBase No. 11. [Internet]. Lyon, France: International Agency for Research on Cancer 2013. [Citated 2015 Sep 30] Available at: http://globo­can.iarc.fr 2. D’Addario G, Früh M, Reck M, Baumann P, Klepetko W, Felip E. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010; 21 (Suppl 5): v116-9. 3. Vansteenkiste J, Ruysscher D De, Eberhardt WEE, Lim E, Senan S, Felip E, et al. Early and locally advanced non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2013; 24 (Suppl 6): vi89-98. 4. Cufer T, Ovcaricek T, O’Brien MER. Systemic therapy of advanced non-small cell lung cancer: major-developments of the last 5-years. Eur J Cancer 2013; 49: 1216-25. 5. Yeh ETH, Bickford CL. Cardiovascular complications of cancer therapy: inci­dence, pathogenesis, diagnosis, and management. J Am Coll Cardiol 2009; 53: 2231-47. 6. Albini A, Pennesi G, Donatelli F, Cammarota R, Flora S De, Noonan DM. Cardiotoxicity of anticancer drugs: the need for cardio-oncology and cardio­oncological prevention. J Natl Cancer Inst 2010; 102: 14-25. 7. Vejpongsa P, Yeh ETH. Prevention of anthracycline-induced cardiotoxicity. J Am Coll Cardiol 2014; 64: 938-45. 8. Lainscak M, Haehling S von, Anker SD. Natriuretic peptides and other bio­markers in chronic heart failure: from BNP, NT-proBNP, and MR-proANP to routine biochemical markers. Int J Cardiol 2009; 132: 303-11. 9. Cardinale D, Sandri MT, Colombo A, Colombo N, Boeri M, Lamantia G, et al. Prognostic value of troponin I in cardiac risk stratification of cancer patients undergoing high-dose chemotherapy. Circulation 2004; 109: 2749-54. 10. Weaver KE, Foraker RE, Alfano CM, Rowland JH, Arora NK, Bellizzi KM, et al. Cardiovascular risk factors among long-term survivors of breast, prostate, colorectal, and gynecologic cancers: a gap in survivorship care? J Cancer Surviv 2013; 7: 253-61. 11. Patane S. Cardiotoxicity: Cisplatin and long-term cancer survivors. Int J Cardiol 2014; 175: 201-2. 12. Bano N, Najam R, Qazi F. Adverse cardiac manifestations of cisplatin - a review. Int J Pharm Sci Rev Res 2013; 18: 80-5. 13. Khan S, Chen CL, Brady MS, Parameswaran R, Moore R, Hassoun H, et al. Unstable angina associated with cisplatin and carboplatin in a patient with advanced melanoma. J Clin Oncol 2012; 30: e163-4. 14. Moore RA, Adel N, Riedel E, Bhutani M, Feldman DR, Tabbara NE, et al. High incidence of thromboembolic events in patients treated with cisplatin­based chemotherapy: a large retrospective analysis. J Clin Oncol 2011; 29: 3466-73. 15. Demkow U, Stelmaszczyk-Emmel A. Cardiotoxicity of cisplatin-based chem­otherapy in advanced non-small cell lung cancer patients. Respir Physiol Neurobiol 2013; 187: 64-7. 16. El-Awady E-SE, Moustafa YM, Abo-Elmatty DM, Radwan A. Cisplatin­induced cardiotoxicity: mechanisms and cardioprotective strategies. Eur J Pharmacol 2011; 650: 335-41. 17. Hussein A, Ahmed AAE, Shouman SA, Sharawy S. Ameliorating effect of DL-.-lipoic acid against cisplatin-induced nephrotoxicity and cardiotoxicity in experimental animals. Drug Discov Ther 2012; 6: 147-56. 18. Schinkel LD van, Willemse PM, Meer RW van der, Burggraaf J, Elderen SGC van, Smit JWA, et al. Chemotherapy for testicular cancer induces acute alterations in diastolic heart function. Br J Cancer 2013; 109: 891-6. 19. Strumberg D, Brugge S, Korn MW, Koeppen S, Ranft J, Scheiber G, et al. Evaluation of long-term toxicity in patients after cisplatin-based chemother­apy for non-seminomatous testicular cancer. Ann Oncol 2002; 13: 229-36. 20. Altena R, Hummel YM, Nuver J, Smit a J, Lefrandt JD, Boer R a de, et al. Longitudinal changes in cardiac function after cisplatin-based chemothera­py for testicular cancer. Ann Oncol 2011; 22: 2286-93. 21. Hardy D, Liu C-C, Cormier JN, Xia R, Du XL. Cardiac toxicity in association with chemotherapy and radiation therapy in a large cohort of older patients with non-small-cell lung cancer. Ann Oncol 2010; 21: 1825-33. 22. Wachters FM, Graaf WTA Van Der, Groen HJM. Cardiotoxicity in advanced non-small cell lung cancer patients treated with platinum and non-platinum based combinations as first-line treatment. Anticancer Res 2004; 24: 2079­ 83. 22 23. Lang RM, Bierig M, Devereux RB, Flachskampf F, Foster E, Pellikka P, et al. Recommendations for chamber quantification. Eur J Echocardiogr 2006; 7: 79-108. 24. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth O A, et al. Recommendations for the evaluation of left ventricular diastolic function by echocardiography. Eur J Echocardiogr 2009; 10: 165-93. 25. Seidman A, Hudis C, Pierri MK, Shak S, Paton V, Ashby M, et al. Cardiac dysfunction in the trastuzumab clinical trials experience. J Clin Oncol 2002; 20: 1215-21. 26. Meinardi BMT, Gietema JA, Veldhuisen DJ Van, Runne MA, Sluiter WJ, Mulder NH, et al. Cardiovascular morbidity in long-term survivors of meta­static testicular cancer. J Clin Oncol 2000; 18: 1725-32. 27. S. Gillespie H, J. McGann C, D. Wilson B. Noninvasive diagnosis of chemo­therapy related cardiotoxicity. Curr Cardiol Rev 2012; 7: 234-44. 28. Daniels LB, Laughlin GA, Clopton P, Maisel AS, Barrett-Connor E. Minimally elevated cardiac troponin T and elevated N-terminal pro-B-type natriuretic peptide predict mortality in older adults: results from the Rancho Bernardo Study. J Am Coll Cardiol 2008; 52: 450-9. 29. Gharib MI, Burnett a K. Chemotherapy-induced cardiotoxicity: current prac­tice and prospects of prophylaxis. Eur J Heart Fail 2002; 4: 235-42. 30. Eschenhagen T, Force T, Ewer MS, Keulenaer GW de, Suter TM, Anker SD, et al. Cardiovascular side effects of cancer therapies: a position statement from the Heart Failure Association of the European Society of Cardiology. Eur J Heart Fail 2011; 13: 1-10. 31. Patil H, Vaidya O, Bogart D. A review of causes and systemic approach to cardiac troponin elevation. Clin Cardiol 2011; 34: 723-8. research article Contribution of diffusion weighted MRI to diagnosis and staging in gastric tumors and comparison with multi-detector computed tomography Harun Arslan1, Mehmet Fatih Özbay2, Iskan Çalli3, Erkan Dogan4, Sebahattin Çelik5, Abdussamet Batur1, Aydin Bora1, Alpaslan Yavuz1, Mehmet Deniz Bulut1, Mesut Özgökçe1, Mehmet Çetin Kotan5 1 YuzuncuYil University DursunOdabas Medical Center, Department of Radiology, Van, Turkey 2 Van Training and Research Hospital,Department of Internal Medicine, Van, Turkey 3 Van Training and Research Hospital,Department of General Surgery, Van, Turkey 4 YuzuncuYil University DursunOdabas Medical Center, Department of Medical Oncology, Van, Turkey 5 YuzuncuYil University DursunOdabas Medical Center, Department of General Surgery, Van, Turkey Radiol Oncol 2017; 51(1): 23-29. Received 7 February 2016 Accepted 24 August 2016 Correspondence to: HarunArslan, M.D., YuzuncuYil University, DursunOdabas Medical Center, Department of Radiology, Van, Turkey. Phone: +9 053 274 688 35; E-mail: harun.ars75@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Diagnostic performance of Diffusion-Weighted magnetic resonance Imaging (DWI) and Multi-Detector Computed Tomography (MDCT) for TNM (Tumor, Lymph node, Metastasis) staging of gastric cancer was compared. Patients and methods. We used axial T2-weighted images and DWI (b-0,400 and b-800 s/mm2) protocol on 51 pre-operative patients who had been diagnosed with gastric cancer. We also conducted MDCT examinations on them. We looked for a signal increase in the series of DWI images. The depth of tumor invasion in the stomach wall (tumor (T) staging), the involvement of lymph nodes (nodal (N) staging), and the presence or absence of metastases (metastatic staging) in DWI and CT images according to the TNM staging system were evaluated. In each diagnosis of the tumors, sensitivity, specificity, positive and negative accuracy rates of DWI and MDCT examinations were found through a comparison with the results of the surgical pathology, which is the gold standard method. In addition to the compatibilities of each examination with surgical pathology, kappa statistics were used. Results. Sensitivity and specificity of DWI and MDCT in lymph node staging were as follows: N1: DWI: 75.0%, 84.6%; MDCT: 66.7%, 82%;N2: DWI: 79.3%, 77.3%; MDCT: 69.0%, 68.2%; N3: DWI: 60.0%, 97.6%; MDCT: 50.0%, 90.2%. The diagnos­tic tool DWI seemed more compatible with the gold standard method (surgical pathology), especially in the staging of lymph node, when compared to MDCT. On the other hand, in T staging, the results of DWI and MDCT were better than the gold standard when the T stage increased. However, DWI did not demonstrate superiority to MDCT. The sensitivity and specificity of both imaging techniques for detecting distant metastasis were 100%. Conclusions. The diagnostic accuracy of DWI for TNM staging in gastric cancer before surgery is at a comparable level with MDCT and adding DWI to routine protocol of evaluating lymph nodes metastasis might increase diagnostic accuracy. Key words: gastric cancer; diffusion weighted imaging; MDCT; staging Introduction third leading cause of death from cancer. When clinically detected, it has already been in the ad-Although there are important geographical differ-vanced stage and made metastasis.1 Indistinct clini­ences worldwide, death due to gastric cancer is the cal presentation of gastric cancer is the main reason FIGURE 1. T4N3M1 C Axial MDCT (A,B,C) and DWI (D,E,D,F,G). (A) Axial contrasted-enhanced MDCT shows gastric corpus tumor and adjacent lymphadenopathies, (B) peripherally contrast enhanced lesions in the liver (metastasis), and (C) an invasive mass extending from the posterior of the gastric corpus to the fatty tissue (arrows). On DWI (D–G): on b800 DWI (D, E) there are diffusion restrictions in the gastric corpus and liver (Gastric tumor + liver metastasis), (F) restrictions that are compatible with lymphadenopathies are seen in the gastro-hepatic ligament, hepatic hilum, and the celiac axis, and (G) invasion of the fatty tissue in the posterior of the gastric corpus (arrow). for late diagnosis. Symptoms are generally non­specific or all symptoms do not appear together.2 Recently, methods such as gastroscopy, endoscop­ic ultrasonography (EUS), computed tomography (CT), and positron emission tomography PET/CT are generally used for the staging of gastric can­cer.3 With multi-dimensional reformatted (MPR) images and technical progress in multi-detector CT (MDCT) technology, which allows 3D imaging of the endoluminal surface of the stomach with thin-section axial images, MDCT is also generally used for the pre-operative evaluation of gastric cancer staging.4 However, in the clinical approach, some patients are not appropriate for contrast-enhanced CT due to renal dysfunction, hypersensitivity reac­tion to iodine-containing contrast agents, or they are avoiding radiation exposure because of the pos­sibility of pregnancy.4,5 In patients with suspected gastric cancer and to whom contrasted CT cannot be applied, MRI has been mostly used for the di­agnosis and staging of gastric cancer as an alter­native imaging method to MDCT in the evaluation of gastric cancer.6,7 Especially, diffusion-weighted magnetic resonance imaging (DWI), whose main feature is to reflect movement of water molecules in tissue and gather information about tissue in­tegration, was investigated in various tumors and the value of DWI was confirmed with various stud­ies in detecting and characterizing gastric cancers. Furthermore, it can provide information for the differentiation of benign lesions from malignant ones, detecting malignant lymphadenopathy or very small peritoneal seeding and tissue cellular­ity, which might be used for monitoring therapeu­tic efficacy. DWI, draws attention as an oncologic imaging tool.8-10 In our study, we tested our hypothesis that DWI might be useful in staging based on pre-operative diagnostic efficacy of DWI and MDCT in gastric cancer patients and correlation analysis of surgical pathology. Patients and methods Between the periods of April 2013 and May 2015, fifty-one patients with malignant gastric adeno­carcinoma diagnosis were included in the current study and their diagnosis was made with endo­scopic biopsy in our hospital. In 51 gastric can­cer patients, who were included in research and whose preoperative clinical TNM (Tumor-Node-Metastasis) staging were made, 37 were male and 14 were female. Their average age was 61 (range: 35 – 82) years old. Approval was taken from a local ethics committee and written consent was obtained from all patients. First, diffusion-weighted magnetic resonance imaging (DWI) and upper and lower abdomen CT were taken pre-operatively from all patients. All examinations were made with multi-slice CT de­vice (Siemens SOMATOM Sensation 16, Germany). In the CT, kV was 120 and mAs was 150. Each pa­tient was examined after eight hours of fasting. Stomach fullness was achieved by giving the pa­tient one liter of water during the examination. By entering the right antecubital vein with an 18G needle, 100 ml of contrast agent was administered (Omnipaque 350 mg, GE) at a rate of 2 ml/s by an automatic injector (Medrad, vision CT injection system). Approximately 70 seconds after the start of contrast medium administration, images were obtained at 2.5 and 5 mm thick axial slices start­ing from the superior of diaphragm to the level of the symphysis pubis while holding their breath. In order to create multi-planar reconstruction images, CT images were reconstituted with 1 mm thick pieces, at a reconstruction interval of 1 mm. For re­construction, thin-piece CT data were transferred to the 3D working station and coronal and sagittal MPR images were constituted with 3 mm intervals and 3 mm slice thickness. MRI Investigations were made with phased-array body coil as routine upper-abdomen MRI in Siemens Magnetom Symphony (Siemens, Erlangen, Germany) device. The field strength of the device is 1.5 Tesla (T), which is considered to have a high field strength. Gradient force of superconductive (Niobium-Titanium) magnetic is 30 mT/m and FOV width is 400 cm. Before the diffusion weighted procedure, T2­weighted True-FISP at axial plane (TR, 4.4s; TE, 2.2s; Average, 2; FlipAngle, 80o; Matrix, 256 x 256; number of sections, 25; thickness of sections, 5mm; FOV, 300; Average, 2; gap between sections, 15%) sequence and after that diffusion-weighted, sin-gle-shot, echo-planar spin sequence, and chemi­cal shift selective fat suppression technique (TR/ TE, 3700/76; Matrix, 128x128; number of sections, 30; FOV, 400; gap between sections, 15%; section thickness, 5 mm; duration, 156s; PAT Factor, 2; PAT mode, parallel imaging (GRAPPA) with modified sensitivity encoding) was applied. In DWI, b val­ues were set as 0,400 and 800 mm2/second. Evaluation of images An examination was conducted after installing diffusion-weighted images to the work station. All abdominal imaging MDCT and DWI results were assessed by two radiologists (HA, AB) who had 12 and 10 years of experience, respectively. Two radiologists were informed about the locali­zation of the lesions, which were diagnosed with gastric cancer due to endoscopic biopsy. However, they were blinded to the pathological findings. MDCT and DWI image analysis were performed. Reviewers were told to determine T(T1,T2,T3,T4) stage and N stage (N1,N2,N3) by classifying as positive or negative and M stage by classifying as MO or M1. In abdominal images, only DWI images were evaluated for staging. Conventional MR im­ages were not assessed in the staging. Surgical pathological analysis Surgery was performed within the following two weeks after cross sectional imaging. Curative or palliative gastrectomy and LN dissection were applied to 48 patients out of total 51 patients who were included in this study. Open-and-closed lap­arotomy was applied to three patients. For each case, pathological TNM staging was reported ac­cording to the Seventh AJCC Guideline.11 Determination of the depth of tumor invasion (T staging) We applied imaging criteria that were based on previous studies about T staging of gastric can­ TABLE 1. Effectiveness of DWI and MDCT in T staging according to surgical pathology result Sensitivity 72.7% 71.0% 55.6% Specificity 77.5% 80.0% 92.9% DWI PPV 47.1% 84.6% 62.5% NPV 91.2% 64.0% 90.7% Kappa (p) 0.419 (0.002) 0.488 (< 0.001) 0.506 (< 0.001) Sensitivity 63.6% 74.2% 66.7% Specificity 77.5% 80.0% 95.2% MDCT PPV 43.8% 85.2% 75.0% NPV 88.6% 66.7% 93.0% Kappa (p) 0.353 (0.009) 0.523 (< 0.001) 0.647 (< 0.001) CT = Computed Tomography; DWI = Diffusion Weighted Imaging; NPV = Negative Predictive Value; PPV= Positive Predictive Value cer and were modified according to the Seventh AJCC Guideline.10,11 For each T stage, imaging cri­teria were as follows: . T2 stage (. muscularis pro­pria invasion), it is an abnormal growing mass or wall thickness with/without ulceration, which has trans-mural involvement and irregular external border. The thin outer layer around the tumor and clear perigastric fatty tissue is preserved. T3 stage (penetrates subserosal connective tissue without involvement of the visceral peritoneum or adja­cent structures), transmural tumor involves the en­tire stomach wall and 1) clear perigastric fat plane around the tumor is maintained, or 2) fine per­igastric oil spicules that extend throughout small and large omentum and do not spread beyond the adjacent perigastric vessels. T4 stage (invasion of serosa (visceral peritoneum) or adjacent structures) is a transmural tumor with irregular borders and perigastric oil infiltration, which involves the en­tire stomach wall and shows invasion beyond adja­cent perigastric vessels. Detection of regional lymph node involvement (N staging) Positive lymph nodes can be detected according to their sizes in MDCT or MRDWI. If the shortest diameter of the largest regional lymph node is . 8 mm, the patient should be considered as N posi­tive; if it is < 8 mm, the patient should be consid­ered as negative. If there is at least one regional lymph node that has a shortest diameter of . 8 mm in DWI or there is any lymph node that shows higher signal intensity than muscle, the patient was classified as N-positive.7 Detection of distant metastasis (M staging) If suspicious lesions were seen in the MDCT or DWI about distant metastasis such as the liver, adrenal gland, distant lymph nodes, and perito­neum involvement, the patient was considered to be M1. Statistical analysis In gastric tumors, the comparison of DWI, MDCT, and surgical pathology was made using SPSS ver­sion 20 (SPSS Inc. Chicago,IL, USA) software. TNM results of two different imaging techniques (DWI and CT) and TNM results of surgical pathology, which is accepted as the gold standard method, was compared and assessed using Pearson’s chi-square (chi-square) or Fisher’s exact test depend­ing on the situation. Conformity between two different imaging techniques (DWI and CT) and surgical pathology was evaluated with the Kappa test. Additionally, sensitivity, specificity, positive predictive value, negative predictive value, and ac­curacy ratio of diagnostic tests were calculated. P values < 0.05 were considered statistically signifi­cant. Results Pathologic TNM staging of gastric cancer A total of 51 cases were included in the analysis. In the results of both surgical pathology and other two diagnostic tools (DWI and MD CT); there were no cases in the T1 stage and N0 stage. Because of this, T staging was made for T2 and higher and N staging was made for N1 and higher stages. T and N staging were confirmed in 51 patients to whom gastrectomy and LN dissection were applied. It was confirmed that in DWI 17 patients and in MDCT, 16 patients were stage T2. In DWI 26 pa­tients and in MDCT 27 patients were stage T3. It was confirmed that in DWI seven patients and in MDCT seven patients were stage T4a and in MDCT one patient was stage 4b. In 51 patients, lymph node was positive. In two patients, peritoneum in­volvement was confirmed by radiologic evaluation and they were evaluated as M1. Other 49 patients were assessed as M0. TABLE 2. Efficiency of DWI and MDCT in N staging according to surgical pathology Diagnostic performance of T staging in result gastric cancer T staging (T2, T3, T4) diagnosis of DWI was differ­ent from surgical pathology result (p values were: Sensitivity 75.0% 79.3% 60.0% 0.03, < 0.001 and 0.002 respectively). Similarly, T Specificity 84.6% 77.3% 97.6% staging of MDCT was also different from the sur- DWI PPV 60.0% 82.1% 85.7% gical pathology results (p values: 0.014, < 0.001, < NPV 91.7% 73.9% 90.9% 0.001, respectively). When we looked for Kappa compatibility test for both diagnostic tools their Kappa (p) 0.549 (< 0.001) 0.563 (< 0.001) 0.649 (< 0.001) compatibility with surgical pathology was gener- Sensitivity 66.7% 69.0% 50.0% ally weak (Table 1). Specificity 82.1% 68.2% 90.2% PPV 53.3% 74.1% 55.6% MDCT Diagnostic performance of N staging in NPV 88.9% 62.5% 88.1% gastric cancer Kappa (p) 0.448 (0.001) 0.367 (0.008) 0.418 (0.003) When we compared results of the DWI and sur­gical pathology in the staging of the lymph node; staging of DWI (N1, N2, N3) was different from the surgical pathology (for the three stages, p < 0.001). Similarly, a significant difference was found between MDCT results and surgical pathology re­sults in N staging (p values; 0.003, 0.009, and 0.009, respectively). When we looked for Kappa compat­ibility test for N staging (Table 2), we detected that DWI is compatible with surgical pathology at a moderate-fine level. However, the compatibility of MDCT was weak. According to surgical pathology result, distant metastasis (M1) was detected in total two cases. For M1, DWI and MDCT had full compliance with the gold standard (Kappa = 1.00 p < 0.001). Sensitivity and specificity of both imaging techniques for de­tecting distant metastasis was 100%. Discussion MRI and CT play an important role in the diag­nosis and preoperative staging of gastric cancer.7,8 Gastric cancer generally does not have any symp­toms when it is at a stage at which a complete cure can be achieved. On the other hand, when clini­cally detected, it is generally at an advanced stage (local advanced or metastatic stage).12 Most of our cases were detected at an advanced stage. An accu­rate assessment of the depth of the tumor invasion into the stomach wall, local lymph node involve­ment, and metastasis presence or absence is very important for the selection of therapeutic strategy. In spite of the development of methods such as computed tomography (CT), positron emission to­mography (PET), magnetic resonance (MR), ultra­sound (US), gastroscopy, double-contrast barium CT = Computed Tomography; DWI = Diffusion Weighted Imaging; NPV = Negative Predictive Value; PPV= Positive Predictive Value X-ray, and endoscopic ultrasound (EUS), there are still difficulties in the diagnosis and staging.13 Preoperative staging of gastric cancers has gained more importance with the recent developments in endoscopic treatment and also minimally invasive treatments of gastric cancers such as laparoscopic surgery.14 Recently, other MRI methods have been put into routine use, as well as conventional MR im­aging. One of these methods is diffusion weighted MR imaging (DWI). Most of the studies about DWI were conducted with the Single Shot EPI (SSEPI) technique. An image is taken in a time unit of less than a second with the SSEPI technique and thus physiological movement is frozen and image arti­facts are reduced.15 We used the SSEPI technique in the current study. Diffusion weighted MR imag­ing makes it possible to obtain information about the perfusion and diffusion of tissue at the same time, so normal and abnormal tissues can be dis­tinguished from each other. Various abnormalities can be detected because of this technique.15 Today, the only imaging method that provides informa­tion about tumor cellularity is DWI.16 This cellu­lar density difference reflects in the DWI. Various studies have reported that diffusion weighted MRI provides an important contribution to the diagno­sis in the characterization of the tumor at differ­ent sites such as the liver, pancreas, ovary, colon, cervix, bladder, prostate, and breast. In association with cellularity of tumors, it was specified that malignant tumors have more diffusion limitations compared to benign lesions.17 So far, very few studies compared diagnostic performance of MDCT and 1.5-3 T MRDWI for the local staging of gastric cancer and showed that DWI is comparable with MDCT in T staging.7,8,18 Many studies have recently shown that using MDCT with MPR imaging in T staging is accurate with a ratio of 83% and 91%, respectively.8,19 The current study demonstrated that DWI did not increase diagnostic performance significantly in T staging. In other studies that compared DWI and MDCT, DWI generally used for staging with conventional MRI and their accuracy values were high.8 In our study, using only DWI images were compared with MDCT in staging. We related low accuracy ratios in T staging with this condition. DWI and MDCT results in T staging can be bet­ter as the T stage increases compared to the gold standard. As T stage progresses, the effectiveness of DWI is related to an increase of size and den­sity of tumor cells and limitation of intracellular diffusion during the impairment of regulation of tumor cells. As a result of these features, this study revealed that DWI could define advanced gastric cancer but it was not an effective method to detect early gastric cancers (T0-T1). We detected that it was possible to have similar results with almost the same accuracy with DWI for detecting wall inva­sion (T2, T3, T4) in 51 patients with gastric cancer when compared to MDCT. Although the results of our work, in terms of N staging, did not have any significant difference for diagnostic accuracy between treatment methods, it was shown that DWI had significantly higher sensi­tivity for evaluating metastatic lymph nodes when compared with MDCT. Lymph node involvement is one of the most important prognostic factors, es­pecially in gastric cancer. Lymph node metastasis is frequent in gastric cancer patients.20 However, as surgery related mortality and complications in­crease in the scope of analysis of the lymph nodes, it has been an issue that has been debated for a long time. In this context, for appropriate treatment strategies, local metastatic lymph nodes should be correctly considered before surgery.21,22 To this day, the diagnostic accuracy of MDCT in the N staging of gastric cancer varies between 46% and 83%.8,22 The weak and variable diagnostic perfor­mance might be a result of current lymph node me­tastasis criteria, which is generally based on lymph node size and shape; however, there can be micro­scopic metastasis in small sized lymph nodes and benign nodes usually can expand in gastric cancer patients when compared with a healthy popula­tion. It should be considered that lymph node size might not be adequate to distinguish metastatic lymph nodes from benign lymph nodes, especial­ly in gastric cancer patients.22,12 DWI also shows more diagnostic accuracy for detecting metastatic lymph nodes when compared to measurements in MRI and CT imaging. In addition to lymph node findings, there is potential to predict lymph node metastases with computerized analysis of tumor characteristics, such as primary tumor localization, diameter, Borrman types, histological types, infil­tration depth, serosa invasion, molecular, and ge­netic markers.7 In the current study, apparent dif­fusion restriction is observed in lymph nodes with millimeter dimension due to diffusion weighted MRI and these were accepted as metastatic lymph nodes were confirmed after surgical pathology results (Figure 2). Malignant lesions are generally more cellular than benign lesions and this feature can provide information about the perfusion and diffusion of a tissue. Because of this high cellular­ity, malignant lesions have higher signal density in DWI and this demonstrates that DWI is more bene­ficial than MDCT to differentiate malignant lesions from benign ones. Moreover, the accuracy of N staging with DWI was statistically higher than the accuracy of N staging with MDCT. Furthermore, when combined with other morphological charac­teristics, DWI reached a higher predictive power than MDCT. In our study, more lymph nodes, es­pecially under 1 cm, were detected when evaluated with DWI. DWI signal changes could have higher predictive power than other morphological factors, including contrast uptake pattern and border ir­regularity for distinguishing metastatic and benign lymph nodes. Distant metastases of gastric cancers include liv­er metastasis and peritoneum involvement. They can be more clearly detected by using DWI without any contrast agent when compared with MDCT. In our study, distant metastasis in two gastric cancer patients included peritoneum involvement and liver metastasis and these were detected with both DWI and MDCT. There was no statistically signifi­cant difference between these two methods. This might be due to the low number of metastatic pa­tients in the study. There were some limitations in this study. First of all, a relatively high proportion of patients in stage T3 might cause a statistical bias. Secondly, we did not detect any T1 stage cancer in the pa­tient population. For this reason, further studies are required with more patients that include T1 stage cancer. Thirdly, as each lymph node was not compared separately for N staging, we could not match imaging and pathology diagnosis for each lymph node. Fourthly, as we used only qualitative assessment in DWI without using any quantita­tive measurement or size criteria, further studies are required to determine optimum diagnostic cri­teria with DWI in TNM staging of gastric cancer. Finally, two radiologists were informed about the localization of lesions but they were blinded to oth­er endoscopic and surgical pathological findings. In conclusion, DWI has a potential clinical use field, especially for the assessment of node involve­ment in gastric cancer. In particular, the conven­tional MRI and DWI combination might produce accurate performance with high sensitivity and specificity in lymph nodes smaller than one cen­timetre. When analyzed from a clinical viewpoint, an accurate lymph node metastasis evaluation be­fore operation facilitates lymphadenectomy sur­gery and may reduce the risk of complications. References 1. Shinya S, Sasaki T, Nakagawa Y, Guiquing Z, Yamamoto F, Yamashita Y. The usefulness of diffusion-weighted imaging (DWI) for the detection of gastric cancer. Hepatogastroenterol 2007; 54: 1378-81. 2. Dan YY, So JB, Yeoh KG. Endoscopic screening for gastric cancer. Clin Gastroenterol Hepatol 2006; 4: 709-16. 3. Smyth E, Schöder H, Strong VE, Capanu M, Kelsen DP, Coit DG, et al. A prospective evaluation of the utility of 2-deoxy-2-[(18) F]fluoro-D-glucose positron emission tomography and computed tomography in staging lo­callyadvanced gastric cancer. Cancer 2012; 118: 5481-88. 4. Kim JI, Kim YH, Lee KH, Kim SY, Lee YJ, Park YS, et al. Type-specific diagnosis and evaluationof longitudinal tumor extent of borrmann type IV gastric cancer: CT versus gastroscopy. Korean J Radiol 2013; 14: 597-606. 5. Makino T, Fujiwara Y, Takiguchi S, Tsuboyama T, Kim T, Nushijima Y, et al. Preoperative T stagingof gastric cancer by multi-detector row computed tomography. Surgery 2011; 149: 672-79. 6. Giganti F, De Cobelli F, Canevari C, Orsenigo E, Gallivanone F, Esposito A, et al. Response to chemotherapy in gastric adenocarcinoma with diffusion-weighted MRI and F-FDGPET/CT: correlation of apparent diffusion coef­ficient and partial volume corrected standardized uptake value with histo­logical tumorregression grade. J Magn Reson Imaging 2014; 40: 1147-57. 7. Liu S, He J, Guan W, Li Q, Zhang X, Mao H, et al. Preoperative T staging of gas­tric cancer: comparison of diffusion- and T2-weighted magnetic resonance imaging. J Comput Assist Tomogr 2014; 38: 544-50. 8. Joo I, Lee JM, Kim JH, Shin CI, Han JK, Choi BI. Prospective comparison of 3T MRI with diffusion-weighted imaging and MDCT for thepreoperative TNM staging of gastric cancer. J Magn Reson Imaging 2015; 41: 814-21. 9. Bonekamp S, Corona-Villalobos CP, Kamel IR. Oncologic applications of dif­fusion-weighted MRI in the body. J Magn Reson Imaging 2012; 35: 257-79. 10. Avcu S, Arslan H, Unal O, Kotan C, Izmirli M. The role of diffusion-weighted MR imaging and ADC values in the diagnosisof gastric tumors. JBR-BTR 2012; 95: 1-5. 11. Edge SB BD, Compton CC, Fritz AG, Greene FL, Trotti A, editors. AJCC cancer staging manual. 7th edition. New York, NY: Springer; 2010. 12. Chen CY, Hsu JS, Wu DC, Kang WY, Hsieh JS, Jaw TS, et al. Gastric cancer: preoperative local staging with 3D multi-detector row CT--correlation with surgical and histopathologic results. Radiology 2007; 242: 472-82. 13. Lian J, Chen S, Zhang Y, Qiu F. A meta-analysis of endoscopic submucosal dissection and EMR for early gastric cancer. Gastrointest Endosc 2012;76: 763-70. 14. Schuhmacher C, Reim D, Novotny A. Neoadjuvant treatment forgastric cancer. J Gastric Cancer 2013; 13: 73-8. 15. Chow LC, Bammer R, Moseley ME, Sommer FG. Single breath-hold dif­fusionweighted imaging of the abdomen. J Magn Reson Imaging 2003; 18: 377-82. 16. Naganawa S, Sato C, Kumada H, Ishigaki T, Miura S, Takizawa O. Apparent diffusion coefficient in cervical cancer of the uterus: comparison with the normal uterine cervix. Eur Radiol 2005; 15: 71-8. 17. Kuroki Y, Nasu K, Kuroki S, Murakami K, Hayashi T, Sekiguchi R, et al. Diffusion-weighted imaging of breast cancer with the sensitivity encoding technique: analysis of the apparent diffusion coefficient value. Magn Reson Med Sci 2004; 3: 79-85. 18. Shen L, Huang Y, Sun M, Xu H, Wei W, Wu W. Clinicopathological features associated with lymph node metastasis in early gastric cancer: analysis of a single-institution experience in China. Can J Gastroenterol 2009; 23: 353-6. 19. Kumano S, Okada M, Shimono T, Kuwabara M, Yagyu Y, Imaoka I, et al. T-staging of gastric cancer of air-filling multidetector-row CT: comparison with hydromultidetector-row CT. Eur J Radiol 2012; 81: 2953-60. 20. Cheng J, Wang Y, Deng J, McCarthy RJ, Wang G, Wang H, et al. Discrimination of Metastatic Lymph Nodes in Patients with Gastric Carcinoma UsingDiffusion-Weighted Imaging. J Magn Reson Imaging 2013; 37: 1436­ 44. 21. Kwee RM, Kwee TC. Imaging in assessing lymph node status ingastric cancer. Gastric Cancer 2009; 12: 6-22. 22. Park HS, Kim YJ, Ko SY, Yoo MW, Lee KY, Jung SI, et al. Benign regional lymph nodes in gastric cancer on multidetector row CT. Acta Radiol 2012; 53: 501-7. research article Electrotransfer of plasmid DNA radiosensitizes B16F10 tumors through activation of immune response Monika Savarin1, Urska Kamensek1, Maja Cemazar1,2, Richard Heller4, Gregor Sersa1,3 1 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Health Sciences, University of Primorska, Izola, Slovenia 3 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 4 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, USA Radiol Oncol 2017; 51(1): 30-39. Received 20 December 2016 Accepted 18 January 2017 Correspondence to: Prof. Gregor Serša, Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Conflict of interest: The authors declare no conflict of interest Background. Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. Materials and methods. The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, prolifera­tion, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. Results. Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in ex­cellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. Conclusions. The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tu­mor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vascu­lature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response. Key words: gene therapy; electrotransfer; plasmid, irradiation, immune response, melanoma Introduction transport.1 It is effective for delivery of smaller molecules, as chemotherapeutics in electrochemo-Electroporation is used as drug delivery system therapy (ECT)1-3 and also for larger molecules, as for molecules with hampered transmembrane are plasmids in gene electrotransfer (GET). GET is recently getting a lot of scientific consideration and it is used for enhanced DNA delivery into various tissue types (i.e. skin, liver, kidney etc.), as well as into tumors.4-6 Its effectiveness was first demon­strated in a wide range of preclinical studies and has thereafter proceeded to clinical oncology, vet­erinary and human. The results of clinical trials of GET of plasmid encoding human IL-127 and anti­angiogenic plasmid AMEP8, are promising. In addi­tion to GET of therapeutic plasmids, some plasmids devoid of therapeutic genes have also resulted in good antitumor effectiveness.9,10 This phenomenon was observed in different tumors models and by using different plasmids. It was attributed to im­mune sensing of the introduced DNA as a DAMP (Damage Associated Molecular Pattern), which switch leads to activation of an immune response.11 Radiotherapy is one of the principal treatment modalities for primary tumors and their metasta­ses.12 Nowadays, irradiation is widely investigated for its associated effects on priming antitumor im­munity.13 There is growing evidence of irradia­tion’s effect on the antitumor immune response, inducing immunogenic cell death and generating danger signals. An important danger signal after irradiation is DNA released from the nucleus of dy­ing cells. This DNA is recognized by the immune system as a DAMP, and can promote the activation of immune response against irradiated cells.14,15 Studies combining tumor irradiation with GET of different therapeutic plasmid demonstrated tu­mor radiosensitization.16-18 A promising approach to target tumors and its microenvironment with a combined treatment modality is through destruct­ing abnormal tumor blood vessels. One of the promising targets, gaining on its value due to dif­ferent signaling pathways from VEGF, is endoglin. It is a TGF-ß coreceptor and has already demon­strated good antitumor and antimetastatic effec­tiveness in different tumor models when targeted with GET of plasmid.18–22 In particular, GET of shRNA for silencing endoglin in B16F10 melanoma mice tumors that express high levels of endoglin, resulted in up to 58% of tumor cures.23 Tissue specific eukaryotic promoters are tightly regulated and mainly drive expression of transgene in specific cell types, although minimal unspecific expression in non-targeted tissue can also occur.24 We constructed a plasmid containing a tissue spe­cific promoter for endothelin and encoding shRNA for silencing endoglin. This plasmid was tested in a previous study, where the effectiveness of the plasmid with tissue specific promoter was com­pared to a plasmid with constitutive promoter in a tumor model that does not express targeted molecule, endoglin. In vivo, the effectiveness of the GET of the plasmids was comparable and resulted in significant radiosensitization, which resulted in prolonged tumor growth delay with nearly 50% of tumor free mice. Thus, the aim of this current study was to determine, whether GET of plasmid, with tissue specific promoter and encoding shRNA for silencing endoglin, can radiosensitize melanoma B16F10 tumors, which express targeted molecule, endoglin, and possibly have also some immu­nomodulatory effectiveness. Materials and methods Cell lines and plasmids Murine melanoma cell line B16F10 (American Type Culture Collection, Manassas, VA, USA) was cultured in advanced minimum essential me­dium (AMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS, Gibco), 10 mM/L L-glutamine (GlutaMAX, Gibco), 100 U/mL penicillin (Grünenthal, Aachen, Germany) and 50 mg/mL gentamicin (Krka, Novo mesto, Slovenia) in a 5% CO2 humidified incubator at 37°C. The plasmid with tissue specific promoter for endothelial cells, encoding shRNA for silencing endoglin (pET-antiCD105; TS plasmid) was used in experiments as the therapeutic plasmid.18 The con­trol plasmid, encoding shRNA with no homology to any gene in the mouse genome and with constitu­tive CMV promoter, was used as a negative control (pControl).25 Amplification of both plasmids was performed in a competent E.coli (JM107; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). All plasmids were isolated using JETSTAR 2.0 ENDOTOXIN-FREE Plasmid MEGA Kit (Genomed, Löhne, Germany) and diluted in endo­toxin free water to a concentration of 1 µg/µL (in vit­ro experiments) and 4 µg/µL (in vivo experiments). Concentrations of plasmids were measured with a spectrophotometer at 260 nm (Epoch Microplate Spectrophotometer, Take3 Micro-Volume Plate, BioTek, Bad Friedrichshall, Germany) and purity of plasmid was determined by agarose gel elec­trophoresis and measurements of the absorbance ratio at 260 and 280 nm. Experimental animals All animal experiments were conducted in accord­ance with the guidelines for animal experiments of TABLE 1. Response of B16F10 melanoma to different treatment modalities CTRL 8 1.2 ± 0.1 0.0 ± 0.1 0 0 - - pControl 8 1.9 ± 0.2 0.7 ± 0.2 0 0 - - TS 8 2.2 ± 0.3 1.0 ± 0.3 0 0 - - 3 × EP 8 3.1 ± 0.3 2.0 ± 0.3 0 0 - - 3 × GET (pControl) 8 9.4 ± 6.5 8.2 ± 2.5 0 0 - - 3 × GET (TS) 9 9.5 ± 3.2 8.6 ± 3.0 4 44 3 75 IR 9 1.8 ± 0.3 0.7 ± 0.3 0 0 - - pControl + IR 9 2.5 ± 0.3 1.3 ± 0.3 1 11 1 100 TS + IR 8 4.0 ± 1.2 2.8 ± 1.2 0 0 - - 3 × EP+IR 9 4.3 ± 1.3 3.2 ± 1.3 2 22 0 0 3 × GET (pControl) + IR 9 36.0 ± n/a 34.9 ± n/a 8 89 5 63 3 × GET (TS) + IR 8 32.0 ± n/a 30.8 ± n/a 7 88 4 57 AM = Arithmetic mean; DT = Tumor doubling time; Groups: (CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET­antiCD105); n = Number of all mice in the group; n/a = Not applicable; SC = Mice resistant to secondary challenge; SEM = Standard error; TF = Tumor free mice; TGD = Tumor growth delay the EU Directive and the permission obtained from the Ministry of Agriculture and the Environment of the Republic of Slovenia (Permission No. 34401­1/2015/16), which was given, based on the approval of the National Ethics Committee for Experiments on Laboratory Animals). Female C57Bl/6 mice, 6-8-week old, purchased from Envigo Laboratories (Udine, Italy), were used in the study. Before the experiment, mice were subjected to an adaptation period of 2 weeks. Animals were maintained under specific patho­gen-free conditions at a constant room tempera­ture, humidity and a 12 h light/dark cycle. Food and water were provided ad libitum. For induction of subcutaneous tumors, a suspension of 1x106 B16F10 cells in 0.1 ml of physiological saline was injected subcutaneously into the back of the mice. The animals bearing tumors of 40 mm3 were ran­domly divided into experimental groups and sub­jected to a specific experimental protocol. The tu­mor measurements were completed when the tu­mors reached 350 mm3, and mice were humanely sacrificed. Experimental groups and the number of animals in each of them were as follows and as described in table Table 1: triple injection of endotoxin-free wa­ter alone (control group; CTRL) or of pControl or TS plasmids alone, or in combination with triple application of electric pulses alone (3 × EP) or com­bined with plasmids (3 × GET (pControl); 3 × GET (TS)). Furthermore, the remaining groups were also the ones in combination with irradiation (IR) and other therapies described above, which are three injections of plasmids (pControl + IR; TS + IR) and in combination with electric pulses (3 × EP + IR; 3 × GET (pControl) + IR; 3 × GET (TS) + IR). In vivo GET In vivo GET of plasmid into subcutaneous tumors was performed 3 times every second day (on days 0, 2 and 4). 12.5 µL (4 µg/µL) of plasmid (150 µg in total) in endotoxin-free H2O was injected intratu­morally 10 min before 8 square electric pulses with a voltage-to-distance ratio of 600 V/cm, a pulse du­ration of 5 ms, and a frequency 1 Hz were applied. Electric pulses were generated by electric pulse generator ELECTRO CELL B10 (Betatech, L’Union, France) and delivered through 2 parallel stainless steel electrodes with 2 or 4 mm distance between them, depending on the tumor volume. After 4 pulses, electrodes were turned for 90° for 4 addi­tional pulses to assure GET to entire tumor. Irradiation of tumors Tumors were locally irradiated with a single dose of 15 Gy on day 1 from the beginning of the experi­ment, at a dose rate of 2.16 Gy/min, using a Darpac 2000 X-ray unit (Gulmay Medical Ltd., Shepperton, UK) operating at 220 kV, 10 mA, with 1.8-mm alu­minum filtration. During irradiation, mice were FIGURE 1. Histological sections of melanoma tumors on day 6 after the beginning of the therapy. CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET-antiCD105 restrained in special lead holders with apertures for irradiation of the tumors. Due to the fixed size of the apertures, some healthy tissue (3 – 5 mm of skin surrounding the tumor ) was exposed to the irradiation as well. Tumor growth The therapeutic potential in vivo was assessed by measuring the tumor size every second day and calculating tumor volume according to the for­ mula for ellipsoid: V=axbxc ./6, where a, b and c represent perpendicular tumor diameters.21,26 The tumor growth curves were drawn as arithmetic means (AM) with bars representing standard er­rors (SEM). The tumor growth delay for each experimen­tal group was calculated as the difference in tu­mor doubling times of experimental and control groups. Tumor doubling time is the number of days in which the initial tumor volume (40-50 mm3) doubles. Mice that remained tumor free for 70 days were termed tumor free and local tumor control was deemed to have been achieved (Table 1). The weight of the mice was followed as a general in­dex of systemic toxicity, and acute skin reaction in the whole irradiated field around the tumor was evaluated as described elsewhere.17 Tumor challenge of tumor free mice After 70 days, when mice were designated as tu­mor free, they were challenged with a subcuta­neous injection of 1 × 106 B16F10 cells in 0.1 ml of physiological saline in the right flank. Mice that at least 20 days after the challenge remained tumor free were marked as resistant to secondary chal­lenge (Table 1, Figure 2). The growing tumors were measured twice a week and when volume of 150 mm3 was reached mice were sacrificed and tumors were collected for further histological analysis as described below. Histology After therapies, at day 6, three mice from each ex­perimental group were sacrificed. The tumors were excised, fixed in IHC zinc fixative (BD Biosciences, San Diego, CA, USA) and embedded in paraffin. Six consecutive 2-µm thick sections were cut from each paraffin block and stained as followed. To estimate the percent of the area of tumor necro­sis, the first section was stained with hematoxylin and eosin. The other five sections were used for immunohistochemical (IHC) staining to evaluate percentage of hypoxic cells, cells in apoptosis, im­mune cells, proliferating cells and the number of blood vessels. To determine hypoxic cells, rabbit polyclonal antibodies against HIF-1-alpha (ab2185, Abcam, Cambridge, MA, USA) at dilution 1:3500, were used. In addition, apoptosis was evaluated with help of cleaved Caspase-3 (Asp175., Cell sign­aling Technology, Danvers, MA, USA) at dilution 1:1500, whereas immune cells (NK and CTL) were stained with help of Granzyme B (ab4059, Abcam) at dilution 1:1250. For staining proliferating cells, rabbit monoclonal antibodies against Ki-67 (clone SP6, Thermo Fisher Scientific) at dilution 1:1200 were used. The last section was stained for deter­mination of the number of blood vessels, by using primary rabbit polyclonal antibodies against CD31 (ab28364, Abcam) at dilution 1:1000. For these sec­tions, a peroxidase-conjugated streptavidin–biotin system (Rabbit specific HRP/DAB detection IHC kit, ab64261, Abcam) was used as the colorogenic reagent followed by hematoxylin counterstaining. From each slide and each feature (apoptosis, hy­poxia, proliferation, vascularisation and immune cells), five randomly selected viable parts of each tumor were observed and captured under the light 34 FIGURE 2. Immune response of melanoma tumors is observed by vitiligo effect. CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET-antiCD105 microscopy, by DP72 CCD camera (Olympus, Hamburg, Germany) connected to a BX-51 micro­scope (Olympus) under 40× magnification (numer­ical aperture 0.85). The viable parts were analyzed in blind fashion and the results were presented as the percent (hypoxia, apoptosis, proliferation) of the cells or the number of cells (immune cells) or the structures (vascularization) positive to IHC staining. The percentage of necrosis was contribut­ed to the tumors as whole and was also evaluated in blind fashion, as previously described.18 In addition, tumors that grew up to 150 mm3 after secondary challenge of tumors, were excised, fixed and embedded in paraffin, as described above. Furthermore, from each tumor sections were cut and stained with hematoxylin and eosin to deter­minate morphological changes of tumor cells. Statistical analysis All data were tested for normality of distribu­tion with the Shapiro-Wilk test. The differences between the experimental groups were statisti­cally evaluated by one-way analysis of variance (one-way ANOVA) followed by a Holm-Sidak test for multiple comparison. A P-value of less than 0.05 was considered to be statistically significant. SigmaPlot Software (Systat Software, Chicago, IL, USA) was used for statistical analysis and graphi­cal representation. Results Gene electrotransfer of plasmid silencing endoglin indicates a vascular targeted effects of the therapy GET of either plasmids (pControl, TS) to melanoma tumors had statistically significant antitumor ef­fectiveness compared to untreated tumors, which resulted in 8.2 ± 2.5 and 8.6 ± 3.0 days of tumor growth delay, respectively (Table 1). However, GET (TS) resulted in 44% of tumor free mice and 75% of them were resistant to secondary challenge, whereas in the GET (pControl) group no tumor free mice were obtained. Histological analysis (Table 2) of GET (TS) group demonstrated reduction of vas­cularization (14.9 ± 1.1%) and proliferating cells (49.5 ± 3.8%), whereas hypoxia (46.3 ± 3.0%) lev­els were increased and statistically significant to at least pertinent control groups (CTRL, pControl, TS and 3 × GET (pControl)). The levels of necrosis, apoptosis and number of infiltrating immune cells in the tumors were comparable in both GET treat­ment modalities (Figure 1) and statistically signifi­cant to at least CTRL, pControl and TS. These re­sults indicated the vascular targeted effects of the GET (TS), used for silencing endoglin. Irradiation, alone or combined either with plasmids injection or electric pulses, had minor effect on radioresistant melanoma tumors Irradiation monotherapy with 15 Gy had minor effects on radioresistant melanoma tumor. The tumor growth delay was moderate in the groups of irradiation alone or in combination with injec­tion of plasmids (from 0.7 ± 0.3 to 2.8 ± 1.2 days) and up to 11% of tumor free mice were obtained in the group of irradiation in combination with in­jection of plasmid pControl (Table 1). The results of histological analysis (Table 2, Figure 1) indicates an immunological effect of the irradiation alone or in combination with plasmids injection, since the number of infiltrating immune cells in the tumors was statistically significantly higher in comparison to control groups (CTRL, pControl, TS) and groups applying electric pulses (alone or combined with injection of plasmids (GET (pControl) and GET (TS)), and the tumor free mice were resistant to sec­ondary challenge. Furthermore, irradiation alone caused the reduction in proliferating cell levels (53.3 ± 3.5%) and in tumor vascularization (30.1 ± 2.3%), whereas apoptosis (18.4 ± 1.8%) and hypox­ia (30.2 ± 2.4%) levels were elevated. All of these results were statistically significant compared to CTRL, pControl and TS groups. The application of electric pulses to tumors in combination with tumor irradiation resulted in bet­ter antitumor effectiveness than observed in each of these two treatment modalities alone. The tumor growth delay in combined treatment was 3.2 ± 1.3 days and 22% of tumor free mice were obtained (Table 1). The histological analysis of combined treatment demonstrated statistically significant higher levels of apoptosis (31.0 ± 2.0%) and hypoxia (51.7 ± 2.6%) in comparison to the groups of irradi­ation and electric pulses alone. Among these three groups no statistically significant differences were observed in the levels of necrosis. Furthermore, the analysis of levels of proliferating cells, the number of tumor blood vessels and immune cells infiltrat­ing in the tumors, resulted in similar and moder­ate effectiveness of the combined treatment (3 × EP + IR) and irradiation (alone or combined with injection of plasmids), which statistically signifi­cantly differed from group of electric pulses alone. Nevertheless, in this combined treatment modality (3 × EP + IR) no tumor free mice were resistant to secondary challenge of tumors. Combination of GET and irradiation exerts pronounced antitumor effects The groups combining GET of plasmids (pControl, TS) and irradiation resulted in pronounced thera­peutic effectiveness, with up to 89% and 88% of tumor free mice, respectively, and from those up to 63% and 57% of mice was resistant to second­ary tumor challenge, respectively (Table 1). The histological analysis, of tumors excised 6 days after treatment (Table 2, Figure 1), indicated on similar mode of action of these combined treatment mo­dalities (3 × GET (pControl or TS) + IR), since the elevation of necrosis (65.0 ± 7.6%, 79.2 ± 4.2%), number of immune cells (45.8 ± 3.0%, 46.5 ± 2.1%), reduction of tumor vascularization (18.9 ± 2.0%, 12.8 ± 1.0%) and proliferation (46.0 ± 4.8%, 46.7 ± 4.8%), did not differ between these groups. Only two statistically significant differences between the therapeutic groups combining GET (pControl, TS) and irradiation were observed which were the level of hypoxia (58.0 ± 2.8%, 66.7 ± 1.6%), and ap­optosis (38.7 ± 3.1%, 50.3 ± 2.4%). The high number of infiltrating immune cells (Table 2) in these tumors indicated on important mode of this therapeutic action; the highest and statistically significantly increased number of im­mune cells was observed in the groups combin­ TABLE 2. Immunohistological analysis of tumors CTRL 25.0 ± 2.6 11.3 ± 1.6 4.7 ± 1.0 92.2 ± 1.3 51.6 ± 3.2 7.9 ± 0.9 pControl 20.0 ± 2.6 11.6 ± 1.0 5.4 ± 1.0 92.1 ± 1.2 47.1 ± 4.7 8.3 ± 1.1 TS 21.7 ± 2.6 11.4 ± 1.5 5.5 ± 0.7 92.7 ± 1.0 50.5 ± 4.2 7.9 ± 0.7 3 × EP 13.3 ± 3.6 18.7 ± 2.7 7.8 ± 0.8 74.4 ± 1.7 39.9 ± 2.6 14.3 ± 1.7 3 × GET (pControl) 53.3 ± 7.9 19.5 ± 3.0 15.3 ± 2.9 75.6 ± 2.4 35.3 ± 4.2 15.1 ± 1.6 3 × GET (TS) 65.8 ± 7.6 26.7 ± 2.2 46.3 ± 3.0 49.5 ± 3.8 14.9 ± 1.1 19.3 ± 1.7 IR 21.7 ± 4.2 18.4 ± 1.8 30.2 ± 2.4 53.3 ± 3.5 30.1 ± 2.3 26.8 ± 2.6 pControl + IR 37.5 ± 8.0 18.7 ± 1.9 32.3 ± 2.5 54.5 ± 4.7 24.5 ± 1.8 26.4 ± 2.1 TS + IR 43.3 ± 8.0 19.9 ± 1.5 31.6 ± 2.3 52.0 ± 2.9 23.8 ± 1.6 26.4 ± 2.3 3 × EP + IR 25.0 ± 4.8 31.0 ± 2.0 51.7 ± 2.6 59.4 ± 4.0 28.9 ± 2.3 29.3 ± 2.1 3 × GET (pControl) + IR 65.0 ± 7.6 38.7 ± 3.1 58.0 ± 2.8 46.0 ± 4.8 18.9 ± 2.0 45.8 ± 3.0 3 × GET (TS) + IR 79.2 ± 4.2 50.3 ± 2.4 66.7 ± 1.6 46.7 ± 4.8 12.8 ± 1.0 46.5 ± 2.1 AM = Arithmetic mean; Groups: (CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET-antiCD105); n = Number of structures or cells; SEM = Standard error ing GET of plasmids and irradiation, followed by all the remaining groups that included irradiation (alone, TS, pControl and 3 × EP). In these groups, the immune cells infiltration was comparable and statistically significant to all of the other remaining groups. Furthermore, the immune cells infiltration was the lowest in the groups that included electric pulses (alone or with plasmids alone). Additionally, all of the mice that were tumor free after the thera­pies including irradiation, either alone or in combi­nation with other modalities, had fur discoloration, known as vitiligo, indicating immune response (Figure 2). This was further confirmed with high number of mice that were resistant to secondary challenge in tumor free mice, with the exception of the group combining electric pulses and irradiation (Table 1). In these groups, in which tumors grew af­ter secondary challenge, the growth rate and histol­ogy of the tumors were the same as after the initial induction of tumors (data not shown). In addition, the safety of the treatment (irradia­tion alone or in combination with other modalities) was proven, since no body weight loss over 10%, or any other side effects were observed, except for the temporary hair loss in the irradiated area, without skin desquamation (data not shown). Discussion The results of this study indicate a dual effect of GET of plasmid encoding shRNA for silencing endoglin, the direct and the indirect, both having a radiosensitizing effect. The direct effect was on the tumor vasculature and also on melanoma tu­mor cells, whereas the indirect was observed with the use of plasmid devoid of therapeutic gene, through boosting the immune response in tumors. Furthermore, irradiation had mainly affected mel­anoma cells, although some effect on vasculature could also be noticed. In addition, the higher num­ber of infiltrating immune cells in all of the groups combined with irradiation indicated an important role of the immune system. All of these effects had synergistic action and, in combined treatment mo­dality of GET and irradiation, resulted in increased radiosensitizing effectiveness of melanoma tumors that resulted in prolonged tumor growth delay, which led to 88% of tumor free mice, of which 57% were resistant to secondary challenge of tumors. Dual effectiveness of GET The first direct effect of GET (TS) was on tumor vasculature, which was significantly reduced after the treatment. This vascular effect can be ascribed to the specificity of the plasmid for endothelial cells and to the endoglin silencing. This direct effect on tumor vasculature was also observed in other stud­ies using GET of plasmid for silencing endoglin on melanoma22,23 and other tumor models.18,19,21 The vascular targeted effects in these studies were first confirmed in non-endoglin expressing tumors, i.e. murine mammary adenocarcinoma, by endotheli­al-specific promoter18,21 and non-specific, constitu­tive promoter18–21, that resulted in pronounced an­titumor effectiveness. In melanoma tumor model, B16F10-luc, the GET of plasmids silencing endog­lin resulted in significant antimetastatic effective­ness.22 Furthermore, GET of plasmid silencing endoglin, with constitutive promoter, performed on small melanoma B16F10 tumor model (4 mm3 at the beginning of therapy) confirmed vascular targeted effects, which resulted in prolonged tu­mor growth delay and tumor free mice (58%), also due to nonspecific nature of constitutive plasmid that was used in this study silenced endoglin also in melanoma cells.23 Nevertheless, in the current study, which was done on bigger tumors (40 mm3), in addition to significant increased level of hypox­ia, also decreased number of proliferating tumor cells was observed. This was attributed to the sec­ond direct effect of GET (TS), that is silencing of endoglin also in melanoma cells, since it is known that plasmid with tissue specific promoters can be leaky and can also be transcribed in non-targeted tissue.27 Therefore, GET (TS) has dual direct effects; primarily by targeting vasculature and secondly by inhibition of proliferation of melanoma cells. These two effects together resulted in 44% of tumor free mice, from which 75% were resistant to secondary challenge of tumor cells. Nevertheless, high level of infiltrating immune cells in the tumors, indicated also an indirect ef­fect of this treatment, through the stimulation of immune response. This is also supported by the data obtained with GET of the plasmid devoid of therapeutic gene (pControl). In comparison to GET of therapeutic plasmid, TS, no tumor cures were obtained, however, in other measured parameters, infiltration of immune cells into the tumors, lev­els of necrosis and apoptosis as well as in tumor growth delay no statistically significant differenc­es were obtained between these groups. Thus, to a certain degree, a similar mode of antitumor ac­tion can be ascribed to the GET of plasmid DNA, mainly because the values of these parameters were statistically different from pertinent control groups. Antitumor effectiveness of GET of non-therapeutic (control) plasmids was also observed in other studies9,10, and the authors indicated that effectiveness was due to involvement of immune system. Namely, the plasmid introduced during GET can act as a DAMP that is recognized through different sensors (Pattern Recognition Receptors), leading to the activation of the signal transduction cascade that ultimately triggers the production of type 1 interferons and other cytokines.28 These act as a link between the innate and adaptive im­mune response29 and can induce the adaptive im­mune response against the introduced DNA and consequently the transfected cells. Furthermore, similar immune response can also be triggered by the stress30 that is produced during the transfection procedure, like mechanical stress, heat, and ROS, that have all been previously reported after elec­troporation.31,32 Priming effect of irradiation To target primary tumors, as was done in this study, irradiation is one of the most used treatment modalities.33 This therapeutic approach alone or in combination with injection of plasmids, in ra­dioresistant melanoma tumor model, resulted in moderate tumor growth delay, which was attrib­uted to decreased proliferation of melanoma cells, increased levels of apoptosis and necrosis. Further histological analysis indicated on the radiation damage of the tumor vasculature, as a second ef­fect of the irradiation, as seen in other studies.34,35 This vascular damage was less pronounced than in group of GET (TS), although in comparison to GET (TS), irradiation monotherapy resulted in high­er infiltration of the immune cells in the tumors. Furthermore, in the group combining irradiation and injection of therapeutic plasmid, one mouse was tumor free, which was also resistant to sec­ondary challenge of tumor. This indicates a prim­ing effect of irradiation combined with introduc­tion of foreign DNA, through boosting the immune response. When irradiated cells die, they release their antigens in the context of the danger signal (DAMPS), which result in the priming of a tumor specific immune response against the released an­tigens. Therefore, irradiated tumors can sometimes act as a powerful individualized in situ vaccine13, which is manifested as the abscopal effect of the ir­radiation. This was confirmed in in vivo studies in­dicating that irradiation can induce in vivo priming of T cells to exogenous model antigens engineered to be expressed by tumors.13 In melanoma tumor model, priming of antitumor T cells to the model antigen ovalbumin, was more effective when sin­gle, 15 Gy dose was used, rather than 3 Gy given in 5 consecutive days.36 Further on, another group al­so showed induction of antitumor T-cell responses with other antigen expression, when single 20 Gy dose was applied, but not by 5 Gy given 4 times.37 The combination of electric pulses and irradia­tion exerted moderate antitumor effectiveness that resulted in 22% of tumor free mice. Similar results were obtained in our previous studies on mu­rine mammary adenocarcinoma18, sarcoma17 and Ehrlich-Lettre ascites38, where also 20%, 27% and 54% of tumor free mice were observed, respective­ly. Therefore we can assume that electroporation of tumors contributes to radioresponsiveness of mel­anoma, most probably due to generation of ROS32,39 after application of the electric pules, which also re­sulted in significantly elevated levels of hypoxia in our histological analysis. Immune boosting and radiosensitization by plasmid DNA The combined treatment modalities (combination of GET (TS or pControl) with irradiation) exerted excellent, highly statistically significant antitumor effect in comparison to control groups, which re­sulted in 88% and 89% of tumor free mice, respec­tively, of which 57% and 63% of mice were resist­ant to secondary challenge of tumors, respectively. The results of histological analysis were similar between groups of combined treatment modality (GET + IR), regardless of the applied plasmid. The only two differences were on the levels of apop­tosis and hypoxia. The analysis of the presence of the immune cells showed on the highest number of the immune cells in the groups of GET of plasmids and irradiation, followed by irradiation groups (alone or combined with injections of plasmids or combined with electric pulses). Therefore, it can be presumed that the priming effect of irradiation can be boosted by GET of the plasmid DNA to fully ex­ert vaccinating effect. Additionally, fur discolora­tion, vitiligo, an immune-mediated destruction of normal melanocytes that has also been recognized as a positive prognostic indicator for treatment re­sponse40,41, was also observed in this study in all the groups where tumor free mice were observed (except 3 × GET (TS)). In addition, majority of the tumor free mice were resistant to secondary chal­lenge of tumors, further indicating on the develop­ment of the immune memory. The tumors in exper­imental groups, which were resistant to secondary challenged of tumor, were therefore radiosensibi­lized through GET of plasmid, since in the group combining electric pulses and irradiation, no mice resulted resistant to secondary challenge of tumor. The GET of plasmids in combination with irradia­tion presumably generated many danger signals that collectively define immunogenic cell death.13 Furthermore, the immunogenicity of tumor might also play an important role in combined treatment modality.42 In the current study we ob­tained significant antitumor effectiveness and up to 89% of tumors free mice, that were up to 63% resistant to secondary challenge of tumors, when combining GET and irradiation in melanoma tu­mor model. Furthermore, in our previous study combining the same treatment modality on a dif­ferent tumor model, murine mammary adenocar­cinoma TS/A, we achieved up to 44% of tumors free mice with therapeutic plasmid silencing en­doglin and 20% tumor free mice with the control plasmid.18 We can presume that the boosting of immune response depends on the tumor type, melanoma being more immunogenic that mam­mary adenocarcinoma TS/A. Nevertheless, the mechanisms of these therapies are not fully elu­cidated. Currently, we presume that there is in­volvement of DNA sensors, ROS and specific im­mune response after irradiation, but further stud­ies are needed. Conclusions The results of this study indicate that irradiation can in radioresistant mice tumors, such as mela­noma, by release of tumor associated antigens serve as the target of the immune response. This can be further boosted by GET of plasmid, with or without therapeutic gene, which was confirmed by the equal radiosensitization resulting in prolonged tumor growth delay and up to 89% of tumor free mice, which kept immune memory to melanoma cells. In addition, GET of therapeutic plasmid si­lencing endoglin has also direct effect on vascu­lature and tumors cells; however in combination with radiotherapy this effect was masked by pro­nounced immune response. Acknowledgements The authors would like to acknowledge M. Lavric and S. Kranjc for their help with cell cultures and tumor transplantation, U. Lampreht Tratar and M. Ota for help with IHC, as well as V. Todorovic for useful tips. The research was supported by Slovenian Research Agency (P3-0003, J3-4211, J3-6793) and conducted in the scope of LEA EBAM (French-Slovenian European Associated Laboratory: Pulsed Electric Fields Applications in Biology and Medicine) and is a result of network­ing efforts within the COST TD1104 Action. 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Nat Rev Cancer 2012; 12: 307-13. doi: 10.1038/nrc3246 research article Comparison between cryoablation and irreversible electroporation of rabbit livers at a location close to the gallbladder Jianying Zeng1,2, Zilin Qin1,2, Liang Zhou2, Gang Fang2, Jibing Chen2, Jialiang Li2, Lizhi Niu1,2, Bing Liang2, Kecheng Xu2 1 School of Medicine, Jinan University, Guangdong Province, Guangzhou, China 2 Fuda Cancer Hospital, Jinan University School of Medicine, Guangdong Province, Guangzhou, China Radiol Oncol 2017; 51(1): 40-46. Received 26 August 2016 Accepted 19 November 2016 Correspondence to: Lizhi Niu, Fuda Cancer Hospital, Jinan University School of Medicine, Guangzhou, China, 510665. E-mail: fudalab@163. com. Bing Liang, Fuda Cancer Hospital, Jinan University School of Medicine, Guangzhou, China, 510665. E-mail: 70404803@qq.com Conflict of Interest: The authors declare that they have no conflict of interest. Jianying Zeng and Zilin Qin contributed equally to this work and share the first authorship. Background. The ablation of liver tumors located close to the gallbladder is likely to lead to complications. The aim of this article is to compare the safety and efficacy of irreversible electroporation (IRE) and cryoablation in rabbit livers at a location close to the gallbladder. Materials and methods. We performed cryoablation (n = 12) and IRE (n = 12) of the area of the liver close to the gallbladder in 24 New Zealand white rabbits in order to ensure gallbladder damage. Serum aminotransferase and serum bilirubin levels were measured before and after the ablation. Histopathological examination of the ablation zones in the liver and gallbladder was performed on the 7th day after the ablation. Results. Seven days after the ablation, all 24 animals were alive. Gallbladder perforation did not occur in the IRE group; only mucosal epithelial necrosis and serous layer edema were found in this group. Gallbladder perforation oc­curred in four rabbits in the cryoablation group. Serum aminotransferase and serum bilirubin levels obviously increased in both groups by Day 3 and decreased gradually thereafter. The elevation in aminotransferase and bilirubin levels was greater in the cryoablation group than the IRE group. Pathological examination revealed complete necrosis of the liver parenchyma from the ablation center to the gallbladder in both groups, but bile duct and granulation tissue hyperplasia were observed in only the IRE group. Full-thickness gallbladder-wall necrosis was seen in the cryoablation group. Conclusions. For ablation of the liver area near the gallbladder, IRE is superior to cryoablation, both in terms of safety (no gallbladder perforation in the IRE group) and efficacy (complete necrosis and rapid recovery in the IRE group). Key words: cryoablation; irreversible electroporation; liver; gallbladder; ablation Introduction Hepatocellular carcinoma (HCC) is the sixth most prevalent malignancy worldwide1, and a large pro­portion of patients with HCC are ineligible for tu­mor resection due to several factors, such as poor hepatic reserve (cirrhosis), multicentric tumors and extrahepatic disease.2-3 In HCC patients who are not candidates for surgery or liver transplanta­tion, the National Comprehensive Cancer Network (NCCN) guidelines recommend the use of locore­gional ablative methods, such as radiofrequency ablation (RFA), microwave ablation (MWA) and cryoablation. However, when the HCC lesion is located in close proximity to structures such as the major bile duct, gallbladder and diaphragm, abla­tion should be performed with caution in order to avoid damaging these structures. According to Lee et al., radiofrequency ablation of the area of the liver abutting the gallbladder can cause substantial complications, including gallbladder perforation, especially when the ablation is performed with­out maintaining a safe distance.4 Furthermore, it has been reported that ultrasound-guided percu­taneous microwave ablation can be safely used for hepatic malignancies adjacent to the gallbladder, only when the ablation is performed under strict temperature monitoring.5 Cryoablation is achieved using the high-voltage-dependent release of argon and helium to induce a cycle of low temperature followed by thawing in order to cause physical damage and treat tumors. Because major vessels in close proximity to the tumor can absorb large amounts of heat during ablation (known as the “heat sink effect”), cryoablation can minimize vas­cular injury during the ablation of liver tumors. In addition, cryoablation is associated with good control, which allows the accurate targeting of the necrotic area and results in few side effects.8 This method might therefore be suitable for the abla­tion of liver lesions located near the gallbladder.6,7 Irreversible electroporation (IRE) is a novel abla­tion technology that utilizes short pulses of high-voltage electrical energy to induce tissue necrosis. IRE has many special advantages, including short ablation time, preservation of the internal struc­ture of vital organs and lack of the heat/cold-sink effect.8 Although not yet included in the NCCN guidelines, IRE, owing to its intrinsic characteris­tics, might be a superior alternative to other abla­tion techniques for the ablation of tumors situated near the gallbladder. Therefore, IRE was selected for comparison with conventional cryoablation techniques. In this animal experimental study, we performed cryoablation and IRE of the liver area located 0.5 cm from the gallbladder in rabbits, and compared and evaluated the safety and efficacy of these two ablation techniques in order to identify the optimal ablation method for liver tumors lo­cated near the gallbladder. Materials and methods Experimental animals In total, 24 healthy female New Zealand white rabbits, weighing (2.5 ± 0.2) kg each, were pro­vided by the Animal Experimental Center of Jinan University. They were maintained in a clean, and mechanically-ventilated environment at a constant temperature. The rabbits were randomly assigned to the cryoablation group (n = 12) and IRE group (n = 12). All animals were fasted for 24 h before sur­gery, and intravenous access was established for intraoperative drug administration. The study was approved by the Research Animal Care and Use Committee of Guangzhou Fuda Cancer Hospital (approval number, LL201402). Cryoablation A cryoablation system (CryocareTM, Endocare, Irvine, CA), composed of the main body, argon/ helium gas container and cryoprobes (CRYO-42; Endocare), was used for cryoablation. Rabbits were anesthetized by the intramuscular injection of 44 mg/kg·bw ketamine, intubated and connected to a respirator for mechanically controlled respiration. Intraoperative anesthesia was maintained with 1.5% – 2% isoflurane. General anesthesia was induced and maintained by the intramuscular injection of 44 mg/kg·bw ket­amine. The surgeon had 17 years of experience in cryoablation. The rabbits were fixed in a supine po­sition on the operating table and shaved. The skin was disinfected with iodine, and an abdominal in­cision was made to expose the liver and gallblad­der under sterile conditions. The gallbladder was pressed on the liver, and a cryoprobe with a diame­ter of 1.4 mm was inserted into the liver until its tip was approximately 0.5 cm away from the gallblad­der. The location of the tip was monitored using an ultrasound system (DP-50Vet, Mindray, Shenzhen, China; Figure 1A). When it was confirmed that the cryoprobe was connected to the main equipment, the argon gas was released to freeze the tissue. The cryoablation protocol was a double freeze-thaw cycle consisting of a 2-min freeze and 1-min thaw; this protocol ensured that part of the gallbladder was included within the ablation zone of the ice ball (Figure 1B). No animal died during the cryoa­blation process. After the cryoablation, the cryo­probe was retrieved. The liver was put back into the abdominal cavity, and conventional abdominal wall sutures were placed to complete the cryoab­lation. The cryoablation tract was filled in with a thrombin-soaked gelatin sponge and closed with a short suture. IRE General anesthesia was induced with an intra­muscular injection of 44 mg/kg·bw ketamine and maintained with 1.5% – 2% isoflurane. Respiration was controlled using a respirator during the op­eration (tidal volume: 30 ml/time, respiratory rate: 30/min, oxygen concentration: 100%). Before the IRE operation, 0.12 mg/kg·bw pancuronium was intramuscularly injected to block muscle contrac­tion. IRE was performed using the Angiodynamics Nanoknife system (AngioDynamics, Latham, NY). The Nanoknife system consists of a main body and electrode probes (catalog no. 20400104, AngioDynamics), which were used for electropora­tion ablation. Monopolar probes were used, which could be changed to achieve 1 – 4 cm of electrode exposure. The size of the ablation zone created de­pended on the exposed length of the applicator as well as the distance between the probes. When the gallbladder was exposed, two monopolar probes were carefully inserted into the liver (Figure 2A). The probes were inserted to a depth of 0.5 cm into the liver parenchyma under ultrasound guidance (DP-50Vet, Mindray, Shenzhen, China), and were kept 1.5 cm apart and 0.5 cm away from the gall­bladder (Figure 3). The monopolar probes were fastened with a spacer device. The ablation pa­rameters consisted of nine groups of ten electrical pulses (total: 90 pulses), and each electrode pulse was 70-µs long; the output voltage was set at 1500 V/cm. This protocol ensured that the electric field covered part of the gallbladder (Figure 2B). Pulse delivery was synchronized with the R waves of the cardiac cycle in case of arrhythmia. One rabbit died during the IRE ablation because of tracheal intuba­tion failure; we replaced this rabbit with another rabbit. After the IRE ablation, muscle relaxation was reversed with intramuscular injections of 5 µg/ kg·bw neostigmine and 5 µg/kg·bw atropine. Postprocedure care The 24 experimental animals returned to conscious­ness under the monitoring of a senior veterinarian and were sent back to the specific pathogen-free experimental room, which was filled with fresh air and maintained at a constant temperature. All the rabbits were under meticulous care, and pain was managed with intramuscular buprenorphine (0.01 mg/kg) and oral meloxicam (0.4 mg/kg). In addition, the rabbits received daily intramuscular injections of 40 mg/kg·bw cephazolin for 3 days af­ter the surgery in order to prevent infection. For 7 days after the surgery, the animals were observed by veterinarians, who assessed whether the rab­bits had suffered any postoperative complications. Feeding was gradually restored over 3 days after the surgery in both groups of animals; however, both food and water intake were lower in the cry­oablation group than in the IRE group. Detection of serum aminotransferases and serum bilirubin We collected blood (2 ml, from an ear marginal vein) 1 day before the operation and on days 1, 3, 5 and 7 after the ablation. The blood samples were centrifuged to obtain serum. Hepatic function was measured using an automatic biochemical analyzer (Hitachi-7100; Hitachi, Tokyo, Japan). Alanine ami­notransferase/aspartate aminotransferase (ALT/ AST) and serum bilirubin levels were determined with kits for in vitro diagnostic use (Biosino Bio­technology and Science Ltd, Beijing, China) by the velocity method. Pathology No animal died after the surgery. At 7 days after the ablation, all the animals were euthanized with an overdose of intravenous pentobarbital sodium (120 mg/kg, Sleepaway; Fort Dodge Animal Health, Fort Dodge, IA). The liver tissues from the ablation tract to the gallbladder were harvested, and the ablation lesions were cut and sectioned from the center. The maximum diameter was measured, and the lesions were photographed. Then, the tissue samples, in­cluding the abnormal and adjacent normal tissues, were fixed in 10% neutral buffered formalin, pro­cessed routinely for histology, embedded in par­affin, cut into 5-µm-thick slices and stained with hematoxylin and eosin. Pathological analyses were performed by an attending surgical pathologist. Statistical analysis All analyses were performed using GraphPad Prism 5 software (GraphPad Inc., San Diego, CA). The size of the ablation lesions and the serum ALT, AST and bilirubin levels were analyzed using the Student t-test. A P value of < 0.05 was considered to indicate a statistical difference, while P values of < 0.01 and < 0.001 indicated significant differences. Results Pathological appearance In the cryoablation group, there were severe in­flammatory adhesions between the ablation sites in the liver and gallbladder, the abdominal wall and the mesentery (12/12); the liver ablation zone was khaki-colored and oval. The lesion area had a narrow and sharp edge of light pink edematous tis­sue, and the ablation range obviously covered the gallbladder. There was a large gray area of necrosis adjacent to the gallbladder, and gallbladder perfo­ration was found in four animals (Figure 4). In the IRE group, there were fewer inflammatory adhe­sions than in the cryoablation group (5/12 vs.12/12). TABLE 1. Sizes of liver and gallbladder lesions at 7 days after the ablation ( ± s) methods Gallbladder side (n = 12) Diaphragm side (n = 12) P value Major diameter (cm) P value Cryo 1.5 ± 0.4 3.8 ± 0.8 > 0.05 1.5 ± 0.4 (n = 8) > 0.05 IRE 1.4 ± 0.4 4.1 ± 0.7 > 0.05 0.8 ± 0.2 (n = 12) FIGURE 6. Pathological changes in the cryoablation (Cryo) and IRE ablation zones 7 days after ablation. (A) The top images show the ablated liver and gallbladder areas (magnification, 40×). The bottom images (A to E) show enlarged versions of the areas marked in the top images (magnification, 400×). (B) The top images and (F) show the ablated liver, gallbladder and common bile duct (magnification, 40×). The bottom images (A to D) show the enlarged versions of the areas marked in the top images (magnification, 400×). The lesion area was yellow and oval with sharp edges. The ablated gallbladder tissue appeared grayish-white, but none of the 12 experimental ani­mals showed gallbladder perforation (Figure 5). Details of the sizes of the ablation lesions are pre­sented in Table 1; there were no differences in le­sion size between the two groups. As gallbladder perforation was found in four animals in the cry­oablation group, we recorded the major diameter of the gallbladder lesions in only eight animals in this group. There were no intact hepatic cells in the liver slices in both the cryoablation and IRE groups. From the cryoablation tract to the liver edge, the cryolesion appeared as a pink-stained area of dis­integrating tissue that included hemorrhagic con­gestion and tissue necrosis, and it showed nuclear disappearance, no intact cellular structures, a band of local bleeding, and abundant inflammatory cell infiltration (Figure 6A). In the IRE group, the main pathological changes consisted of small bile duct, acute granulation tissue hyperplasia, rare congestion, hemorrhage band and a small amount of inflammatory cell infiltration (Figure 6B). The most conspicuous difference between the two groups was the appearance of the gallbladder: in the cryoablation group, full-thickness gallbladder wall necrosis, muscular collapse and mucosal loss were found, and the necrotic area was large and uniformly stained red. In the IRE group, there was mucosal epithelial necrosis but no full-thickness gallbladder wall necrosis, and serous layer edema were largely concentrated in the area of the gall­bladder wall near the liver. Changes in serum aminotransferase and serum bilirubin levels In both groups, the serum aminotransferase and bilirubin levels obviously increased on day 3, gradually decreased thereafter and recovered to normal in a week. At each time point, the serum aminotransferase and serum bilirubin levels were higher in the cryoablation group than in the IRE group (Table 2). TABLE 2. Changes in aminotransferase and serum bilirubin levels over time ( ± s) ALT IRE 38.35 ± 3.22 167.05 ± 19.50 214.83 ± 10.37 119.60 ± 12.99 68.25 ± 10.08 (U/L) Cryo 39.20 ± 3.90 195.03 ± 9.75** 289.58 ± 12.81** 142.35 ± 57.85* 94.90 ± 9.78* IRE 34.10 ± 0.86 47.64 ± 0.69 60.21 ± 2.55 46.78 ± 1.71 31.05 ± 0.43 AST(U/L) Cryo 35.26 ± 2.45 60.11 ± 3.44** 79.12 ± 2.58** 52.80 ± 3.41* 37.41 ± 2.50* Serum bilirubin IRE 8.08 ± 2.19 26.01 ± 3.68 35.15 ± 5.70 29.09 ± 3.27 21.41 ± 3.39 (µmol/L) Cryo 7.59 ± 2.01 32.72 ± 4.08** 37.34 ± 6.14* 35.88 ± 4.28** 25.86 ± 5.37* *P < 0.05 and **P < 0.01, compared with the IRE group Discussion In the study by Fairchild et al., the ice ball extended into the gallbladder lumen (up to a mean distance of 6 mm), but no cases of gallbladder perforation occurred, even when the iceball had extended as far as 1.8 cm into the gallbladder lumen.9 This may be explained by the theory that the bile within the gallbladdder may dissipate the cooling effect of the cyroprobe around the gallbladder fossa. However, in our study, perforation occurred in one-third of the animals, and inflammatory adhesions occurred in all the animals in the cryoablation group. We at­tribute this difference to the fact that in our study, the ice ball extended to a far greater distance into the gallbladder lumen, and thus, the cryodamage to the gallbladder was more severe. Furthermore, as more of the gallbladder wall was cryoablated, the “dissipating effect” of the bile was extremely weakened, and a local self-limited inflammatory response resulted in gallbladder wall edema and reactive fluid accumulation, which in turn in­creased the severity of the gallbladder damage.9 In contrast, IRE caused a color change in the gall­bladder, but no perforation occurred 7 days after ablation. In addition, full-thickness wall necrosis occurred only in the cryoablation group; in the IRE group, the damage to the gallbladder was con­fined to the mucous layer. This is because IRE is a promising, non-thermal technology that takes ad­vantage of electric fields instead of thermal energy to induce cell death. The most distinctive feature of IRE is that it has little influence on the adjacent large vessels and can preserve the collagen matrix of tissue structures.10-13 In addition, it does not lead to significant ductal damage if the electrodes are not placed very close to the ducts.14 In our study, the IRE probes were 0.5 cm away from the gall­bladder, and the damage to the gallbladder caused by IRE was acceptable. Thus, our findings indicate that IRE is a potentially safe modality for the abla­tion of hepatic tumors adjacent to the gallbladder. Furthermore, although the lesion sizes and changes in the serum aminotransferase and serum bilirubin levels were similar in the two groups, the damage to the liver cells was more severe in the cryoablation group. This phenomenon may be explained as follows: Cryodamage mainly leads to cell necrosis, and secondary injuries can be me­diated by the resultant inflammatory reaction.15 According to current studies, thermal injury may require time to cause gallbladder perforation16 and perforations are usually found in the acute phase, not the immediate phase.4 In contrast, electrical injury (IRE) mainly causes cell apoptosis and tis­sue damage, and repair following IRE is rapid.8 At 7 days after cryoablation, tissue destruction was still ongoing (areas of cell disruption with hemor­rhage), while at the same time point, tissue repair was obvious in the IRE group (areas of bile duct hyperplasia). Collectively, the above findings indicate that IRE is unlikely to destroy the gallbladder wall and offers the advantage of faster repair of the dam­aged tissue. Thus, in this study, IRE was found to be superior to cryoablation in terms of protecting the gallbladder, and was a safer procedure than cryoablation. Complete necrosis of tumor cells with no re­sidual tumor is the main goal of ablation. During cryoablation, ice ball formation can be precisely monitored using ultrasonography or computed tomography. It is impossible to induce tumor cell necrosis in the periphery of the ice ball because the peripheral temperature of the ice ball is 0°C17, and the temperature that is lethal to cells is at least –20°C.18,19 To ensure efficacious cryoablation, the edge of the ice ball is usually kept 1 cm beyond the edge of tumor, and the double freeze-thaw cy­cle protocol is used. In our study, the efficacy of cryoablation of the liver area near the gallbladder reached the expected levels, as determined based on the appearance (gray-red lesion spread to the gallbladder ) as well as histopathological examina­tion (no live cells between the cryoprobe and gall­bladder ) of the cryozones. IRE ablation has been reported to achieve nearly 100% necrosis in the ab­lation zone at 24 h in a swine liver model.8,20 The ef­fectiveness of IRE ablation over a longer time is one of the focuses of this study. Seven days after IRE ablation, not only were the target cells destroyed completely, but the area of bile duct hyperplasia was far greater than the area of pink, disintegrating tissue. All in all, although there was a great differ­ence between the abilities of the two therapies to destroy the liver cells, the long-term effects of both treatments were good. The major limitation of this article is the lack of short- and long-term studies. Analysis of the pathological appearance of the gallbladder at dif­ferent time points after ablation would help pro­vide a stronger basis for the clinical application of the two ablation methods. Future experiments should use cholangiography to observe changes in the gallbladder and determine the integrity of the main bile duct. In conclusion, we compared the safety and ef­ficacy of cryoablation and IRE in rabbit livers, at a location close to the gallbladder. IRE provided complete ablation of the target tissue and com­pletely retained the entire gallbladder structure. In the case of liver tumors situated close to the gallbladder, cryoablation must be performed care­fully to avoid direct trauma to the gallbladder. The comparison in this article has a certain meaning in terms of the selection of instruments for liver tu­mor ablation. However, the clinical value of our findings and long-term curative effect of IRE re­main to be confirmed. Funding This study was funded by the Medical and Health Fund of Guangdong Province (2013kw051), China. The funders had no role in study design, data col­lection and analysis, decision to publish, or prepa­ration of the manuscript. Acknowledgements We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript. References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin 2015; 65: 87-108. DOI: 10.3322/caac.21262. 2. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol 2012; 56: 908-43. DOI: 10.1016/j.ejca.2011.12.021. 3. Grandhi MS, Kim AK, Ronnekleiv-Kelly SM, Kamel IR, Ghasebeh MA, Pawlik TM. Hepatocellular carcinoma: From diagnosis to treatment. Surg Oncol 2016; 25: 74-85. DOI: 10.1016/j.suronc.2016.03.002. 4. Lee J, Rhim H, Jeon YH, Lim HK, Lee WJ, Choi D, et al. Radiofrequency abla­tion of liver adjacent to body of gallbladder: histopathologic changes of gallbladder wall in a pig model. AJR Am J Roentgenol 2008; 190: 418-25. DOI: 10.2214/ajr.07.2526. 5. Li M, Yu X, Liang P, Dong B, Liu F. Ultrasound-guided percutaneous micro­wave ablation for hepatic malignancy adjacent to the gallbladder. Int J Hyperthermia 2015; 31: 579-87. DOI: 10.3109/02656736.2015.1014869 6. McGregor HC, Saeed M, Surman A, Ehman EC, Hetts SW, Wilson MW, et al. Gallbladder cryoablation: Proof of concept in a swine model for a percuta­neous alternative to cholecystectomy. Cardiovasc Intervent Radiol 2016; 39: 1031-5. DOI: 10.1007/s00270-016-1343-0 7. Kim GM, Won JY, Kim MD, Park SI, Lee do Y, Shin W, et al. Cryoablation of hepatocellular carcinoma with high-risk for percutaneous ablation: Safety and efficacy. Cardiovasc Intervent Radiol 2016; 39: 1447-54. DOI: 10.1007/ s00270-016-1384-4 8. Wagstaff PG, Buijs M, van den Bos W, de Bruin D M, Zondervan P J, de la Rosette JJ, et al. Irreversible electroporation: state of the art. Onco Targets Ther 2016; 9: 2437-46. DOI: 10.2147/OTT.S88086 9. Fairchild AH, Tatli S, Dunne RM, Shyn PB, Tuncali K, Silverman SG. Percutaneous cryoablation of hepatic tumors adjacent to the gallbladder: assessment of safety and effectiveness. J Vasc Interv Radiol 2014; 25: 1449­ 55. DOI: 10.1016/j.jvir.2014.04.023 10. Ben-David E, Ahmed M, Faroja M, Moussa M, Wandel A, Sosna J, et al. Irreversible electroporation: treatment effect is susceptible to local environ­ment and tissue properties. Radiology 2013; 269: 738-47. DOI: 10.1148/ radiol.13122590 11. Lee YJ, Lu DS, Osuagwu F, Lassman C. Irreversible electroporation in porcine liver: short- and long-term effect on the hepatic veins and adjacent tissue by CT with pathological correlation. Invest Radiol 2012; 47: 671-75. DOI: 10.1097/RLI.0b013e318274b0df 12. Niessen C, Beyer LP, Pregler B, Dollinger M, Trabold B, Schlitt HJ, et al. Percutaneous ablation of hepatic tumors using irreversible electroporation: A prospective safety and midterm efficacy study in 34 patients. J Vasc Interv Radiol 2016; 27: 480-6. DOI: 10.1016/j.jvir.2015.12.025 13. Kos B, Voigt P, Miklavcic D, Moche M. Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutane­ous irreversible electroporation (IRE). Radiol Oncol. 2015;49(3):234-41. DOI: 10.1515/raon-2015-0031 14. Choi JW, Lu DS, Osuagwu F, Raman S, Lassman C. Assessment of chrono­logical effects of irreversible electroporation on hilar bile ducts in a porcine model. Cardiovasc Intervent Radiol 2014; 37: 224-30. DOI: 10.1007/s00270­013-0731-y 15. Forest V, Peoc’h M, Campos L, Guyotat D, Vergnon JM. Effects of cryo­therapy or chemotherapy on apoptosis in a non-small-cell lung cancer xenografted into SCID mice. Cryobiology 2005; 50: 29-37. DOI: 10.1016/j. cryobiol.2004.09.007 16. Livraghi T, Solbiati L, Meloni MF, Gazelle GS, Halpern EF, Goldberg SN. Treatment of focal liver tumors with percutaneous radio-frequency abla­tion: complications encountered in a multicenter study. Radiology 2003; 226: 441-51. DOI: 10.1148/radiol.2262012198 17. Goel R, Anderson K, Slaton J, Schmidlin F, Vercellotti G, Belcher J, et al. Adjuvant approaches to enhance cryosurgery. J Biomech Eng 2009; 131: 074003. DOI: 10.1115/1.3156804 18. Shurrab M, Wang H, Kubo N. The cooling performance of a cryoprobe: Establishing guidelines for the safety margins in cryosurgery. Int J Refrig 2016; 67: 308-18. DOI: 10.1016/j.ijrefrig.2016.03.007 19. Littrup PJ, Jallad B, Vorugu V, Littrup G, Currier B, George M, et al. Lethal isotherms of cryoablation in a phantom study: effects of heat load, probe size, and number. J Vasc Interv Radiol 2009; 20: 1343-51. DOI: 10.1016/j. jvir.2009.05.038 20. Charpentier KP, Wolf F, Noble L, Winn B, Resnick M, Dupuy DE. Irreversible electroporation of the pancreas in swine: a pilot study. HPB (Oxford) 2010; 12: 348-51. DOI: 10.1111/j.1477-2574.2010.00174.x research article Cancer burden in Slovenia with the time trends analysis Vesna Zadnik, Maja Primic Zakelj, Katarina Lokar, Katja Jarm, Urska Ivanus, Tina Zagar Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Ljubljana, Slovenia Radiol Oncol 2017; 51(1): 47-55. Received 6 January 2017 Accepted 14 January 2017 Correspondence to: Assoc. Prof. Vesna Zadnik, M.D., Ph.D., Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 587 9451; Fax: +386 1 587 9400; E-mail: vzadnik@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of our study was to describe cancer burden and time trends of all cancers combined, the most frequent as well as the rare cancers in Slovenia. Patients and methods. The principal data source was the population-based Cancer Registry of Republic of Slovenia. The cancer burden is presented by incidence and prevalence for the period 1950–2013 and by mortality for years 1985–2013. The time trends were characterized in terms of an average annual percent change estimated by the log-linear joinpoint regression. The Dyba-Hakulinen method was used for estimation of incidence in 2016 and the projections of cancer incidence for the year 2025 were calculated applying the Globocan projection software. Results. In recent years, near 14,000 Slovenes were diagnosed with cancer per year and just over 6,000 died; more than 94,000 people who were ever diagnosed with cancer are currently living among us. The total burden of cancer is dominated by five most common cancer sites: skin (non-melanoma), colon and rectum, lung, breast and prostate, together representing almost 60% of all new cancer cases. On average the incidence of common cancers in Slovenia is increasing for 3.0% per year in last decade, but the incidence of rare cancers is stable. Conclusions. Because cancer occurs more among the elderly, and additionally more numerous post-war genera­tion is entering this age group, it is expected that the burden of this disease will be growing further, even if the level of risk factors remains the same as today. Key words: cancer burden; cancer incidence; time trend; Cancer Registry of Republic of Slovenia Introduction Cancer is a growing public health challenge in Europe, with substantial and persistent inequali­ties in the incidence, mortality and survival within and between Member States.1 There were 14.1 mil­lion new cancer cases, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis) in 2012 worldwide.2 Europe comprises only one eight of the total world pop­ulation but has around one quarter of the global total number of cancer cases.1,3 Without effective interventions (comprehensive cancer prevention and control policies) the burden of cancer will in­crease dramatically.4 Almost 200 cancer registries are established only in Europe with the same goal of monitoring the burden of disease and the effec­tiveness of cancer control measures.5,6 Slovenia has a long tradition in establishing can­cer control and it has one of the oldest population-based cancer registries in Europe – Cancer Registry of Republic of Slovenia (CRS) that was founded in 1950 at the Institute of Oncology Ljubljana. Notification of cancer has been compulsory in Slovenia since the foundation of the CRS and is prescribed by law.7 The main source of data are notifications of cancer, gathered from all hospitals and diagnostic centres in Slovenia, exceptionally also from primary health care centres in case the patient has not been referred for further diagnostic investigations and/or treatment. Since 2010, regu­lar annual reports7 of CRS are supported by web­portal SLORA (www.slora.si/en), where on-line analyses of CRS data are available.8 The data on cancer, that are collected by the CRS, incidence, survival and prevalence, serve together with mortality data, collected by the National Institute of Public Health as the basis for assessing the cancer burden in the country. They are important for planning and evaluation of all levels of cancer control: primary prevention, diag­nosis, treatment, palliation and rehabilitation, for planning facilities and funding needed for can­cer control (personnel, equipment and hospital capacities). Furthermore, they are basis for clini­cal and epidemiological research in Slovenia and in international multicentre studies as well as for evaluation of effectiveness of cancer screening pro­grams.5-7 In Slovenia, all patients with cancer have equal access to quality treatment, which is completely covered by obligatory health insurance. Primary care is provided by health care centres and gen­eral practitioners included in the public network. Secondary health care is provided by 11 general hospitals, two tertiary Clinical Centres, in Ljubljana and Maribor and Golnik Clinic. The Institute of Oncology Ljubljana, founded in 1938, is the only national comprehensive centre for cancer diseases, involved also in research and education. Common cancers are treated in general hospitals also, but the Institute is the referral centre for rare cancers; furthermore, radiotherapy is administered only there.9 It was estimated that health-care costs of cancer per person in Slovenia is around 70% of the European average cancer case costs.10 Since 2010, the National Cancer Control Programme is aimed to improve all aspects of cancer prevention and care in Slovenia.11,12 In this study we are presenting the incidence, mortality and prevalence data collected by the Cancer Registry of Republic of Slovenia in order to describe cancer burden together with the exist­ing time trends and projections in Slovenia, com­plementing the Annual Reports and SLORA web portal data. Patients and methods The basic data source of our analysis is the pop-ulation-based Cancer Registry of the Republic of Slovenia. The mortality data are provided by the National Institute of Public Health. All the data re­fer to all cancer patients, residents of Republic of Slovenia at the time of diagnosis, irrespective of where they have been treated or where they have died. The CRS’ quality and completeness indices suggest that cancer registration in Slovenia ad­equately covers the entire population. To assure the completeness and to obtain additional infor­mation on registered cancer cases, CRS is linked with several governmental and health databases. Synchronisation of data between different sources is based on comparing unique personal identifica­tion number which is assigned to every resident in Slovenia and recorded in every state registry including CRS. Using unique personal identifica­tion number guaranties data integrity, data qual­ity and prevents data duplication. CRS links with the Central Register of Population instantaneously through secure on-line connection (24/7 availabil­ity) and daily updates information on vital status and address for each person registered by CRS. The electronic linkage to the national Mortality Database and to the breast, colorectal and cervical screening registries is performed several times per year.13 The data on gender, date of diagnosis, age at diagnosis, code of primary site according to the International classification of Diseases 10th edition, morphological code according to International Classification of Disease for Oncology 3rd edition, vital status and cause of death for deceased were extracted from the CRS’ database for all cancer cas­es. The cancer burden is presented by three basic epidemiological indicators: incidence, prevalence and mortality. Incidence and mortality are abso­lute numbers of all newly diagnosed cancer cases or number of deceased from cancer in individu­als with permanent residence in Slovenia in one calendar year. New primary cancers of the same histology in paired organs, e.g. in the left and right breast, are not comprised in the incidence figures, neither are any new cancers of the same histology appearing in the same organ, e.g. multiple lesions of the colon. Prevalence is the number of all cancer patients that are alive on a given date, regardless of when they were diagnosed with cancer. Lifetime cancer prevalence is defined as all persons living and had ever been diagnosed with cancer, while partial prevalence counts only those patients, di­agnosed with cancer within a defined period of time e.g. 1, 5 or 10 years before the date on which prevalence is calculated. Incidence and prevalence measures are available for the period from 1950 to 2013, the mortality measures since 1985. Incidence rates were age standardised using the world standard population14 and represent the hy­pothetical crude rate assuming that the age struc­ture in the observed (Slovenian) population is the same as in the standard population, i.e. if the age structure of the population would remain the same over the time. The time trends were analysed using the log-linear joinpoint regression on age standard­ised incidence and mortality rates, implemented in the Joinpoint Regression program.15 The trends were characterized in terms of an average annual percent change (APC) of the incidence and mortal­ity rates, assuming the rates change at a constant percentage every year in the analysed time period. The method by Dyba and Hakulinen16 was used for estimation of incidence in 2016. This model in­cludes patients according to the period of diagnosis and their age at diagnosis for the last ten registered years. The population age structure was fitted for the current year (2016), provided by Statistical Office of the Republic of Slovenia. Projections of cancer incidence for the year 2025 were calculated applying the Globocan projection software.2 The expected number of new cancer cases was com­puted by multiplying the age-specific incidence rates estimated for 2012, by the expected popula­tion (size and age structure) for 2025. This analysis assumes that recent trends in cancer incidence will continue and that predictions of the size and age structure of the future population of Slovenia are accurate. Results and discusion Cancer burden In Slovenia in the year 2013 there were 13,717 (666/100,000) patients newly diagnosed with cancer, 7,442 (730/100,000) males and 6,275 (604/100,000) females. According to the EUCAN estimates Slovenia ranked in the 8th place among 40 European countries in 2012 considering all can­cer sites incidence.3 At the end of December 2013, there were 94,073 people (41,607 males and 52,466 females) alive who were diagnosed with one or more cancers at any time since the foundation of the CRS. One-year partial prevalence which in­cludes patients, diagnosed with cancer within one year before the date of calculation was 11,101. This reflects the number of patients which are cur­rently in the process of their primary oncological management. Further on, there were 31,148 pa­tients included in the regular follow-up (1-4 years prevalence). According to the official data on cancer mortality 6,075 (295/100,000) patients died from cancer in 2013 in Slovenia; 3,392 (333/100,000) males and 2,679 (258/100,000) females. According to the EUCAN estimates Slovenia ranked in the 8th place among 40 European countries in 2012 consid­ering all cancer sites mortality.3 The total burden of cancer is dominated by five most common cancer sites: skin (non-melanoma), colon and rectum, lung, breast and prostate, repre­senting almost 60% of all new cancer cases. Cancers of these sites are associated with unhealthy life­style, such as excessive sunbathing, unhealthy diet, smoking and excessive alcohol consumption; the aim of primary prevention is to lower the risk associated with these factors. Figure 1 shows ten leading cancer sites with percentage distribution in three consecutive periods in Slovenia, separately for males and females. In 2013 1,485 males (20%) were diagnosed with prostate cancer in Slovenia, being the most common diagnosis in males, com­pared to the 6th place and less than 8% two decades ago. Lung cancer represents nowadays only 12% of all cancers in males (ranks 3rd; 869 new cases), but in previous periods it was the most common male cancer representing more than 20% of all can­cers. In females in all three time periods breast is the leading cancer site with 20% of all cancer cases. In 2013 1,252 females were diagnosed with breast cancer in Slovenia. Non-melanoma skin cancer is gaining its share in female population and it is ap­proaching breast cancer in the most recent period. Lung cancer incidence in females is disturbingly climbing – from 8th place and having a share of 4.5% two decades ago it now represents 6.6% of all newly diagnosed cases and occupying 4th place. Ten leading cancer sites according to the mortal­ity shares are presented in Figure 2. The non-mel­anoma skin cancer is rarely fatal and is not part of this list, although it is among four most common cancer diagnoses. Almost one quarter (23%) of all cancer deaths in males in Slovenia are due to lung cancer (748 deaths in 2013 in Slovenia). Another quarter of cancer death is attributed to colorec­tal (468 deaths in 2013) and prostate cancer (371 deaths in 2013); other cancer types cause much less cancer deaths. In females 40% of cancer deaths are attributed to breast (410 deaths in 2013 in Slovenia), lung (340 deaths in 2013) and colorectal (327 deaths in 2013) cancer. Supplementary material provides further epidemiological data for all cancers com­bined and for 15 most common cancer sites for the period 2009–2013 in Slovenia (SupMat 1-16). Cancers of other sites are quite rare. In the contrast to the common definition of rare disease (prevalence less than 50/100,000), the experts from the project Surveillance of Rare Cancers in Europe (RARECARE)17 defined the rare cancer as any cancer with crude incidence rate less than 6 per 100,000 inhabitants. According to this definition 198 groups of rare cancer were identified. They are further joined into 65 groups, which are listed in the supplementary material (SupMat 17), and further into 15 categories as defined by the RARECARE project, the later are shown on Figure 3. All rare cancers combined account for 23% of all cancers in Slovenia in period 2004–2013: annually there are around 2,880 new rare cancers (142/100,000). The rare cancer is most frequently diagnosed in digestive organs, following by head and neck, fe­male genital organs and haematological tissue. In comparison to common cancers, rare cancers occur more often in childhood and adolescence. As in any rare disease also in rare cancers the low inci­dence is the main obstacle to conduct clinical trials needed to investigate and develop effective treat­ments. One way to overcome this obstacle would be to precisely monitor the rare cancer burden on population level and establish centres of excellence for diagnosis and treatment.17 The burden of cancer varies with age. Cancer is mostly the disease of the elderly since for the vast majority of cancers, incidence increases with age. 51 From all patients diagnosed in 2013 61% males and 58% females were aged 65 or more at diagnosis. From those born in 2013, one in two males and one in three females are expected to develop cancer by their age of 74 in Slovenian population. Cancer is a rare disease until the age of 19 years (0.6% of all cancer cases in 2013). In childhood and ado­lescence, the most frequent type of cancer is leu­kaemia. Only 1.7% of all cancers in 2013 occurred among people in the age group 20–34 years, where the most frequent malignancies are testicular can­cer in males and cervical cancer in females. Among cancer patients, there is a predominance of females in the age group 35–49 years, while this ratio is reversed for the age group 50–74. Between 35–49 years, the most frequent types of malignancies in males are oral and lung cancers, while in females, breast and cervical cancers. Colorectal cancer be­comes the predominant form of cancer for both sexes in the age group 50-74 years, while among older males (75+ years) prostate cancer is most fre­quently diagnosed. After the age of 75 years, there is a higher proportion of cancers among females, partially because of their longer life expectancy, with breast, colorectal and stomach cancers being the most common at this age. Time trend analysis The time trends of cancer burden in Slovenia for the last 64 years are summarized in tables and graphs available as the supplementary material (SupMat 1). Since 1950, the crude cancer incidence rate in Slovenia increased by 700% in males and by 470% in females. In the last 10 years, the average annual increase in males was 2.8% and 1.6% in females. Correspondingly, the average annual increase in age standardised incidence rate in males was 0.8% and 0.6% in females in the last 10 years. By com­paring crude incidence rate to the age standard­ised rate where the standard population is taken to be the age structure of the Slovenian population in first year for which the graph is prepared (1961) we can assume that more than half of the crude incidence rate increase is a consequence of ageing of the population. The increase in crude mortality rate in the last 10 years was substantially slower; on average by 1.5% per year in males and by 1.7% in females. When we calculate the hypothetical rates in case, the Slovenian population would not age, the trend becomes reverse as the age standardised mortality rate is decreasing on average by 1.2% an­nually in males and by 0.5% in females in the last 10 years. The decreasing mortality despite increas- FIGURE 3. Rare cancers categorized in 15 groups, Slovenia 2004-2013. ing incidence indicates improvement in treatment of cancer patients. It is a proof that more and more are cured of or living with and not dying from can­cer. The long-term time trends of the incidence for most common cancers by gender are plotted in Figure 4. The APCs in last 10 years are included. In males the lung cancer incidence was increasing till the beginning of the nineties, while it became stable onwards (85/100.000) and is slowly decreas­ing (on average by 1.3% per year ) and was finally overcome by prostate cancer (average increase by 6.3% per year ). The huge increase in prostate cancer incidence rate in the last years is not due to some new risk factor, but more frequent use of prostate specific antigen (PSA) test as an opportun­istic screening method9, resulting in detection of many cancers that would have never progressed or would even not have been detected during lifetime without screening, because the person would not develop any health problems. The changes in the colorectal cancer incidence should be interpreted taking into consideration the changes in the diagnostic practice – in Slovenia the National colorectal cancer screening programme (SVIT) has been introduced in 2009.18 The aver­age annual increase in colorectal cancers in males in the period 2004–2011 was 5.3%, but there was a decrease of 10.5% in the following years 2011–2013, attributable to treatment of precancerous polyps that would have progressed to cancer if person would not respond to the screening invitation. In females, the incidence rate of breast, lung and non-melanoma skin cancer is increasing. Like in males also in females the colorectal cancer incidence time trends have been changed because the national screening programme SVIT has been introduced: in the period 2004–2010 the incidence increased for 3.6% annually, but in the years 2010–2013 it de­creased by 6.4%. The effect of population-based screening is even more pronounced in the case of cervical cancer. Slovenia is one of the European countries with the highest historical incidence of cervical cancer and also one of the countries with the highest decrease of cervical cancer incidence over time.19 As pre­sented in Figure 5, the highest peak of the cervical cancer incidence was registered in the population based CRS in year 1962 when 286 new cases were diagnosed and age standardised incidence rate was 27.5/100,000. The incidence was then decreasing till the end of the eighties by 2.6% per year on aver­age, most likely due to the effect of the opportun­istic screening. At the beginning of the nineties, the cervical cancer incidence trend has changed again with a second peak in 1997, which triggered the in­troduction of organised population-based cervical cancer screening programme ZORA. In 1998 and 2001 two pilot programmes of organised screen­ing were implemented in Central and Coastal-Karst Slovenian region. The steepest decrease of incidence was observed after the implementation of programme ZORA at the national level in 2003 with APC being 5.8 in the period 2003.2015. The lowest incidence of cervical cancer to this year was registered in 2014 when 114 new cases were diagnosed and age standardised incidence rate was 6.8/100,000, which presents a difference of .20.7/100,000 in comparison with the highest re­corded age standardised incidence rate in 1962.20 On average the incidence of common cancers in Slovenia is increasing for 3.0% each year. On the contrary, in the group of rare cancers the inci­dence in stable – for all rare cancers combined the APC for the period 2004–2013 is 0.5 (SupMat 18). Nevertheless, also among rare cancers there are some specific cancer types with significant vari­ation in incidence rate over time. One of them is malignant mesothelioma. Till the end of 2013 there were 729 mesothelioma cases registered in the population based CRS, 518 (71%) of them among men. In the period 2009–2013 there were 1.9 meso­thelioma cases per 100,000 inhabitants in Slovenia; 2.8/100,000 in male population. The age stand­ardised mesothelioma incidence rates with 5-year moving averages for the period 1961–2014 are shown in Figure 6. From 1970 on the mesothelioma incidence is irregularly increasing: the steepest in­crease can be observed from 1998 to 2003 (APC = 13.5). After the year 2004 the mesothelioma time trend curve is stable (APC = -0.4). In a recent age-period-cohort analysis of the presented data the highest mesothelioma risk was detected for the co­hort born between 1940 and 1944.21 The peak value of asbestos importation in 197322 corresponds to the peak of mesothelioma incidence curve exactly 30 years later.Considering the latency between as­bestos exposure and mesothelioma development, which is around 40 years23, these results imply that the mesothelioma peak has already been reached in Slovenia. Projections As CRS is using many data sources the data collec­tion and preparation is delaying the report of can­cer burden figures for 2 to 3 years. The numbers for 2013 are currently available and presented in our results. However, the estimation of the num­ber of cancer cases was prepared for the current year, 2016. There will be 14,785 new cancer cases registered in the year 2016; 8,259 males and 6,527 females. The 2016 estimations with 95% confidence intervals for specific cancer sites are available in the supplementary material (SupMat 19). In males the number of head and neck, oesophageal, testicular and thyroid cancers is expected to be stable or even smaller. In females a smaller number is expected only for cervical cancer. Long-term prediction of cancer incidence can help to plan cancer services and cancer control measures for the future. Due to the aging popula­tion the overall number of new cancer cases is pre­dicted to increase further. The expected additional number of cases for some of common cancers in 2025 in Slovenia is presented in Figure 7, consider-ing only the ageing of population. The incidence of all cancers in males will increase for 32.7% till 2025 and for 13.3% in females. In 10 years in Slovenia more than 17,000 new cancer cases are expected. It is important to note that this number does not include non-melanoma skin cancers, which is es­timated to more than 2,000 additional incident cases. The increase of more than 30% is predicted for prostate, stomach, colorectal and lung cancers in males. There will be more than 600 additional prostate cancers and more than 300 additional colorectal and lung cancer cases in Slovenian male population in 2025. In females there will be only slight increase (8%) in the number of melanoma cases. More than 100 additional cases are expected in breast and colorectal cancer. The majority of predicted additional cases will be diagnosed in the elderly. Therefore, the median age at diagnosis will increase at population level. It is expected that in 2025 more the 70% of patients will be diagnosed at age 65 or more compared to 59% in 2013. They will on average have more con­comitant diseases and less favourable health relat­ed quality of life, which render specific oncological treatment more difficult or impossible to carry out. Hence, better palliative and adjusted specific on­cological treatment should be the major priority in Slovenian health care system in the next years. However, when predicting the future cancer burden, it is not enough to consider the ageing of population only. The number of incident cancer cases depends considerably also on the risk or pro­tective factors to which the population is exposed. In the next ten years in Slovenia we expect that the results of the effective national smoking cessation programmes will manifest. In male population the proportion of adult male smokers has almost halved in the last thirty years24, which in turn will lead to less smoking related cancers among men. In females the smoking cessation is not evident in Slovenian population so far and increasing num­ber of adolescents started smoking is of concern. National policy on healthy diet and physical activ­ity should further contribute to lower risk of com­mon cancers.9 In addition to success of primary preventions, the national organised cancer screen­ing policy will have major impact on the number of new cancer patients as well as mortality from cancers for which population based screening programmes are established. Besides cervical and colorectal cancer screening, breast cancer screen­ing will cover the whole female population in the target age group till the end of 2020, so decrease in breast cancer mortality is expected in the next decade. Considering the population ageing as well as the effects of primary and secondary prevention measures the cancer incidence is aimed to remain below 15,500 new cases in 2020 (9,000 in males and 6,500 in females) in the existing Slovenian National Cancer Control Plan.12 Conclusion In 1981, Doll and Peto25 estimated the proportion of cancer deaths attributable to known risk fac­tors. They judged that more than a third of cancer deaths were attributable to risk factors as conse­quence of unhealthy lifestyle. The most important are those that are the consequence of western way of life: obesity, diet with too much energy and low in vegetables and fruit, sedentary lifestyle together with smoking and high alcohol consumption. Changing political and economic circumstances over the past decades, resulted in very different lifestyle for most Slovenes and their exposure to carcinogens in the working and living environ­ment, but also led to changing health care organi­zation. Since latent period in cancer is generally longer than 20 years, it can be concluded that the effects of these changes are not fully developed yet.26 In recent years, near 14,000 Slovenes were di­agnosed with cancer per year and just over 6,000 died; more than 94,000 people who were ever diag­nosed with cancer are currently living among us. Slovenian population is ageing - because cancer oc­curs more among older, and additionally more nu­merous post-war generation is coming to this ages, it is expected that the burden of this disease will be increasing further, even if the level of risk factors remained the same as today. Control of the cancer burden can be estab­lished only by clear objectives, well-planned and financially supported interventions and constant monitoring of the quality and effectiveness of these actions. They should be summarised in national cancer control plans with well-established priori­ties.27 Cancer registries, a core component of can­cer health information systems, have a major role in providing data to prioritize programmes in na­tional cancer control plans as well as to monitor the progress of their implementation. In Slovenia, we are following these recommendations by the sec­ond National Cancer Control Programme, as well as with long-standing and good-quality cancer reg­istry. Acknowledgements The authors thank the staff of the Cancer Registry of Republic of Slovenia whose efforts to collect ac­curate and complete data have made this report possible. We express a sincere thankfulness to professors Matej Bračko, Marko Hočevar, Vera Pompe-Kirn, Primož Strojan and Branko Zakotnik for their care­ful review and commenting on the Slovenian can­cer burden indicators issued in the Annual Report of the Cancer Registry of Republic of Slovenia. References 1. Bray F. The burden of cancer in Europe. In: Coleman MP, Alexe D, Albreht T, McKee M, eds. Responding to the challenge of cancer in Europe. Ljubljana: Institute of Public Health of the Republic of Slovenia; 2008. p. 7-40. 2. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. 2013 [cited 25.12.2016]. Available from: http://globocan.iarc.fr. 3. Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 2013; 49: 1374-403. doi: 10.1016/j.ejca.2012.12.027 4. Coleman MP, Alexe D, Albreht T, McKee M, eds. Responding to the challenge of cancer in Europe. Ljubljana: Institute of Public Health of the Republic of Slovenia; 2008. 5. Forsea AM. Cancer registries in Europe-going forward is the only option. Ecancermedicalscience 2016; 10: 641. doi: 10.3332/ecancer.2016.641 6. Siesling S, Louwman WJ, Kwast A, van den Hurk C, O’Callaghan M, Rosso S, et al. Uses of cancer registries for public health and clinical research in Europe: Results of the European Network of Cancer Registries survey among 161 population-based cancer registries during 2010-2012. Eur J Cancer 2015; 51: 1039-49. doi: 10.1016/j.ejca.2014.07.016 7. CRRS. Cancer in Slovenia 2013. Ljubljana: Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Cancer Registry of Republic of Slovenia; 2016. 8. SLORA: Slovenia and Cancer [Internet]. Institute of Oncology Ljubljana, Epidemiology and Cancer Registry. 2010 [cited 15.12.2016]. Available from: http://www.slora.si/en. 9. Primic Zakelj M, Zagar T. The burden of cancer in Europe. In: Coleman MP, Alexe D, Albreht T, McKee M, eds. Cancer control in Slovenia: achievements, shortcomings and opportunities. Ljubljana: Institute of Public Health of the Republic of Slovenia; 2008. p. 279-96. 10. Luengo-Fernandez R, Leal J, Gray A, Sullivan R. Economic burden of cancer across the European Union: a population-based cost analysis. Lancet Oncol 2013; 14: 1165-74. doi: 10.1016/S1470-2045(13)70442-X 11. Ministry of Health RS. Slovene national cancer control programme 2010­2015. Ljubljana: Ministry of Health, Republic of Slovenia; 2010. 12. Ministry of Health RS. Slovene national cancer control programme 2017­2021. Ljubljana: Ministry of Health, Republic of Slovenia; 2017. 13. Zadnik V, Primic Zakelj M, Zagar T. Linkage to supplementary registration sources: key lessons learned in the last 30 years in Slovenia. In: Benider A, Zanetti R, Siesling S, Bray F, eds. The 38th Annual IACR Conference. Marrakech: IACR; 2016. 14. Segi M. Cancer mortality for selected sites in 24 countries (1950-57). Sendai: Department of Public Health, Tohoku University of Medicine; 1960. 15. JPR. Joinpoint Regression Program, Version 4.0.4. Bethesda: Statistical Research and Applications Branch, National Cancer Institute; 2013. 16. Dyba T, Hakulinen T, Paivarinta L. A simple non-linear model in incidence prediction. Stat Med 1997; 16: 2297-309. 17. Gatta G, van der Zwan JM, Casali PG, Siesling S, Dei Tos AP, Kunkler I, et al. Rare cancers are not so rare: the rare cancer burden in Europe. Eur J Cancer 2011; 47: 2493-511. doi: 10.1016/j.ejca.2011.08.008 18. Tepeš B, Kasesnik K, Novak Mlakar D. Smernice programa SVIT. Slovenske smernice za zagotavljanje kakovosti presejanja raka debelega črevesa in danke. [Guidelines of Svit programme. National guidelines for quality insur­ance in colorectal screening; in Slovene]. Ljubljana: National Institute for Public Health; 2015. 19. Elfstrom KM, Arnheim-Dahlstrom L, von Karsa L, Dillner J. Cervical cancer screening in Europe: Quality assurance and organisation of programmes. Eur J Cancer 2015; 51: 950-68. doi: 10.1016/j.ejca.2015.03.008 20. Ivanus U, Primic Zakelj M, eds. 6. izobraževalni dan programa ZORA. [6th educational day of the ZORA programme; in Slovene]. Ljubljana: Institute of Oncology Ljubljana; 2015. 21. Zadnik V, Primic Zakelj M, Jarm K, Zagar T. Time trends and spatial patterns in the mesothelioma incidence in Slovenia, 1961–2014. Submitted to Eur J Cancer Prev. 2016. 22. Dodic Fikfak M, Sesok J. Nacionalne smernice za azbest. Zaključno poročilo projekta. [National guidelines for asbestos. Final report, in Slovene]. Ljubljana: Institute of Public Health of the Republic of Slovenia; 1999. 23. Robinson BM. Malignant pleural mesothelioma: an epidemiological per­spective. Ann Cardiothorac Surg 2012; 1: 491-6. doi: 10.3978/j.issn.2225­319X.2012.11.04 24. Maucec Zakotnik J, Tomsic S, Kofol-Bric T, Korosec A. Zdravje in vedenjski slog prebivalcev Slovenije. Trendi v raziskavi CINDI 2001-2004-2008. [Health and lif style in Slovenian population. Trends from CINDI researches 2001­2004-2008; in Slovene]. Ljubljana: Institute of Public Health of the Republic of Slovenia; 2012. 25. Doll R, Peto R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 1981; 66: 1191­ 308. 26. Zadnik V, Primic Zakelj M, Krajc M. Breme raka v Sloveniji v primerjavi z drugimi evropskimi državami = Cancer burden in Slovenia in comparison with the burden in other European countries. Zdrav Vestn 2012; 81: 407­ 12. 27. Albreht T, Martin-Moreno JM, Jelenc M, Gorgojo L, Harris M, eds. European Guide for Quality National Cancer Control Programmes. Ljubljana: National Institute of Public Health; 2015. research article Leiomyosarcoma of the renal vein: analysis of outcome and prognostic factors in the world case series of 67 patients Marko Novak1, Andraz Perhavec1, Katherine E. Maturen2, Snezana Pavlovic Djokic3, Simona Jereb4, Darja Erzen1 1 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Radiology, University of Michigan Hospitals, Ann Arbor, Michigan, USA 3 Department of Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Department of Radiology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2017; 51(1): 56-64. Received 11 June 2016 Accepted 5 October 2016 Correspondence to: Marko Novak, M.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +38615879949; Fax: +38615879998; E-mail: mnovak@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Leiomyosarcoma is a rare malignant mesenchymal tumour. Some cases of leiomyosarcoma of the renal vein (LRV) have been reported in the literature, but no analysis of data and search for prognostic factors have been done so far. The aim of this review was to describe the LRV, to analyse overall survival (OS), local recurrence free survival (LRFS) and distant metastases free survival (DMFS) in LRV world case series and to identify significant predictors of OS, LRFS and DMFS. Methods. Cases from the literature based on PubMed search and a case from our institution were included. Results. Sixty-seven patients with a mean age of 56.6 years were identified; 76.1% were women. Mean tumour size was 8.9 cm; in 68.7% located on the left side. Tumour thrombus extended into the inferior vena cava lumen in 13.4%. All patients but one underwent surgery (98.5%). After a median follow up of 24 months, the OS was 79.5%. LRFS was 83.5% after a median follow up of 21.5 months and DMFS was 76.1% after a median follow up of 22 months. Factors predictive of OS in univariate analysis were surgical margins, while factors predictive of LRFS were inferior vena cava luminal extension and grade. No factors predictive of DMFS were identified. In multivariate analysis none of the factors were predictive of OS, LRFS and DMFS. Conclusions. Based on the literature review and presented case some conclusions can be made. LRV is usually lo­cated in the hilum of the kidney. It should be considered in differential diagnosis of renal and retroperitoneal masses, particularly in women over the age 40, on the left side and in the absence of haematuria. Core needle biopsy should be performed. Patients should be managed by sarcoma multidisciplinary team. LRV should be surgically removed, with negative margins. Key words: leiomyosarcoma; renal vein; surgery, outcome Introduction Leiomyosarcoma (LMS) is a rare malignant mes­enchymal tumour of smooth muscle origin. It represents only 5–7% of soft tissue sarcomas.1 Approximately 2.0% of LMS originate from the smooth muscle of vessel walls, predominantly veins and 60.0% of these originate from inferior vena cava (IVC).1 According to Gage et al. 1, the most common location of extracaval venous LMS is the renal vein, followed by the great saphenous, pulmonary and femoral vein. Leiomyosarcoma of the renal vein (LRV) is extremely rare. There have been some cases reported in the literature, but no 57 analysis of data and search for prognostic factors have been done so far. The first case was reported by Lopez Varela and Pereira Garro in 1967.2 We present an additional case, world literature over­view and the outcome of these patients. Patients and methods Literature overview and data collection The search criteria in PubMed were “leiomyosar­coma“ and “renal vein“. In the literature 62 articles were identified describing cases of LRV. Fourteen of the articles were in Japanese, 7 in French, 4 in Spanish, 1 in Polish and 36 in English. Data from 49 articles only2-50 were merged into a database, be­cause out of 18 Japanese cases only 4 were reported in English articles 23,37,40,44 and the rest in Japanese articles, not accessible to us (Figure 1). The last re­view of Japanese cases by Kato et al.42 was trans­lated and these data included in the study. Three times the patient was discussed as different case report by two different authors.3-4,13-14,20-26 That low­ered the total number of reported cases in the last review by three.50 In some articles more than one case was reported.14,19,26,42,46 The authors were from the fields of urology (18/49; 36.7%), surgery (15/49; 30.6%), radiology (8/49; 16.3%), pathology (5/49; 10.2%) and internal medicine (3/49; 6.1%). A retro­spective review was performed to evaluate patient demographics, tumour site, clinical presentation, operative details, tumour thrombus IVC exten­sion, neoadjuvant and adjuvant treatment, tumour size, tumour grade, surgical margin status, time to local recurrence, time to dissemination, time to death and status at last follow up. To the authors or coauthors 18 emails were sent around the world to update the data and follow up, we received 4 replies. Illustrative case A 46-years old female presented in January 2014 to University Hospital Ljubljana with upper abdomi­nal pain of 6 months duration and weight loss. Her past medical history was unremarkable. On physical examination there was a palpable mass in the left upper abdomen. Gastroscopy was not diagnostic, but computed tomography (CT) re­vealed a left retroperitoneal mass, 11 x 10 x 9 cm in size, interposed between the aorta and hilum of the left kidney (Figure 2). The tumour surrounded the left renal artery and the vein was not identified. Ultrasound guided fine needle aspiration biopsy (FNAB) was performed. The sample was suspi­cious for LMS. She was referred to the Institute of Oncology Ljubljana in February 2014 for manage­ment and treatment. A dynamic renal scintigraphy was performed for evaluation of kidney function. Excretory function of the left kidney was 47% and of the right kidney 53%. Thoracic CT revealed no metastases. After discussing the case at the multi­disciplinary team (MDT) we decided to perform surgery, without preoperative core needle biopsy (CNB). Tumour was removed en bloc with left co­lon, left kidney, adrenal gland and psoas fascia. A suspicious node 3 cm in size was found intraopera­tive in psoas muscle close to the vertebra. It was removed separately. The main specimen weighed 1152 g. Histology confirmed a LMS, according to Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC) grading system grade 3, 12 cm in largest diameter, originating from the left renal vein, not infiltrating the surrounding organs. Surgical margins were negative. The spindle tu­mour cells stained positive for smooth muscle ac­tin, desmin and focally for CD34. The separately removed node was an LMS satellite, margins were positive (Figure 3). She received adjuvant radio­therapy (RT), 59 Gy. There were no surgical or radiotherapy-related complications. In December 2014, 10 months postoperatively, liver metastases were detected on CT. After subsequent magnetic resonance imaging treatment was planned at the MDT. All 4 liver metastases, 4 to 17 mm in size were removed surgically with clear margins. She received no adjuvant treatment. In October 2015 lung metastases were detected on both sides, the largest 17 mm. She is receiving chemotherapy (ChT) with adriamycin, ifosfamide and mesna at the time of this report. Written informed consent for all diagnostic and therapeutic procedures was obtained from the pa­tient. Statistical analysis On the basis of limited data, univariate analysis was used to evaluate the following potential prog­nostic factors for overall survival (OS), local recur­rence free survival (LRFS) and distant metastases free survival (DMFS): age, gender, tumour site, dis­semination, weight loss, palpable mass, operation type, tumour thrombus IVC luminal extension, tumour size, grade, margin status, number of mi­toses and neoadjuvant or adjuvant treatment. OS and LRFS were compared using log-rank test. All comparisons were two sided. P-value of 0.05 was considered statistically significant. Survival curves were calculated and plotted using Kaplan-Meier method. Cox’s multivariate regression was used to identify independent prognostic variables of OS, LRFS and DMFS. The statistical program SPSS® version 22 was used for analysis. Results Patient and tumour characteristics In total 67 cases were identified. The tumour pre­dominantly occurred in women (76.1%; 51/67) and on the left side (68.7%; 46/67). The mean age at diag­nosis was 56.6 years (range 27–93 years). Detailed patient and clinicopathologic characteristics are presented in Table 1, Figure 4 and 5. Histological biopsy before treatment was performed in 9 pa­tients (13.4%; 9/67); 1 patient had biopsy during exploration9, 4 patients had CT guided CNB19,20,27,50, the biopsy type for 2 patients was not specified in the article33,41 and 2 patients had biopsy through femoral approach during cavography.36,43 FNAB before operation was performed in 1 patient34 and in our case (3.0%; 2/67). The mean tumour size was 8.9 cm, described in 54 cases (80.6%; 54/67). System used for sarcoma grading was defined in single article.10 Tumour grade was described in 28 cases (41.8%; 28/67), surgical margin status in 18 cases (26.9%; 18/67) and number of mitoses in 18 cases TABLE 1. Patients data and histologic variables, treatment modalities and disease (26.9%; 18/67). Tumour cells stained positive for smooth muscle actin in 23 cases (34.3%; 23/67), for desmin in 22 cases (32.8%; 22/67) and for vimentin in 6 cases (9.0%; 6/67). Intraluminal caval tumour thrombus was reported in 9 cases (13.4%; 9/67), IVC mural invasion in 3 cases (4.5%; 3/67), the renal parenchyma invasion in 8 cases (11.9%; 8/67) and the adrenal gland invasion in a single case (1.5%; 1/67). The data about IVC mural invasion were tak­en as stated in the articles.11,15,16 Surgery All patients but one underwent surgery (98.5%; 66/67). Four patients had tumorectomy (6.0%; 4/67) and 60 had nephrectomy (89.6%; 60/67). One pa­tient had attempt of laparoscopic tumorectomy, two had laparoscopic nephrectomy and one had robotic laparoscopic nephrectomy. Two patients (3.0%; 2/67) had compartment resection, tumour removed en bloc with (at least) adjacent segment of colon, kidney and psoas. Adrenalectomy was performed in 11 patients (16.4%; 11/67) and lymph node dissection in 6 patients (9.0%; 6/67). Tumour thrombus extended into the lumen of IVC in 9 pa­tients (13.4%; 9/67), in 4 cases tumour was on the left side and in 5 cases on the right. In two of these patients there was also invasion of the caval wall. IVC was resected in 5 patients (7.5%; 5/67), once li­gated and without reconstruction, once oversewn, once reconstructed with venous patch and once with allograft. There are no data about the type of operation on IVC for the fifth patient. Cavotomy and extraction of the tumour thrombus was per­formed in 3 patients (4.5%; 3/67). One patient had locally advanced tumour, with tumour extension into the right atrium and received palliative ChT only. In a patient with tumour caval wall invasion progression Age at diagnosis (year) Gender Side Size (cm) Tumour thrombus extension Preoperative biopsy Tumour grade Surgical margins Operation Preoperative treatment Intraoperative treatment Postoperative treatment Disease progression Site of dissemination Mean Range Female Male Left Right Mean Range IVC Histology Fine needle aspiration No biopsy G1 G2 G3 Unknown Negative Positive Unknown Nephrectomy Tumorectomy Compartment resection No operation Embolization ChT RT + ChT RT RT ChT ChT + RT Immunotherapy LR M LR + M Total Liver Lungs Bone Soft tissue 56.6 27–93 51 16 46 21 8.9 3.5–25 9 9 2 56 6 8 14 39 15 3 49 60 4 2 1 3 1 2 1 7 9 1 1 3 20 10 33 17 16 8 4 76.1 23.9 68.7 31.3 13.4 13.4 3.0 83.6 9.0 11.9 20.9 58.2 22.4 4.5 73.1 89.6 6.0 3.0 1.5 4.5 1.5 3.0 1.5 10.4 13.4 1.5 1.5 4.5 29.9 14.9 49.3 25.4 23.9 11.9 6.0 ChT = chemotherapy; G = grade; IVC = inferior vena cava; LR = local recurrence; M = metastases; RT = radiotherapy TABLE 2. Univariate analysis of overall, local recurrence free and distant metastases free survival (log-rank) Age . 50 50 13 26 5 12 0.285 15 30 3 8 0.519 Gender F M 32 7 15 2 0.812 38 7 9 2 0.993 Side L R 29 10 9 8 0.448 31 14 7 4 0.987 Weight loss Y N 5 34 6 11 0.414 9 36 2 9 0.734 Palpable mass Y N 9 30 6 11 0.581 12 34 4 9 0.562 Operation Intracaval luminal extension Nephrectomy Tumorectomy Compartment Y N 35 2 2 5 34 15 2 0 3 14 0.543 (nephrectomy or tumorectomy vs. compartment) 0.340 41 2 2 4 41 9 2 0 4 7 0.562 0.016 . 10 cm 23 10 0.485 28 5 0.219 Tumour size Grade > 10 cm Unknown 1 2 or 3 Unknown 11 5 4 13 22 3 4 2 6 9 (. 10 cm vs. > 10 cm) 0.265 (1 vs. 2 or 3) 11 6 6 12 27 3 3 0 7 4 (. 10 vs. > 10 cm) 0.05 (1 vs 2 or 3) Margins Mitoses/10hpf Negative Positive Unknown <10 .10 Unknown 14 1 24 8 2 29 1 1 15 2 1 14 0.014 (neg. vs. pos.) 0.782 (<10 vs. .10) 12 0 33 8 1 36 3 2 6 2 2 7 0.096 (neg. vs. pos.) 0.244 (<10 vs. .10) Neoadjuvant/ adjuvant chemotherapy Neoadjuvant/ adjuvant raditherapy Y N Y N 8 31 6 33 3 14 4 13 0.987 0.165 7 38 8 37 4 7 2 9 0.337 0.975 F = female; L = left; LR = local recurrence; M = male; N = No; R = right; Y = Yes . 50 9 9 marginal and R1. The operation would be optimal if both specimens would be removed en bloc, but the margins on the vertebra would probably be Age > 50 22 16 0.805 positive anyway. Because reoperation with clear F 27 20 0.221 margins on vertebra in case of local recurrence Gender M 45 L 19 19 would probably not be possible, we decided for Side 0.138 R 12 6 adjuvant RT. Y 29Weight loss 0.087 According to magnetic resonance imaging liver N 29 16 Y 5 10 Palpable mass 0.277 metastases were small and resectable and that was N 26 15 0.336 the reason at the MDT to decide for metastasec- Nephrectomy 27 23 (nephrectomy or Operation Tumorectomy 3 1 tomy. tumorectomy vs. Compartment 1 1 compartment) LRV is very rare. Cases from the last literature Intracaval luminal Y 4 4 extension N 27 21 0.284 overview in 2010 50, cases from nonenglish litera­. 10 cm 21 12 0.210 ture, new reports from 2010–2015 and present case Tumour size > 10 cm 7 7 (. 10 cm vs. Unknown 3 6 > 10 cm) were summarised. From data gathered from these 1 42 0.131 case reports, subsequent analysis and with respect Grade 2 or 3 9 10 (1 vs. 2 or 3) Unknown 18 13 to sarcoma guidelines, several observations can be Negative 10 5 0.815 made. Margins Positive 1 1 (neg. vs. pos.) Unknown 20 19 From the clinical point of view, LRV presents <10 9 1 Mitoses/10hpf .10 1 2 0.266 difficulties in making diagnosis, because it is un­ (<10 vs. .10) Unknown 21 22 common, has no specific symptoms and no pathog- Neoadjuvant/ Y 56 0.683 nomonic radiological features. It predominantly adjuvant N 26 19chemotherapy occurs in women (76.1%), on the left side (68.7%) Neoadjuvant/ Y 55 adjuvant 0.087 and affects older population, with the peak occur- N 26 20radiotherapy ring at age 60–69 years. Presenting symptoms are DM = distant metastases; F = female; L = left; M = male; N = No; R = right; Y = Yes unspecific, abdominal pain was reported in 49.3%. Hematuria was reported in a single case (1.5%) of LRV patients, but is present in more than one third of the cases (34.8%) of renal cell carcinoma (RCC) with venous extension.51 Genetic predisposition may play a role in development of primary LRV, with two patients being treated for retinoblastoma and one patient having Li Fraumeni syndrome. From the point of imaging, location of LRV is more important than the size of the tumour. It can overlap with much more common RCC with venous extension. LRV is usually located in the hilum of the kidney. The bulk of the tumour lies predominantly or entirely outside the hilar paren­chyma or the tumour is limited to the renal vessels [46]. The mean tumour size in this LRV group is 8.9 cm. In a study group of 1192 patients with RCC with extension into the renal vein (23.0%) and IVC (7.0%) the mean tumour size was 8.9 cm as well.52 It may not be possible to distinguish between these two entities by imaging. Other diagnoses consid­ered in this location are metastatic lymph node in a patient with a history of malignancy, renal pel­vis leiomyosarcoma, extremely rare as well, with around 10 cases reported in the literature53, lym­phoma, adrenal gland tumour, upper tract urothe­lial carcinoma, granulomatous disease and renal vein thrombus.46 With regard to biopsy, retroperitoneal mass is usually detected on abdominal CT scans. When imaging is not diagnostic of a retroperitoneal li­posarcoma, image-guided CNB of retroperitoneal tumour is strongly recommended to obtain the sample for diagnosis. Correct diagnosis may sig­nificantly affect surgical decision and neo/adjuvant therapy.54,55 Wilkinson et al.56 from Royal Marsden, London reported, that preoperative CNB for retro­peritoneal sarcoma (RPS) is safe and does not af­fect oncological outcome. Patients with intermedi­ate and high-grade RPS were included. There were no intra-abdominal complications requiring early operation. The group of 90 patients with preopera­tive CNB was compared to a group of 60 patients, who did not have preoperative CNB. There was no significant difference in local recurrence (p = 0.101) or OS (p = 0.191). FNAB in retroperitoneal tumours rarely yields diagnostic information and should be avoided55, but it can be performed in RCC in spite of danger of haemorrhage. In the present review preoperative histological biopsy was performed in 13.4% of cases only and FNAB in 3.0%. With regard to treatment, the only potentially curative treatment for RPS is surgery with mac­roscopically complete resection.54,55,57 The role of ChT and RT in RPS is not proven and still under investigation. It is generally recommended, that in case of RT administration, it should be delivered in the preoperative setting and possibly within a clinical trial.54 Postoperative RT should not be ad­ministered routinely in R0 and R1 resections.54 ChT is an option in the preoperative setting of resect-able disease, is an option after surgery in case of R2 resection and is an option in case of unresectable or metastatic disease.54 Because of complex evaluation and treatment options patients with RPS should be managed by sarcoma MDT in a specialized reference center.54,55 Histologic subtype is one of the major deter­minants of the oncologic outcome in RPS. The most common location of LMS is the retroperito­neum58, where it represents the second most com­mon histological subtype after liposarcoma, ac­counting for 14–36% of patients in major series.59,60 Retroperitoneal LMS has a high propensity for dis­tant recurrence. The reported rate of distant metas­tases for retroperitoneal LMS at 5 years is around 40–50% and for local recurrence at 5 years around 5%.61 Similar results are present in the present review, with the rate of local recurrence of 4.5% (3/67), distal metastases of 29.9% (20/67) and both in 14.9% (10/67), but in much shorter period of fol­low up. And finally, in the present review of the litera­ture 79.5% of the LRV patients survived at 2 years. A 5-year OS, LRFS and DMFS was not performed because of the inadequate sample size at that length of follow up. Retrospective comparisons of series of RPS patients have demonstrated 5-years OS rates of 50–70% and 5-years local control rates of 40–80%.57 In the IVC LMS series 5-year survival has been reported between 33.0% and 53.0%.26 Data from different large series of RPS patients have demonstrated tumour grade and surgical margin status as independent prognostic factors of OS and LRFS.62,63 Cases from this review are dispersed world wide and through half of the century, lack­ing data for tumour grade (58.2%; 39/67), surgical margin status (73.1%; 49/67) and follow up (16.4%; 11/67). Because of insufficient histologic data and truncated follow up, we were not able to identify prognostic factors of OS, LRFS and DMFS in mul­tivariate analysis. As a retrospective analysis this study has limita­tions. Most of the information collected was from case reports, without significant follow up and lacking histological data. As a consequence, there was a limitation in the statistical analysis and the conclusions that could be drawn from it, particu­larly in patients’ outcome. However, to our knowl­edge, this is the largest study on this topic, and even this limited survey expands our understand­ing of the natural history of this rare sarcoma. Conclusions LRV is usually located in the hilum of the kidney. It should be considered in differential diagnosis of renal and retroperitoneal masses, particularly in women over the age of 40, on the left side and in the absence of hematuria. Core needle biopsy should be performed. Patients should be managed by sarcoma MDT. For optimal clinical outcomes, LRV should be surgically removed, with negative margins. After a median follow up of 24 months OS was 79.5%, LRFS was 83.5% after a median fol­low up of 21.5 months and DMFS was 76.1% after a median follow up of 22 months. Factors predictive of OS in univariate analysis were surgical margins, while factors predictive of LRFS were inferior vena cava luminal extension and grade. No factors pre­dictive of DMFS were identified. Because of insuf­ficient histologic data and follow up, we were not able to identify prognostic factors of OS, LRFS and DMFS in multivariate analysis. Acknowledgements The authors gratefully acknowledge Dr. Gideon Adam Blecher, Dr. Mark Frydenberg, Dr. Yosuke Ikegami, Dr. Wojciech Wysocky, Dr. Zbigniew Darasz and Dr. Raghu Vikram for assistance with patients data collection. Sincere thanks to Matjaž Musek, Ksenija Žmavc and the Library of the Institute of Oncology Ljubljana for providing the articles, to Jure Čižman for preparing the figures and to Ikue Nishi for translation from Japanese. References 1. Gage MJ, Patel AV, Koenig KL, Newman E. Non-vena cava venous leiomyo­sarcoma: a review of the literature. Ann Surg Oncol 2012; 19: 3368-74. 2. Lopez Varela EA, Pereira Garro C. 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Ann Surg Oncol 2014; 21: 2150-8. research article Prognostic significance of uPA/PAI-1 level, HER2 status, and traditional histologic factors for survival in node-negative breast cancer patients Nina Fokter Dovnik1, Iztok Takac1,2 1 Maribor University Clinical Center, Maribor, Slovenia 2 Faculty of Medicine, University of Maribor, Maribor, Slovenia Radiol Oncol 2017; 51(1): 65-73. Received 13 December 2015 Accepted 3 April 2016 Correspondence to: Nina Fokter Dovnik, M.D., Department of Gynecologic Oncology and Oncology of the Breast, Maribor University Clinical Center, Ljubljanska 5, 2000 Maribor, Slovenia. Phone: +386 2 321 2178; Fax: +386 2 321 2085; E-mail: nfokter@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The association of HER2 status with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) levels raises the question whether uPA/PAI-1 level carries additional clinically relevant prognostic information independently from HER2 status. The aim of our study was to compare the prognostic value of uPA/PAI-1 level, HER2 status, and traditional prognostic factors for survival in node-negative breast cancer patients. Patients and methods. A retrospective analysis of 858 node-negative breast cancer patients treated in Maribor University Clinical Center, Slovenia, in the years 2000–2009 was performed. Data were obtained from patient medical records. The median follow-up time was 100 months. Univariate and multivariate analyses of disease-free (DFS) and overall survival (OS) were performed using the Cox regression and the Cox proportional hazards model. Results. In univariate analysis, age, tumor size, grade, lymphovascular invasion, HER2 status and UPA/PAI-1 level were associated with DFS, and age, tumor size, grade, and uPA/PAI-1 level were associated with OS. In the multivariate model, the most important determinants of DFS were age, estrogen receptor status and uPA/PAI-1 level, and the most important factors for OS were patient age and tumor grade. The HR for death from any cause in the multivariate model was 1.98 (95% CI 0.83–4.76) for patients with high uPA and/or PAI-1 compared to patients with both values low. Conclusions. uPA/PAI-1 level clearly carries an independent prognostic value regardless of HER2 status in node-neg­ative breast cancer and could be used in addition to HER2 and other markers to guide clinical decisions in this setting. Key words: node-negative breast cancer; adjuvant systemic treatment; survival; uPA/PAI-1; HER2 status Introduction One of the greatest challenges in the treatment of node-negative breast cancer is deciding in which patients the benefit from adjuvant cytotoxic chem­otherapy would outweigh its adverse effects. Traditionally, this decision has been based on clini­cal and histomorphologic prognostic factors, such as patient age, tumor size, tumor grade, presence of lymphovascular invasion, and steroid hormone receptor status. Human epidermal growth factor receptor 2 (HER2) status, first used as a prognostic marker, became an important factor for predicting response to anti-HER2 therapy and is now a crucial part of this decision-making. The serine protease urokinase-type plasmino­gen activator (uPA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) are markers of tumor invasion and metastasis that have reached the highest level of evidence for clinical utility as prog­nostic factors in breast cancer.1,2 Node-negative patients with high values of uPA and/or PAI-1 have been shown to benefit from adjuvant chemo­therapy in a prospective randomized multicenter therapy trial.3,4 In addition, independent prognos­tic value of uPA/PAI-1 was confirmed in a pooled analysis of 8377 breast cancer patients that showed high levels of uPA and PAI-1 to be the strongest predictors of relapse-free survival and overall sur­vival apart from lymph node status.5 In spite of this evidence, these biomarkers are still not widely used in the clinic.1 Unfortunately, none of the large trials investi­gating the prognostic value of uPA and PAI-1 in­cluded HER2 status in the survival analysis. Only limited information is available on the relative prognostic impact of these factors when considered along with traditional prognostic markers in the same group of breast cancer patients. Therefore, it is still uncertain whether uPA and PAI-1 can give additional clinically relevant prognostic informa­tion after traditional prognostic factors and HER2 status have been taken into account. To address this issue, we undertook the present study with the aim of comparing the prognostic impact of HER2 status, uPA, PAI-1, and traditional prognostic factors tumor size, grade, histological subtype, lymphovascular invasion, steroid hor­mone receptor status, and patient age on disease-free, overall, and breast cancer specific survival in node-negative breast cancer patients. Patients and methods Our retrospective analysis included all patients with lymph node-negative invasive breast cancer without distant metastases who underwent pri­mary surgical treatment in Maribor University Clinical Center, Slovenia, in the ten-year period between January 1 2000 and December 31 2009. Exclusion criteria were neoadjuvant chemotherapy and presence of another active malignancy during breast cancer treatment. Considering the Helsinki Declaration principles the Slovenian National Medical Ethics Committee approved this study (Approval No. 55/11/13). Clinical information on diagnosis, treatment, and follow-up was obtained from patient medical records. Survival data were completed with up­dated information from Slovenian Cancer Registry. Data on tumor size, histological subtype, grade, lymph node status, steroid hormone receptor sta­tus, and HER2 immunohistochemistry were ob­tained from original histology reports from the pri­mary surgery. HER2 gene amplification, uPA and PAI-1 levels were obtained from our institution’s Medical Genetics Laboratory. Due to economical limitations, uPA and PAI-1 could not be assessed in all patients. Some other histological data were missing in a small fraction of patients due to incon­sistent hospital guidelines on histology reports in the past. All patients underwent either modified radi­cal mastectomy or breast conserving surgery and radiotherapy. Adjuvant systemic treatment was given according to the guidelines followed at our institution at the time and was not influenced by uPA and/or PAI-1 values. Patients who completed primary treatment were followed-up at our institu­tion at regular intervals. HER2 status was determined immunohisto­chemically and additional fluorescent in situ hy­bridization (FISH) with PathVysion HER-2 DNA Probe Kit (Abbott Molecular, Abbott Park, IL, USA) was performed in samples with an immuno­histochemical result of 2+. Since the patients were diagnosed before the publication of new ASCO/ CAP guideline recommendations for immunohis­tochemical testing of estrogen and progesterone receptors6 and human epidermal growth factor receptor 2 testing in breast cancer7 and treated ac­cordingly, steroid hormone receptor status and HER2 status were determined using the old guide­line recommendations in order to avoid cases with diagnostic-therapeutic mismatch. uPA and PAI-1 were analyzed prospectively from representative pieces of tumor tissue that were frozen in liquid nitrogen after histologic eval­uation. The frozen samples were pulverised us­ing a micro-dismembrator, suspended in a buffer (pH 8.5) containing 0.02 M Tris-HCl, 0.125 M NaCl and 2% Triton X-100, and shaken for three hours at 4°C. The obtained suspension was centrifuged for 30 minutes at 100,000 x g. Protein content was determined with the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and uPA and PAI-1 content was determined with commercially available ELISA assay kits (American Diagnostica, Greenwich, CT, USA). uPA and PAI-1 content was expressed as nanograms of analyte per milligram of tissue protein. The correlations between different variables were tested with Spearman’s rank correlation, Kruskal-Wallis test, Mann-Whitney U test and chi-square test, depending on the type of variables. The continuous variables uPA and PAI-1 were coded as binary variables using the previously optimized cutoffs of 3 ng uPA/mg protein and 14 ng PAI-1/mg protein to distinguish between low- and high-risk patients8, and combined into one variable (both low versus one or both high) as previously shown to be of greatest clinical relevance.9 Because adju-TABLE 1. Distribution of traditional prognostic factors, HER2 status, uPA and PAI-1 in the study group of node-negative breast cancer patients (N = 858) vant trastuzumab was not available for the whole cohort, the patients were divided into three groups based on HER2 status and adjuvant trastuzumab treatment for the survival analyses: HER2 nega­ tive, HER2 positive who were not treated with ad­ < 40 years 37 4.3 juvant trastuzumab, and HER2 positive who were Pathological tumor size 846 treated with adjuvant trastuzumab. Disease-free < 2 cm 474 56.0 . 2 cm 372 44.0 survival (DFS) was calculated from the date of pri- Pathological tumor type 858 mary surgery until the date of disease recurrence Ductal invasive 720 83.9 or death from any cause, or the date of the last Other invasive 138 16.1 follow-up visit in case of no recurrence or death. Histological grade 799 G1-2 557 69.7 Overall survival (OS) was calculated from the date G3 242 30.3 of diagnosis until the date of death from any cause, Lymphovascular invasion 795 No 720 90.6 or the date of the last follow-up visit. Breast can- Yes 75 9.4 cer specific survival (BCSS) was calculated from Estrogen receptor status 854 the date of diagnosis until the date of death from Positive 674 78.9 Negative 180 21.1 breast cancer, or the date of the last follow-up visit Progesterone receptor status 803 or death from other causes for censored patients. Positive 466 58.0 Kaplan-Meier method was used to calculate sur-Negative 337 42.0 vival curves and univariate Cox regression was HER2 status 761 Negative 610 80.2 used to assess the differences between the curves Positive, without adjuvant trastuzumab 97 12.7 in univariate analysis. Multivariate analyses were Positive, with adjuvant trastuzumab 54 7.1 uPA and PAI-1 level 332 performed by applying the multivariate Cox pro- Both low 159 47.9portional hazards model. All variables regardless One or both high 173 52.1 of univariate analysis results were initially in­cluded in the Cox model and the method used for the model was backward stepwise likelihood ratio (LR). Model if term removed was reported sepa­rately. All tests were performed at a significance level of p=0.05 and a confidence interval (CI) of 95%. All p values were two-sided. Statistical analy­sis was performed using the SPSS software pack­age v. 21 (IBM, Armonk, NY, USA). Results Patient characteristics Eight hundred fifty-eight node-negative distant metastasis-free breast cancer patients who under­went primary surgery with curative intent were included in the study. The median age of the pa­tients was 62 years (range, 24-95 years). The distri­bution of the traditional prognostic factors, HER2 status, uPA and PAI-1 in the study group is pre­sented in Table 1. 787 (91.7%) patients received some kind of adjuvant systemic therapy. Of these, 132 received adjuvant chemotherapy, 522 adjuvant hormone therapy, and 133 a combination of both. Among patients who were given adjuvant chemo­therapy, 79.2% received anthracycline-based ther­apy and the majority of the others received CMF (cyclophosphamide, methotrexate, 5-fluorouracil). Some factors could not be assessed in all tumors. Among patients who were given hormone therapy, 28.9% received tamoxifen, 52.7% received an aro­matase inhibitor, and the rest received a combina­tion of both. Adjuvant trastuzumab was given in combination with chemotherapy in 6.4% of all pa­tients and in 35.8% of HER2-positive patients. The median follow-up time was 100 months (range, 49–181 months). Associations between traditional prognostic factors, HER2 status, uPA and PAI-1 Positive HER2 status was observed more frequently in younger patients (p = 0.008), in larger tumors (p < 0.001), tumor types other than ductal invasive (p = 0.035), tumors of a higher differentiation grade (p < 0.001), with lymphovascular invasion (p = 0.018), those with a lower expression of estrogen (p < 0.001) and progesterone receptors (p = 0.002), as well as in tumors with higher levels of uPA (p = 0.026) and PAI-1 (p = 0.023). uPA and PAI-1 levels correlated positively with each other (r = 0.553, p < 0.001). Higher uPA and PAI-1 values were seen in larger tumors (p < 0.001 for uPA; p = 0.004 for PAI-1), his­ TABLE 2. Univariate and multivariate analysis of disease–free survival in lymph node–negative breast cancer patients with a median follow–up time of 100 months. Multivariate analysis was performed in the 273 patients for whom complete data were available Age (continuous, unit = 10 years) 1.80 (1.60–2.02) < 0.001 1.53 (1.20–1.96) 0.001 1.51 (1.19–1.93) 0.001 < 0.001 Tumor size (.2 vs. <2 cm) 1.41 (1.08–1.83) 0.012 1.01 (0.56–1.80) 0.982 – – 0.982 Tumor type (other invasive vs. ductal invasive) 1.08 (0.76–1.55) 0.658 1.28 (0.51–3.20) 0.598 – – 0.606 Grade (G3 vs. G1–2) 1.37 (1.03–1.81) 0.031 1.54 (0.75–3.17) 0.240 – – 0.240 Lymphovascular invasion (present vs. absent) 1.54 (1.03–2.29) 0.035 1.43 (0.65–3.15) 0.377 – – 0.393 Estrogen receptors (negative vs. positive) 1.19 (0.87–1.61) 0.271 1.81 (0.79–4.16) 0.165 2.25 (1.24–4.09) 0.008 0.158 Progesterone receptors (negative vs. positive) 1.10 (0.84–1.45) 0.496 1.03 (0.48–2.23) 0.935 – – 0.935 HER2 status 0.005 0.355 0.379 positive NT vs. negative 1.73 (1.21–2.47) 0.003 1.66 (0.83–3.31) 0.150 – – positive T vs. negative 0.70 (0.35–1.43) 0.332 1.16 (0.46–2.91) 0.758 – – uPA/PAI–1 (one or both high vs. both low) 2.16 (1.23–3.72) 0.005 1.76 (0.89–3.49) 0.106 1.99 (1.05–3.77) 0.035 0.098 NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab FIGURE 1. Effect of uPA/PAI-1 level and HER2 status on disease-free survival (DFS) in lymph-node negative breast cancer patients. (A) uPA/PAI-1 low (19 of 159 relapsed or died) versus uPA/PAI-1 high (43 of 173 relapsed or died). (B) HER2 negative (154 of 610 relapsed or died) versus HER2 positive not treated with adjuvant trastuzumab (NT) (37 of 97 relapsed or died) and HER2 positive treated with adjuvant trastuzumab (T) (8 of 54 relapsed or died). NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab tologic types other than ductal invasive (p < 0.001 addition, uPA but not PAI-1 was higher in tumors for uPA; p = 0.048 for PAI-1), less differentiated tu-with lymphovascular invasion (p = 0.047). There mors (p < 0.001 for both), and tumors with lower were no associations between uPA or PAI-1 values estrogen receptor expression (p < 0.001 for both). In and patient age or progesterone receptor status. TABLE 3. Univariate and multivariate analysis of overall survival in lymph node-negative breast cancer patients with a median follow-up time of 100 months. Multivariate analysis was performed in the 273 patients for whom complete data were available Age (continuous, unit = 10 years) 2.18 (1.88–2.52) < 0.001 1.61 (1.19–2.17) 0.002 1.68 (1.25–2.27) 0.001 0.001 Tumor size (.2 vs. <2 cm) 1.38 (1.02–1.88) 0.036 0.94 (0.47–1.88) 0.865 – – 0.865 Tumor type (other invasive vs. ductal invasive) 1.24 (0.83–1.85) 0.298 2.24 (0.76–6.59) 0.142 – – 0.165 Grade (G3 vs. G1-2) 1.40 (1.01–1.94) 0.042 2.39 (0.95–6.02) 0.066 2.69 (1.34–5.40) 0.005 0.064 Lymphovascular invasion (present vs. absent) 1.34 (0.83–2.16) 0.236 1.36 (0.46–4.01) 0.582 – – 0.594 Estrogen receptors (negative vs. positive) 1.24 (0.88–1.75) 0.224 1.80 (0.67–4.81) 0.242 – – 0.233 Progesterone receptors (negative vs. positive) 1.01 (0.74–1.38) 0.955 0.62 (0.25–1.54) 0.299 – – 0.279 HER2 status 0.190 0.843 0.837 positive NT vs. negative 1.37 (0.89–2.09) 0.152 0.76 (0.31–1.90) 0.562 – – positive T vs. negative 0.63 (0.26–1.55) 0.318 0.90 (0.26–3.18) 0.871 – – uPA/PAI-1 (one or both high vs. both low) 2.73 (1.33–5.58) 0.006 1.98 (0.83–4.76) 0.126 – – 0.114 NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab Disease-free survival A total of 228 events occurred in the DFS analysis. Univariate and multivariate analyses of DFS are presented in Table 2. In univariate analysis, older age, tumors larger than 2 cm, grade 3, with evident lymphovascular invasion, and high uPA and/or PAI-1 (Figure 1A) were associated with worse DFS. HER2 positive patients who were not treated with adjuvant trastuzumab had significantly worse DFS than HER2 negative patients. DFS of HER2 positive patients who received adjuvant trastuzumab was not significantly different than DFS of HER2 nega­tive patients (Figure 1B). In backward stepwise LR multivariate model, the three variables that re­mained significant for DFS after the final step were patient age at diagnosis, estrogen receptor status and uPA/PAI-1 level. uPA/PAI-1 level was the sec­ond most important variable after patient age in the model if term removed (Table 2). In multivariate analysis, the HR for disease re­currence or death from any cause in estrogen recep­tor positive patients with high uPA and/or PAI-1 was 2.78 (95% CI, 1.28–6.03; p = 0.010) compared to those with both values low. The corresponding HR values for estrogen receptor negative, HER2 nega­tive, HER2 positive patients who did not receive adjuvant trastuzumab, HER2 positive patients who received adjuvant trastuzumab, and triple negative patients, were 1.11 (95% CI, 0.33–3.76; p = 0.863), 1.60 (95% CI, 0.70–3.68; p = 0.268), 9.25 (95% CI, 1.06–80.82; p = 0.044), 3.31 (95% CI, 0.10–109.67; p = 0.503), and 1.08 (95% CI, 0.30–3.86; p = 0.902), re­spectively. Overall survival A total of 172 events occurred in the OS analy­sis. Univariate and multivariate OS analyses are shown in Table 3. Patient age at diagnosis, tumor size, grade, and uPA/PAI-1 levels (Figure 2a) were found to have a significant impact on OS in uni­variate analyses. HER2 (Figure 2B) and the other traditional prognostic factors were not found to influence OS in our series of node-negative breast cancer patients. In backward stepwise LR mul­tivariate model, the remaining variables signifi­cantly associated with OS after the last step were patient age and tumor grade (Table 3). uPA/PAI­1 level was the third most important factor in the model if term removed and was removed from the model at the last step. Breast cancer specific survival A total of 65 events occurred in the BCSS analy­sis. Univariate and multivariate BCSS analyses TABLE 4. Univariate and multivariate analysis of breast cancer specific survival in lymph node-negative breast cancer patients with a median follow-up time of 100 months. Multivariate analysis was performed in the 273 patients for whom complete data were available Age (continuous, unit = 10 years) 1.16 (0.95–1.41) 0.148 0.76 (0.51–1.12) 0.162 – – 0.160 Tumor size (.2 vs. <2 cm) 2.44 (1.45–4.13) 0.001 1.72 (0.55–5.38) 0.350 – – 0.340 Tumor type (other invasive vs. ductal invasive) 1.20 (0.63–2.29) 0.587 3.33 (0.64–17.23) 0.152 – – 0.196 Grade (G3 vs. G1-2) 3.58 (2.12–6.05) < 0.001 7.10 (1.23–40.86) 0.028 10.34 (2.33–45.97) 0.002 0.014 Lymphovascular invasion (present vs. absent) 1.48 (0.70–3.12) 0.305 1.43 (0.36–5.65) 0.608 – – 0.618 Estrogen receptors (negative vs. positive) 2.21 (1.33–3.66) 0.002 1.24 (0.28–5.36) 0.778 – – 0.776 Progesterone receptors (negative vs. positive) 1.74 (1.06–2.86) 0.028 0.81 (0.17–3.77) 0.789 – – 0.788 HER2 status 0.031 0.663 0.614 positive NT vs. negative 2.18 (1.21–3.93) 0.009 1.29 (0.39–4.31) 0.683 – – positive T vs. negative 0.96 (0.30–3.10) 0.944 0.46 (0.06–3.68) 0.460 – – uPA/PAI-1 (one or both high vs. both low) 6.46 (1.47–28.43) 0.014 2.79 (0.57–13.67) 0.205 – – 0.166 NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab FIGURE 2. Effect of uPA/PAI-1 level and HER2 status on overall survival (OS) in lymph-node negative breast cancer patients. (A) uPA/PAI-1 low (10 of 159 died) versus uPA/PAI-1 high (31 of 173 died). (B) HER2 negative (118 of 610 died) versus HER2 positive not treated with adjuvant trastuzumab (26 of 97 died) and HER2 positive treated with adjuvant trastuzumab (5 of 54 died). NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab are shown in Table 4. Tumor size, grade, estrogen the final multivariate model using the backward and progesterone receptor status, HER2 status and stepwise LR method was tumor grade. uPA/PAI­uPA/PAI-1 levels were associated with BCSS in 1 level was the third most important factor in the univariate analyses. The only variable retained in model if term removed. Discussion The results of our study indicate an important prognostic value of uPA/PAI-1 level in node-neg­ative breast cancer patients. Even though uPA and PAI-1 values were associated with most of the prognostic factors currently in use for clinical decision-making in the adjuvant setting, multivari­ate analysis showed that uPA/PAI-1 level carries additional, independent prognostic information. This was particularly true for DFS analysis where uPA/PAI-1 level was the second most important variable in the survival model after age and was retained in the backward LR model after the final step along with age and estrogen receptor status. In OS analysis, the final model included only age and tumor grade, which is probably a reflection of the very strong association between higher uPA and PAI-1 values and less differentiated tumors. However, in the multivariate model that included all variables, the HR for death from any cause was 1.98 (95% CI 0.83–4.76) for patients with high uPA and/or PAI-1 compared to patients with both val­ues low, which shows a substantial possibility of an important effect on OS that would have prob­ably remained statistically significant in a larger sample. The same three variables were the most important in the multivariate model of BCSS as well. Here, the singular prognostic importance of tumor grade was even more evident. As opposed to DFS and OS which both included deaths from other causes as events in the analysis, younger age seemed associated with worse BCSS. It is important to emphasize that BCSS analysis must be interpret­ed with caution due to the small number of events. Considering the HRs and confidence intervals, it is probable that both age and uPA/PAI-1 level im­portantly influence BCSS and would have retained statistical significance in a larger sample. On the other hand, HER2 status only showed prognostic significance in univariate DFS and BCSS analyses when comparing HER2 negative to HER2 positive patients who were not treated with adjuvant tras­tuzumab. It did not remain an important determi­nant of DFS and BCSS in multivariate analyses and lost all prognostic value in OS analysis. A subgroup analysis of DFS according to estro­gen receptor and HER2 status showed that apart from the no longer relevant group of HER2 posi­tive patients who did not receive adjuvant trastu­zumab treatment, uPA/PAI-1 level was prognosti­cally by far the most important in estrogen receptor positive tumors. In contrast, its prognostic value in multivariate analysis was practically null in HER2 positive patients treated with adjuvant trastuzum­ab and in triple negative patients. Conveniently, estrogen receptor positive patients are the ones with the highest uncertainty regarding the benefit of adjuvant chemotherapy, while the other two subgroups are generally recommended adjuvant chemotherapy ± anti-HER2 therapy regardless of other prognostic factors. Our findings confirm those of numerous other studies that have reported uPA and PAI-1 to be sta­tistically independent prognostic factors in lymph node-negative breast cancer patients.10-18 However, these studies did not include HER2 status which is now an important part of the decision about ad­juvant systemic therapy. Because of the observed associations between overexpression of the HER2/ neu protein and tumor proteolytic factors in breast and in other cancers19-21, HER2/neu has been sug­gested to up-regulate the proteolytic enzymes, in­cluding uPA and PAI-1, and thus play a direct role in tumor invasion and metastasis.19 This possible association might be one of the additional reasons besides methodological difficulties why uPA and PAI-1 testing is still not that frequently used in the clinic in spite of the excellent evidence of its clinical utility.1 Even so, the main reason for this inconsistency are practical issues. uPA and PAI-1 are determined using validated ELISA assay kits as described above. This measurement requires relatively large amounts of fresh or freshly fro­zen tumor tissue, which is not practical for clinical use, especially in needle or surgical biopsies and in small tumors. Besides, the results may not be available at the time of the histology report due to sample pooling. However, attempts to develop im­munohistochemistry assays on formalin-fixed and paraffin-embedded tissues are ongoing.1 Among the few studies that considered the clini­cal relevance of both HER2 and proteolytic enzymes for survival in breast cancer patients, the results are somewhat conflicting. Harbeck et al.22 showed that PAI-1 was the only independent prognostic fac­tor for DFS and OS when considered along with tumor size, tumor grade, steroid hormone recep­tor status, uPA, HER2 status, MIB1, SPF, p53, and cathepsin D in 100 node-negative breast cancer patients. Similarly, Bouchet et al.23 found no addi­tional prognostic information of HER2/neu protein levels when evaluated in multivariate analysis to­gether with uPA, PAI-1 and traditional histologic factors in the subgroup of 226 node-negative pa­tients. They reported DFS to be independently in­fluenced by PAI-1 and tumor size and OS by PAI-1 and uPA levels. Recently, Buta et al.24 also reported superior DFS in 73 node-negative, postmenopau­sal, steroid hormone receptor positive breast can­cer patients with smaller tumors and low PAI-1, independent of HER2 status. On the other hand, Konecny et al.19 reported that both uPA/PAI-1 level and HER2 status independently influenced DFS in addition to tumor size and nodal status in a group of 542 patients with a short follow-up not selected by nodal status. The same factors were found to be important in multivariate OS analysis as well, but this time HER2 status did not quite reach statistical significance. Interestingly, among 118 node-nega­tive patients with long-term follow-up, Zemzoum et al.25 found uPA/PAI-1 to be the only variable in­dependently influencing DFS, and HER2 status to be the most important factor in the multivariate analysis of OS. uPA/PAI-1 and tumor grade were of borderline significance for OS. Complicating the matter even further, without reference to uPA and/ or PAI-1, Schmidt et al.26 reported HER2 status to be prognostically significant in node-negative breast cancer patients only when determined by FISH and not when determined immunohistochemical­ly. In our group of 273 node-negative patients with long-term follow-up who were available for multi­variate analysis, the combination of uPA and PAI-1 levels clearly carried independent prognostic value for DFS and was important although not formally statistically significant in multivariate analysis of OS as well, while HER2 status determined with the usual combination of immunohistochemistry and FISH did not prove to be an important determinant of DFS or OS in multivariate analyses regardless of adjuvant anti-HER2 treatment. Most of the studies confirming the prognostic value of uPA and PAI-1 in breast cancer patients have focused on patients who received no adjuvant systemic treatment. In fact, the prognostic informa­tion from these biomarkers seems diminished in patients who receive adjuvant treatment, particu­larly adjuvant hormone therapy27,28, indicating a possible predictive role of uPA and PAI-1 for re­sponse to therapy. However, the prognostic impor­tance of uPA/PAI-1 level was evident in our group of patients although the vast majority received ad­juvant hormone therapy and a significant fraction were given adjuvant chemotherapy. Contrastingly, based on our results, HER2 status is clearly more important in its predictive than in its prognostic role. Our study has several limitations, the princi­pal one being its retrospective character and the associated possibility of bias. A major problem is missing data, particularly on uPA and PAI-1 values which could only be determined in about 40% of the patients because of our institution’s economi­cal limitations and the fact that these markers still largely serve only academic purposes. Because of inconsistent histology reports in the past, some other information is missing in a fraction of pa­tients. The total number of patients with available information was used for each univariate analysis and multivariate analyses were performed in the 273 patients in whom complete data were avail­able. Even so, we are aware of only one study comparing the prognostic values of uPA, PAI-1, HER2, and traditional prognostic factors that in­cluded a slightly larger subgroup of 283 lymph node-negative patients.19 Another possible limita­tion is the comparatively high proportion of deaths from causes other than breast cancer, which may have somewhat obscured the results of both DFS and OS analysis and is probably also the cause of such a marked association of older age and worse prognosis, an assumption confirmed by the fact that older age was associated with improved breast cancer specific survival. Furthermore, our results must be considered in the light of the unavoida­ble multiple analyses and the possibility of a type I statistical error. To facilitate interpretation, 95% confidence intervals and not just the p values have been stated wherever possible. On the other hand, one of the main strengths of our study in addition to the combination of analyzed prognostic factors is the long follow-up which was in the range of 49­181 months, the median of 100 months being more than three times the median follow-up of the larger study by Konecny et al.19 Moreover, we believe that the scientifically sound and straightforward statis­tical analysis is a strength of our study as well. Based on the results from our retrospective analysis of node-negative breast cancer patients with long-term follow-up, we conclude that the combined uPA/PAI-1 level carries important addi­tional prognostic information, particularly for DFS, even after all traditional prognostic factors as well as HER2 status have been taken into account. We believe that routine use of uPA/PAI-1 level would further improve risk stratification and adjuvant therapy decisions in this setting, especially in es­trogen receptor positive patients. Acknowledgements The authors would like to thank prof. Lara Lusa, Ph.D., from the Institute for Biostatistics and Medical Informatics, Faculty of Medicine, University of Ljubljana, for advice regarding sta­tistical methods. We would also like to thank Assoc. Prof. Darja Arko, M.D., Ph.D., and Nina Čas Sikošek, M.D., from Maribor University Clinical Center, for coordinating patient treatment and follow-up. References 1. 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Malmström P, Bendahl PO, Boiesen P, Brünner N, Idvall I, Fernö M. S-phase fraction and urokinase plasminogen activator are better markers for distant recurrences than Nottingham Prognostic Index and histologic grade in a prospective study of premenopausal lymph node-negative breast cancer. J Clin Oncol 2001; 19: 2010-9. 16. Meo S, Dittadi R, Peloso L, Gion M. The prognostic value of vascular en­dothelial growth factor, urokinase plasminogen activator and plasminogen activator inhibitor-1 in node-negative breast cancer. Int J Biol Markers 2004; 19: 282-8. 17. De Cremoux P, Grandin L, Diéras V, Savignoni A, Degeorges A, Salmon R, et al. Urokinase-type plasminogen activator and plasminogen-activator-in­hibitor type 1 predict metastases in good prognosis breast cancer patients. Anticancer Res 2009; 29: 1475-82. 18. Rabi ZA, Todorović-Raković N, Vujasinović T, Milovanović J, Nikolić­Vukosavljević D. Markers of progression and invasion in short term follow up of untreated breast cancer patients. Cancer Biomark 2015; 15: 745-54. 19. Konecny G, Untch M, Arboleda J, Wilson C, Kahlert S, Boettcher B, et al. Her-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer. Clin Cancer Res 2001; 7: 2448-57. 20. Berney CR, Yang J, Fisher RJ, Russell PJ, Crowe PJ. Correlates of urokinase-type plasminogen activator in colorectal cancer: positive relationship with nm23 and c-erbB-2 protein expression. Oncol Res 1998; 10: 47-54. 21. Allgayer H, Babic R, Gruetzner KU, Tarabichi A, Schildberg FW, Heiss MM. c-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumor-associated protease systems. J Clin Oncol 2000; 18: 2201-9. 22. Harbeck N, Dettmar P, Thomssen C, Henselmann B, Kuhn W, Ulm K, et al. Prognostic impact of tumor biological factors on survival in node-negative breast cancer. Anticancer Res 1998; 18: 2187-97. 23. Bouchet C, Ferrero-Poüs M, Hacene K, Becette V, Spyratos F. Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma. Int J Biol Markers 2003; 18: 207-17. 24. Buta M, Džodić R, Đurišić I, Marković I, Vujasinović T, Markićević M, et al. Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up. Tumour Biol 2015; 36: 8193-200. 25. Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, et al. Invasion factors uPA/PAI-1 and HER2 status provide independent and complemen­tary information on patient outcome in node-negative breast cancer. J Clin Oncol 2003; 21: 1022-8. 26. Schmidt M, Lewark B, Kohlschmidt N, Glawatz C, Steiner E, Tanner B, et al. Long-term prognostic significance of HER-2/neu in untreated node-negative breast cancer depends on the method of testing. Breast Cancer Res 2005; 7: R256-66. 27. Harbeck N, Alt U, Berger U, Krüger A, Thomssen C, Jänicke F, et al. Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plas­minogen activator inhibitor 1, and cathepsin B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy. Clin Cancer Res 2001; 7: 2757-64. 28. Cufer T, Borstnar S, Vrhovec I. Prognostic and predictive value of the uroki­nase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer. Int J Biol Markers 2003; 18: 106-15. research article Safety and efficacy of doxorubicin-eluting superabsorbent polymer microspheres for the treatment of liver metastases from neuroendocrine tumours: preliminary results Lawrence Bonne1, Chris Verslype2, Annouschka Laenen3, Sandra Cornelissen1, Christophe M. Deroose4, Hans Prenen2, Vincent Vandecaveye1, Eric Van Cutsem2, Geert Maleux1 1 Department of Radiology, University Hospitals Leuven, Belgium 2 Department of Hepatology and Gastroenterology, University Hospitals Leuven, Belgium 3 Interuniversity Centre for Biostatistics and Statistical Bioinformatics, Catholic University of Leuven and University Hasselt, Belgium 4 Department of Nuclear Medicine, University Hospitals Leuven, Belgium Radiol Oncol 2017; 51(1): 74-80. Received 23 December 2016 Accepted 12 January 2017 Correspondence to: Geert Maleux, M.D., Ph.D., Department of Radiology, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium. Phone: +32 16 34 37 82; Fax: +32 16 34 37 65; E-mail: geert.maleux@uzleuven.be Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to retrospectively evaluate the symptom control, tumour response, and complication rate in patients with liver-predominant metastatic neuroendocrine tumours treated with transarterial chemoembolization using doxorubicin-eluting superabsorbent polymer (SAP) microspheres. Patients and methods. Patients with neuroendocrine liver metastases who underwent hepatic transarterial chem­oembolization using doxorubicin-eluting SAP-microspheres (50–100 µm Hepasphere/Quadrasphere Microsphere® par­ticles, Merit Medical, South Jordan, Utah, USA) were included in this study. Pre- and post-procedure imaging studies were evaluated to assess short and intermediate-term tumour response using modified RECIST criteria. Symptom relief and procedure-related complications were evaluated. Results. A total of 27 embolization procedures were performed on 17 patients. Twelve of 17 patients (70%) were symptomatic, including carcinoid syndrome (n = 8) and severe, uncontrollable hypoglycemia (n = 4). Eight of 12 patients (67%) had complete symptom relief, and the remaining 4 (33%) had partial relief. One patient developed ischemic cholecystitis (6%). No other hepatobiliary complications occurred. Short-term and intermediate-term imag­ing follow-up was available for 15/17 patients (88%) and 12/14 patients (86%) respectively. At short-term follow-up (< 3 months), 14 patients (93%) showed partial response and the remaining patient had progressive disease (7%). At intermediate-term imaging follow-up (> 3 months), partial response, stable disease and progressive disease were found respectively in 7 (58%), 3 (25%) and 2 (17%) patients. Conclusions. Chemoembolization with doxorubicin-eluting SAP-microspheres is a safe and effective treatment op­tion for neuroendocrine liver metastases and is associated with a low complication rate. In particular, no clinically evident liver necrosis or bile duct complications were encountered. Key words: neuroendocrine tumour; chemoembolization; drug-eluting beads; procedure-related complications Introduction at initial presentation.1,2 The metastases may be responsible for hormone-related symptoms, in-Among patients with neuroendocrine tumours cluding carcinoid syndrome and uncontrollable (NET) hepatic metastases are present in 46%–93% hypoglycemia in case of insulinoma. These clinical symptoms can be treated effectively with somato­statin analogues in more than 70% of patients, but efficacy can decrease with time.3 Although surgi­cal resection of NET liver metastases can be cu­rative and remains the treatment of choice, only 10% of patients are candidates for liver surgery.4,5 Liver-directed transarterial therapies in nonsurgi­cal candidates include transcatheter arterial embo­lization6,7, chemo-embolization6,8 and Yttrium-90 radioembolization.9,10 These techniques have been used both to palliate hormone-related symptoms as well as to reduce metastatic tumour burden. Conventional transarterial chemoembolization us­ing ethiodized oil and cytotoxic drugs has been the most popular locoregional, catheter-directed ther­apy to treat unresectable NET liver metastases for more than 20 years.3,11 Drug-eluting microspheres have been used recently for both hepatocellular car­cinoma12,13 and metastases to the liver14-16, and are associated with sustained efficacy and less systemic toxicity.17,18 In contrast to its use in hepatocellular carcinoma19, a high complication rate was found when using drug-eluting beads (DC/LC-beads, Biocompatibles, Farnham, UK) in neuroendocrine liver metastases, including a high risk for postpro­cedural formation of liver necrosis and biloma.20-23 We used superabsorbent polymer (SAP) micro-spheres (50–100 µm Hepasphere/Quadrasphere Microspheres®, Merit Medical, South Jordan, Utah, USA) loaded with doxorubicin as an alternative drug-eluting microsphere technology to treat NET liver metastases and assessed the feasibility, safety, clinical and radiological response rates in a retro­spective study design. Patients and methods The institutional interventional radiology database was queried for patients undergoing transarte­rial chemoembolization using doxorubicin-eluting SAP microspheres for the treatment of liver metas­tases from histologically proven neuroendocrine tumours. Demographic, clinical, radiologic and laboratory data were collected. All patients were discussed at the Multidisciplinary tumour board, including medical, surgical and radiation oncolo­gists as well as pathologists and diagnostic and interventional radiologists. All patients gave in­formed consent for the chemoembolization proce­dure and this retrospective study was approved by the local ethics committee. Inclusion criteria for chemoembolization were symptomatic patients with liver-only or liver-pre­dominant neuroendocrine metastases, unrespon­sive under chemotherapy and not a candidate for surgical resection or radiofrequency ablation or patients with progression of disease, despite opti­mal chemotherapeutic management. Additionally, the residual liver function allowed chemoemboli­zation: bilirubin levels less than 2.0 mg/dl, alanine amino transferase (ALT) and aspartate aminotrans­ferase (AST) less than five times the upper limit of normal, albumin more than 2 mg/dl. Contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) was per­formed within one month before the first chem­oembolization procedure. Postprocedure imaging interval was variable, depending on whether a unilobar or bilobar infusion was performed within 1 session, and ranged from 1–3 months for short-term follow-up and from 5–6 months for interme­diate-term follow-up. Radiologic tumour response was assessed by size based on modified Response Evaluation Criteria in Solid Tumors (m-RECIST). All chemoembolization procedures were per­formed using a standard technique as described previously for the treatment of unresectable hepa­tocellular carcinoma treated with doxorubicin­eluting SAP microspheres.13 Briefly, after admin­istration of local anaesthesia in the right groin, a 4 French (F) sheath (Boston Scientific, Natick, MA, USA) was introduced into the right common femoral artery. Selective catheterization of the ce­liac trunk and the superior mesenteric artery was performed with use of a 4F catheter (Impress diag­nostic catheter, Merit Medical, South Jourdan, UT, USA) and followed by superselective catheteriza­tion of the right and left hepatic arteries using a mi­crocatheter (EmboCath Plus or Maestro 2.8, Merit Medical, South Jourdan, UT, USA). Depending on number and distribution of the metastatic lesions the drug-eluting microspheres were delivered to segmental or lobar hepatic artery/arteries. If tumour burden was more than 50% of total liver volume, 2 separate lobar infusions 1 month apart were performed. No antibiotics were administered before or during the procedure. All patients had intact biliary sphincters; analgesics or antiemetics were administered if needed. All patients received somatostatin analogues (Sandostatin, Novartis Pharma, Vilvoorde, Belgium) before the start of the procedure. For each procedure, one vial of 25 mg dry SAP microspheres with a nominal dry diam­eter of 50-100 microns (Hepasphere/Quadrasphere Microspheres, Merit Medical, South Jordan, UT, USA), was prepared in the hospital pharmacy. The SAP microspheres were mixed with doxorubicin FIGURE 1. A 63-year-old male patient presented with a carcinoid of the lung and diffuse bilobar liver involvement. (A) Portal venous phase contrast-enhanced CT-scan confirms diffuse metastatic involvement of both liver lobes (white arrowheads at the level of the largest metastasis in the left liver lobe; black arrows at the level of multiple smaller lesions in the right liver lobe); Selective angiogram of the left hepatic artery (B) before and (C) after chemoembolization with doxorubicin-eluting SAP-microspheres (arrow at the level of the micro-catheter in the left hepatic artery); Selective angiogram of the celiac trunk (D) before and (E) after chemoembolization with doxorubicin-eluting SAP-microspheres (arrow shows stasis of contrast at the level of the right hepatic artery); (F) Portal venous phase contrast-enhanced CT-scan 10 weeks after initial chemoembolization shows marked decrease in volume and enhancement of most of the metastatic lesions in left and right liver lobes. (Pharmachemie BV, Haarlem, Netherlands) and suspended in 10 ml sodium chloride 0.9% and 10 ml iodinated contrast medium (Visipaque 270, GE Healthcare, Oslo, Norway). The standard doxo­rubicin dose was 75 mg/m2 (square metre of body surface area), which was reduced to 50 mg/m2 or 25 mg/m2 in cases of elevated bilirubin or cytope­nia. The injection of the doxorubicin-eluting SAP microspheres was stopped once the feeding ar­teries were completely occluded as confirmed by hand-injected hepatic angiography at completion. If residual hypervascular blush was identified on confirmation angiography after chemoemboliza­tion, an additional vial of bland polyvinyl alco­hol particles (nsPVA) of 250–355 micron caliber (Contour, Boston Scientific Corporation, Natick, MA, USA) was injected. Patients were followed clinically every 2 months on an outpatient basis; early and late adverse events were noted in the patients’ electronic hospital re­cords. Chemo-embolization-related toxicity was as­sessed according to the National Cancer Institute – Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0. Assessment of symptom control after chemoembolization was performed 4–6 months after the initial chemoembolization by the referring medical oncologist. Overall survival probabilities were estimated by the Kaplan-Meier method. Results Seventeen patients (9 women, 8 men) with histo­logically proven gastrointestinal, pancreatic or pulmonary neuroendocrine tumours metastastic to the liver were included. Patients’ age, site and type of the primary tumour, tumour grade based on the Ki-67 index, extrahepatic tumoural disease, and previous surgical and medical therapies are summarized in Table 1. No patient presented with portal vein thrombosis or bile duct dilatation at the time of chemoembolization procedure. In 5 patients (29%), chemoembolization of right and left liver lobes was performed in separate ses­sions. Both right and left liver lobes were treated in 8 patients (47%) during the same session (Figure 1). For the remaining 4 patients (24%), isolated right hepatic lobe chemoembolization was performed. In all 27 chemoembolization procedures the whole vial of doxorubicin-loaded SAP micro-spheres could be injected. The total median dose of doxorubicin injected per session was 133 mg (min 25 mg – max 150 mg). In 4 of 27 procedures (15%), one additional vial of PVA particles was injected after the SAP microspheres. Procedure-related toxicity is summarized in Table 2. Management of these complications included percutaneous gall­bladder drainage (n = 1), medical management of carcinoid and insulin storm (n = 2) and analgesics and antiemetics (n = 14). No procedure-related liv­er necrosis or biloma was found during immediate or late follow-up. Short-term and intermediate-term imaging fol­low-up was available for 15/17 patients (88%) and 12/14 patients (86%) respectively. On short term follow-up, partial response and progressive dis­ 77 TABLE 1. Patients’ demographic data Age (years) Mean SD Range Sex M F Primary tumour Intestinal NET Pancreatic NET Pulmonary NET Pancreatic insulinoma Unknown Surgical resection of primary tumour Intestinal NET Pancreatic NET Pulmonary NET Pancreatic insulinoma Total Tumour grade (Ki-67 index) Ki-67 < 2% Ki-67 2–20% Ki-67 > 20% Unknown Tumour burden (% of total liver volume) 0–10% 10–20% 20–50% > 50% Extrahepatic metastatic disease Lymphadenopathy Bone Lung Spleen Adrenal Brain Ovary Pancreas Peritoneum Skin Previous treatment Surgical resection of liver metastases Radiofrequency ablation of liver metastases Radiotherapy for brain metastases Radiotherapy for bone metastases Interferon Everolimus m-TOR-inhibitor Sunitinib Somatostatine analogue Diazoxide 56 13.9 18–82 8 9 6 2 2 4 3 5 2 2 1 10 4 6 4 3 4 3 5 5 16 7 5 FIGURE 2. (A) Kaplan-Meier overall survival estimate shows an overall survival (with 3 95% confidence interval) at 12, 24 and 36 months after TACE of 67% (lower limit 38% 1 - upper limit 85%), 47% (lower limit 21% - upper limit 69%) and 32% (lower limit 11% - 3 upper limit 56%); (B) Kaplan-Meier overall survival estimate shows an overall survival 1 (with 95% confidence interval) at 5, 10 and 15 years after diagnosis of 53% ( lower 2 limit 26% - upper limit 74%), 18% (lower limit 3% - upper limit 41%) and 18% (lower limit 2 3% - upper limit 41%). 1 1 ease according to m-RECIST criteria were found in 1 14 (93%) and 1 (7%) patients respectively. On inter­1 mediate term follow-up, m-RECIST-based partial 1 response, stable disease and progressive disease 1 was found respectively in 7 (58%), 3 (25%) and 2 3 (17%) patients. 7 Eight of 17 patients (47%) presented with carci­1 noid syndrome-related symptoms including diar­4 rhea (n = 7; 41%) and flushing (n = 7; 41%); 5 of 15 these 8 patients (63%) had complete symptom re­3 lief after chemoembolization. The remaining 3 pa- TABLE 2. Procedure-related toxicity Ischemic cholecystits 3 1 6 Carcinoid storm 2 1 6 Insulin storm 2 1 6 Postembolization syndrome 2 14 82 Grading of procedure-related toxicity is based on National Cancer Institute – Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0. tients had moderate relief of symptoms with per­sisting signs of diarrhea (n = 2) and flushing (n = 3). Three of 4 patients with liver metastases related to primary insulinoma of the pancreas (pre-inter­ventional mean glycemia values of 57 mg/dl (range 39–83 mg/dl)) had complete normalisation of their glycaemic values after the chemoembolization pro­cedure. One patient had a partial clinical response with a residual glycemic value of 100 mg/dl. One patient with predominant symptoms of abdominal pain did not respond to the chemoembolization treatment. Overall survival of the studied patients is summarized in Figure 2 with an estimated sur­vival at one year after TACE of 67%, and an esti­mated survival at five years after diagnosis of 53%. Discussion This retrospective observational study shows a high rate of symptomatic relief after chemoembo­lization for neuroendocrine liver metastases using doxorubicin-eluting SAP-microspheres: 5 of 8 pa­tients with carcinoid syndrome-related symptoms showed complete symptom relief after the first session of chemoembolization and 3 of 4 patients with preinterventional hypoglycemia presented with complete normalisation of their glycemic val­ues. These results are consistent with symptomatic responses after conventional chemoembolization showing symptom relief in 52%–86% of cases. These encouraging results are even higher when the treatment is performed as a first line therapy, with up to 70% complete symptomatic response and 20% partial response.3,6,8,11 This study also demonstrates substantial ra­diologic response rates, with 93% of patients showing partial response or stable disease at 3 month follow-up, based on modified RECIST cri­teria. These results are consistent with response rates using doxorubicin-eluting DC Beads (BTG-Biocompatibles, Farnham, UK) or using conven­tional ethiodized oil-based chemoembolization, which range between 57% and 80%.8,20,21 These high response rates potentially can also be achieved in patients with high tumour burden, as suggested in the present study, with tumour burden ranging from 8% to 58%. Bland embolization without the addition of any chemotherapeutic agent also has demonstrated high response rates similar to those of chemoembolization.3,6,11 Although most of the studies dealing with catheter-directed liver inter­ventions for neuroendocrine metastases are based on chemo-embolization, however, no retro- or prospective comparative study has demonstrated a significant difference in response rate or other benefit for chemoembolization compared to bland embolization. Toxicity related to SAP microsphere-based chemoembolization for neuroendocrine liver me­tastases is mainly limited to the post-embolization syndrome which was observed in 82% of cases. Only one case of post-embolization cholecystitis was observed, which was treated by percutaneous drainage, and one instance each of post-emboliza­tion carcinoid (n = 1) and insulin (n = 1) storm oc­curred despite prophylactic administration of so­matostatin analogues before chemoembolization. Importantly, we did not observe any cases of post­embolization liver necrosis, liver abscess or biloma. Post-chemoembolization liver abscess formation is a risk in patients with biliodigestive anastomosis or biliary stents in situ, despite the prophylactic administration of broad spectrum antibiotics.8,24 In this series there were no patients with previous bile duct surgery or intervention. In other studies post-embolization liver necrosis and intrahepatic biloma-formation were found in nearly half of the cases using small calibre (100–300 µm) doxoru­bicin-loaded DC Beads, but not after conventional, ethiodized oil-based chemoembolization.20-22,25 It is unclear why this high number of biloma cases was found after DC/LC Bead chemoembolization for neuroendocrine liver metastases; it is hypothesized that small calibre microspheres will penetrate deeper in the (normal) residual liver parenchyma and thereby induce irreversible ischemia to the liver and biliary tree tissues.20 This serious side ef­fect was seen more often in patients with a smaller tumour burden compared to patients with a higher tumour load, stressing the fact that the residual normal liver parenchyma is sensible to ischemic changes when small calibre microspheres are in­jected.20 SAP microspheres used in this study had a diameter of 50–100 µm in their dry state, swell­ing to 200-400 µm once hydrated in saline solution. This mechanism of expansion and subsequent larg­er diameter, thereby resulting in a more proximal embolization when injected into the liver26-28, might prevent any necrotic complication. Finally, we did not observe procedure-related liver failure, even in patients with considerable tumour burden, bilobar treatment, or in patients heavily pretreated by somatostatin analogues and different types of other drugs. Limitations of this study are multiple. First, this study deals with a very heterogeneous patient population presenting with both symptomatic and asymptomatic neuroendocrine liver metastases who were previously treated with various types of chemotherapeutics and other medical treat­ments. Second, the patient sample is too small to draw firm conclusions with regard to symptom and tumour responses. Third, late response rates as well as overall survival also depend on prior, concomitant and further treatment options and not on the chemoembolization procedures alone. Last, no comparative analysis with other locoregional or medical treatments was performed. In conclusion, this study suggests that doxoru­bicin-eluting SAP microspheres appear to be a safe and potentially effective treatment option in the treatment of neuroendocrine liver metastases and this interventional technique might be proposed as an alternative locoregional treatment option for conventional ethiodized oil-based chemoemboliza­tion. References 1. Chamberlain R, Canes D, Brown K, Saltz L, Jarnagin W, Fong Y, et al. Hepatic neuroendocrine metastases: does intervention alter outcome? J Am Coll Surg 2000; 190: 432-45. doi: http://dx.doi.org/10.1016/S1072­7515(00)00222-2 2. Tomassetti P, Migliori M, Lalli S, Campana D, Tomassetti V, Corinaldesi R. Epidemiology, clinical features and diagnosis of gastroenteropancreatic endocrine tumors. Ann Oncol 2001; 12 (Suppl 2): S95-9. 3. Madoff D, Gupta S, Ahrar K, Murthy R, Yao JC. Update on the management of neuroendocrine hepatic metastases. J Vasc Interv Radiol 2006; 17: 1235­49; quiz 1250. doi: 10.1097/01.RVI.0000232177.57950.71 4. Mayo S, De Jong M, Bloomston M, Pulitano C, Clary B, Reddy S, et al. Surgery versus intra-arterial therapy for neuroendocrine liver metastases: a multicenter international analysis. Ann Surg Oncol 2011; 18: 3657-65. doi: 10.1245/s10434-010-1343-2 5. Arrese D, McNally ME, Chokshi R, Feria-Arias E, Schmidt C, Klemanski D, et al. Extrahepatic disease should not preclude transarterial chemoemboliza­tion for metastatic neuroendocrine carcinoma. Ann Surg Oncol 2013; 20: 1114-20. doi: 10.1245/s10434-012-2786-4 6. Ruutiainen A, Soulen M, Tuite C, Clark T, Mondschein J, Stavropoulos S, et al. Chemoembolization and bland embolization of neuroendocrine tumor metastases to the liver. J Vasc Interv Radiol 2007; 18: 847-55. doi: 10.1016/j. jvir.2007.04.018 7. Ho A, Picus J, Darcy MD, Tan B, Gould JE, Pilgram TK, et al. Long-term out­come after chemoembolization and embolization of hepatic metastatic le­sions from neuroendocrine tumors. AJR Am J Roentgenol 2007; 188: 1201­ 7. doi: 10.2214/AJR.06.0933 8. Hur S, Chung J, Kim H, Oh D, Lee S, Bang Y, et al. Survival outcomes and prognostic factors of transcatheter arterial chemoembolization for he­patic neuroendocrine metastases. J Vasc Interv Radiol 2013; 24: 947-56. doi: 10.1016/j.jvir.2013.02.030 9. Devcic Z, Rosenberg J, Braat A, Techasith T, Banerjee A, Sze D, et al. The efficacy of hepatic 90Y resin radioembolization for metastastic neuroendo­crine tumors: a meta-analysis. J Nuc Med 2014; 55: 1404-10. doi: 10.2967/ jnumed.113.135855 10. Memon K, Lewandowski R, Mulcahy M, Riaz A, Ryu R, Sato K, et al. Radioembolization for neuroendocrine liver metastases: safety, imaging and long-term outcomes. 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A randomized phase II study of drug-eluting beads versus transarterial chemoembolization for unresectable hepatocellular carcinoma. Onkologie 2011; 34: 368-76. doi: 10.1159/000329602. 18. Varela M, Real M, Burrel M, Forner A, Sala M, Brunet M, et al. Chemoembolization of hepatocellular carcinoma with drug eluting beads: efficacy and doxorubicin pharmacokinetics. J Hepatol 2007; 46: 474-81. doi: 10.1016/j.jhep.2006.10.020 19. Vogl T, Lammer J, Lencioni R, Malagari K, Watkinson A, Pilleul F, et al. Liver, gastrointestinal and cardiac toxicity in intermediate hepatocellular carcinoma with PRECISION TACE with drug-eluting beads: results from the PRECISION V randomized trial. AJR Am J Roentgenol 2011; 197: W562-70. doi: 10.2214/AJR.10.4379 20. Bhagat N, Reyes D, Lin M, Kamel I, Pawlik T, Frangakis C, et al. Phase II study of chemoembolization with drug-eluting beads in patients with hepatic neuroendocrine metastases: high incidence of biliary injury. Cardiovasc Intervent Radiol 2013: 36: 449-59. doi: 10.1016/j.jvir.2012.08.028 21. Guiu B, Descamps F, Aho S, Munck F, Dromain C, Boige V, et al. Liver/biliary injuries following chemoembolization of endocrine tumours and hepatocel­lular carcinoma: lipiodol versus drug-eluting beads. J Hepatol 2012: 56: 609-617. doi: 10.1016/j.jhep.2011.09.012 22. Joskin J, de Baere T, Auperin A, Tselikas L, Guiu B, Farouil G, et al. Predisposing factors of liver necrosis after transcatheter arterial chem­oembolization in liver metastases from neuroendocrine tumor. Cardiovasc Intervent Radiol 2015; 38: 372-380. doi: 10.1016/j.jhep.2011.09.012 80 23. De Baere T, Deschamps F, Teriitheau C, Rao P, Conengrapht K, Schlumberger M, et al. Transarterial chemoembolization of liver metastases from well differentiated gastroenteropancreatic endocrine tumors with doxorubicin­eluting beads: preliminary results. J Vasc Interv Radiol 2008; 19: 855-61. doi: 10.1016/j.jvir.2008.01.030 24. Sakamoto I, Iwanaga S, Nagaoki K, Matsuoka Y, Ashizawa K, Uetani M, et al. Intrahepatic biloma formation (bile duct necrosis) after transcatheter arterial chemoembolization. AJR Am J Roentgenol 2003; 181: 79-87. doi: 10.2214/ajr.181.1.1810079 25. Gaur S, Friese J, Sadow C, Ayyagari R, Binkert C, Schenker M, et al. Hepatic arterial chemoembolization using drug-eluting beads in gastrointestinal neuroendocrine tumor metastatic to the liver. Cardiovasc Intervent Radiol 2011; 34: 566-72. doi: 10.1007/s00270-011-0122-1 26. Loewe C, Schindl M, Cejna M, Niederle B, Lammer J, Thurnher S. Permanent transarterial embolization of neuroendocrine metastases of th eliver using cyanoacrylate and lipiodol: assessment of mid- and long-term results. AJR Am J Roentgenol 2003; 180: 1379-84. doi: 10.2214/ajr.180.5.1801379 27. Bilbao J, de Luis E, García de Jalón JA, de Martino A, Lozano MD, de la Cuesta AM, et al. Comparative study of four different spherical embolic particles in an animal model: a morphologic and histologic evaluation. J Vasc Interv Radiol 2008; 19: 1625-38. doi: 10.1016/j.jvir.2008.07.014 28. De Luis E, Bilbao JI, de Ciércoles JA, Martínez-Cuesta A, de Martino Rodríguez A, Lozano MD. In vivo evaluation of a new embolic spherical particle (HepaSphere) in a kidney animal model. Cardiovasc Intervent Radiol 2008; 31: 367-76. doi: 10.1007/s00270-007-9240-1 research article Long-term outcomes of high dose treatment and autologous stem cell transplantation in follicular and mantle cell lymphomas – a single centre experience Lucka Boltezar1, Karlo Pintaric2, Jože Pretnar3, Maja Pohar Perme4, Barbara Jezersek Novakovic1 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Slovenia 3 Department of Hematology, University Clinical Centre Ljubljana, Slovenia 4 Department of Biostatistics and Medical Informatics, University of Ljubljana, Slovenia Radiol Oncol 2017; 51(1): 81-87. Received 6 January 2016 Accepted 24 May 2016 Correspondence to: Assoc. Prof. Barbara Jezeršek Novaković, M.D., Ph.D., Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 631; Fax: +386 1 5879 305; E-mail: bjezersek@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Advanced follicular lymphoma (FL) and mantle cell lymphoma (MCL) are incurable diseases with conventional treatment. The high dose treatment (HDT) with autologous stem cell transplantation (ASCT), however, offers a certain proportion of these patients the prospect of a prolonged disease-free and overall survival. The aim of this study was to investigate the event free survival (EFS) and overall survival (OS) in patients with FL and MCL treated with ASCT. Patients and methods. Seventeen patients with FL and 29 patients with MCL were included, 15 of them were trans­planted to consolidate the response to second line treatment and 24 to consolidate their first remission, respectively. All were conditioned with total body irradiation (TBI) and high dose cyclophosphamide between 2006 and 2014 and all were transplanted with peripheral blood stem cells. Results. The estimated 5-year OS for FL was 87.8% (95% confidence interval [CI] 59.5%–96.8%) and for MCL 79.3% (95% CI 56.1%–91.1%), respectively. The estimated 5-year EFS for FL was 76.0% (95% CI 48.0%–90.3%) and for MCL 69.8% (95% CI 45.5%–84.8%), respectively. There were no secondary hematological malignancies observed in either group. Conclusions. Based on above results, the ASCT with TBI is a good treatment option in terms of long-term survival for patients with follicular and mantle cell lymphoma demonstrating a relatively low rate of late toxicities and secondary malignancies. Key words: follicular lymphoma; mantle cell lymphoma; autologous stem cell transplantation; overall survival; hema­tological malignancies Introduction Follicular lymphoma (FL) is nowadays still an in­curable disease using standard chemotherapy.1 Even though it is very sensitive to chemo- and radi­otherapy relapses remain the main treatment fail­ure. Significant changes were made in the past two decades, a great gain was the addition of rituximab to the standard CHOP regimen (cyclophospha­mide, doxorubicin, vincristine, and prednisone) to prolong progression-free survival (PFS).2,3 Rituximab is now the golden standard in the first line treatment2, it also improves the overall sur­vival (OS) in relapsed FL patients.4 In a Cochrane review in 2012, the authors demonstrated that high dose treatment (HDT) with autologous stem cell transplantation (ASCT) improves the progression-free survival in comparison with chemotherapy or immuno-chemotherapy in previously untreated patients with FL, but does not prolong the OS.5 There is also evidence that HDT with ASCT brings benefits to patients with relapsed FL.5,6 A consen­sus was made in 2013 by the European Group for Blood and Marrow Transplant stating that the SCT is appropriate in patients with first chemo-sensi­tive relapse to consolidate remission, especially in patients with a short response after immuno-chem­otherapy or with high-risk follicular lymphoma international prognostic index (FLIPI). They also pointed out that the HDT with ASCT is appropriate in second or subsequent chemo-sensitive relapses.1 Allogeneic transplantation and autologous transplantation are both possible. A higher relapse rate was observed in autologous SCT, whereas no significant difference in the disease-free survival or OS was found.7,8 Allogeneic SCT showed a lower risk for disease recurrence9,10, and that also ap­plies for the reduced intensity conditioning SCT.8 However, higher treatment-related mortality after allogeneic SCT than after autologous was objecti­fied. One of the factors which contributed signifi­cantly to the higher treatment-related mortality was the total body irradiation (TBI).9 The mantle cell lymphoma (MCL) is also an in­curable disease and successful treatment is still a challenge. In the past few years, many induction regimens were tested for their efficacy.11-14 Since 2013, we use in our centre the alternation of ritux-imab-CHOP (R-CHOP) regimen with R-high dose cytarabine-based regimen for younger patients, as it was shown that it gives a better OS and a higher proportion of partial remission (PR) to complete remission (CR) conversions than other regimens.15 Nevertheless, the HDT and ASCT remain an attrac­tive option for those with chemo-sensitive disease regardless of the induction regimen applied.12,14-16 In relapsed or refractory disease, long-term dis-ease-free intervals have not been established, the reduced intensity conditioning transplantation is here an option.16 Secondary hematological malig­nancies after the SCT remain an important issue with an estimated 5 year risk of 3.8%17, although some authors report of minimum hematological malignancies or none at all.15 The allogeneic SCT offers a lower relapse rate but a higher non-re­lapse mortality resulting in OS similar to ASCT.18 However, it was also shown by Romera et al. that intensive chemotherapy with R-hyper-CVAD (cy­clophosphamide, vincristine, doxorubicin, and dexamethasone) alternating with R-high dose methotrexate-cytarabine is also a suitable treat­ment option for treatment of MCL without ASCT.19 The aim of this study was to investigate the event free survival (EFS) and OS in patients with FL and MCL treated with ASCT. Patients and methods The study population includes 17 patients with FL and 29 patients with MCL who underwent ASCT after HDT conditioning with TBI (fractionated 6 x 200 cGy during three days’ period) and high dose cyclophosphamide (2 x 60 mg/kg) between 2006 and 2014 and whose conventional treatment and the TBI were performed at the Institute of Oncology Ljubljana and the transplantation procedure at the University Clinical Centre Ljubljana, Department of Hematology. The study was conducted accord­ing to the Helsinki Declaration, all patients were aged 18 or above and patient’s informed consent was obtained. According to national guidelines, FL patients were transplanted in their second remission and only three patients with adverse prognostic fac­tors were consolidated for their first remission. Conversely, MCL patients were transplanted al­ready in their first remission and exceptionally on­ly five patients (with low risk international prog­nostic index for MCL [MIPI]) were consolidated for their second or later remission. All patients in­cluded in the study were transplanted with stem cells collected from peripheral blood. All patients were also treated with maintenance rituximab post ASCT concordant with the latest studies20,21 and were treated with rituximab containing regimens in their induction treatments. Twenty-six MCL pa­tients received R-CHOP regimen as their first in­duction treatment and 3 patients received R-CHOP as the second line treatment. In the FL group, only 10 patients started with R-CHOP as the first line in­duction treatment and 7 patients received R-CHOP after failure of first line treatment without rituxi­mab. The disease status prior to ASCT was evalu­ated according to revised Cheson’s criteria.22 The OS was measured from the time of HDT/ASCT to death from any cause. The EFS was measured from the time of HDT/ASCT to progression, recurrence of the disease, or death from any cause. The non-relapse mortality was defined as death from any cause without progression or relapse of lympho­ma. The EFS and OS were calculated according to the Kaplan-Meier method and differences between TABLE 1. Patients’ characteristics Sex (F/M) 7 (41.2%) / 10 (58.8%) 6 (20.7%) / 23 (79. 3%) Age at diagnosis in years, median 46 (range 29–62) 54 (range 38–65) FL grade I/II IIIa 13 (76.5%) 4 (23.5%) / Pleomorphic/blastoid MCL / 10 (34.5%) Distribution according to FLIPI or MIPI low risk/intermediate/high risk 4 (23.5%) / 9 (52.9%) / 4 (23.5%) 24 (82.8%) / 4 (13.8%) / 1 (3.4%) HDT/ASCT After first line treatment* After second line treatment or later* 3 (17.6%) 14 (82.4%) 24 (82.7%) 5 (17.2%) Disease status prior HDT/ASCT CR PR 5 (29.4%) 12 (70.6%) 13 (44.8%) 16 (55.2%) Documented rituximab treatment before HDT/ ASCT 17 (100%) 29 (100%) * according to national guidelines; CR = complete remission; F = female; FL = follicular lymphoma; FLIPI = follicular lymphoma international prognostic index; HDT/ASCT = high dose treatment/autologous stem cell transplantation; M = male; MCL = mantle cell lymphoma; MIPI = international prognostic index for mantle cell lymphoma; N = number; PR = partial remission subgroups were analyzed by the log-rank test and by the Cox proportional hazards model. The non-relapse mortality was estimated using the cumula­tive incidence function. The program used was R Statistical software (R for Windows, version R-3.1.2, University of Auckland) and GraphPad Prism pro­gramme (version 3.02, GraphPad Software, USA). The survival outcomes of three patients with histologically confirmed transformation of FL into the diffuse large B-cell lymphoma at the time of HDT/ASCT were analyzed separately. Results Patients’ characteristics and disease status prior to ASCT Patients’ characteristics are shown in Table 1. In the FL group, there was a slight male predomi­nance. Median age at diagnosis of lymphoma was 46 years. Thirteen patients had FL grade I/II and 4 patients had FL grade IIIa. None of the patients had FL grade IIIb. Four of them were considered as low risk patients (0–1 points) according to FLIPI, 9 were in the intermediate group (2 points) and 4 of them in the high risk group (3–5 points). Three patients (17.6%) were transplanted to consolidate their first remission, 14 (82.4%) were transplanted as a consolidation after treatment for their first re­lapse. Five (29.4%) of them were in CR before trans­plantation, 12 (70.6%) of them had PR. In the MCL group, there were 23 males and 6 females. Median age at diagnosis was 54 years. There were 10 pleo­morphic/blastoid variants of MCL in the study population. Twenty-four patients had MIPI below 5.7 (low risk group), 4 patients between 5.7 and 6.2 (intermediate risk group) and one patient was in the high risk group (MIPI above 6.2). Twenty-four patients (82.7%) were transplanted in their first remission, 3 patients (10.3%) were transplanted to consolidate their second remission and 2 patients (6.9%) for later than second remission. Thirteen (44.8%) of them had CR before transplantation and 16 (55.2%) of them had PR. Overall and event free survival The estimated 5-year OS for FL was 87.8% (95% confidence interval [CI] 59.5%–96.8%) and the median OS has not been reached yet with a me­dian follow-up time of 57.5 months (range 15–102 months) (Figure 1A). The FLIPI score did not influ­ence the OS (p = 0.365, data not shown) and also the disease status had no significant impact on the OS (p = 0.570, hazard ratio (HR) 2.188, 95% CI HR 0.120–47.29). The estimated 5-year OS for MCL was 79.3% (95% CI 56.1%–91.1%), and the median OS has not been reached yet with a median follow-up time of 31.5 months (range 2.5 months–95.5 months) (Figure 1B). The MIPI index had no impact on OS (p = 0.776, data not shown) and the same is true for studies, for example a study of Khouri et al, where they reported the 11-year OS of 78% achieved by the addition of 90Y to the fludarabine and cyclo­phosphamide conditioning regimen.24 Van Besien et al. conducted a study comparing allogeneic and autologous SCT. The TBI conditioning regi­men was more used for allogeneic transplantation, which differs from our study. Their 5-year adjust­ed probabilities of survival after allogeneic trans­plantation were 51% and TBI was associated with higher transplant related mortality but a lower re­currence of the disease.9 Our study confirms that the TBI including regimen is also appropriate for ASCT with a low rate of secondary malignancies and late toxicities. The estimated 5-year EFS of our patients was 76.0% (95% CI 48.0%–90.3%) which is also better than in El-Najjar et al. study, where the 5-year EFS for TBI-based HDT was 58%. Like in case of OS, the EFS was also significantly worse in patients with BEAM-based HDT (49%, p < 0.001).23 Eighty-two point four percent of our patients were trans­planted for their first relapse, with the HDT and ASCT being an established treatment regimen for relapsed disease in the rituximab era.25-27 Sebban et al. namely showed no benefit of HDT and ASCT after first line treatment regarding the EFS and OS in comparison to standard chemotherapy.25 The significant difference in EFS between our patients with FL and those whose lymphomas transformed to the diffuse large-B cell lymphoma (p = 0.0034) is interesting despite the fact that our study groups were relatively small. These results emphasize the importance of appropriate patient selection for transplantation procedure as the out­come of the patients with transformation cannot compare to our FL group (at least partially on ac­count of a small number of transformations but al­so on account of a significantly worse prognosis of patients having the transformation), showing that FL patients with a transformation to the diffuse large-B cell lymphoma are not ideal candidates for TBI and high dose cyclophosphamide followed by ASCT. However, the transformation cannot be pre­dicted and for these patients an alternative salvage option should be sought - for example a different conditioning regimen. Mantle cell lymphoma In our study population, the estimated 5-year OS for MCL was 79.3% (95% CI 56.1%–91.1%) and the median OS has not been reached. Also in this population, our results are comparable to the ones reported by other authors11,28,29, for example by Dreyling et al. where they compared survival after TBI and ASCT versus maintenance with .-interferon after completion of induction therapy. The 3-year OS was 83% after ASCT versus 77% in the IFN group (p = 0.18).11 The prospective trial of Nordic Lymphoma Group, however, reports of a 4-year OS of 81% and a 4-year EFS of 63%, but their patients were conditioned with the BEAM regimen.30 We can also estimate the 7-year OS for our MCL as 79.3% since there were no additional events, but that estimation is not as reliable as the 5-year OS. In a study by Oinonen et al., the median EFS was set at 39 months31, while in our group the median has not been achieved yet with a median observa­tion period of 31.5 months. Delarue et al. revealed the same, comparing the BEAM treatment to TBI - the median EFS of patients receiving BEAM was 55 months, whereas the median EFS for the TBI regi­men group has not been reached (p = 0.05).15 This again proves that TBI conditioning regimen is safe and appropriate for ASCT in MCL. The ASCT performed in line of first line treat­ment to consolidate the first remission was docu­mented to have a significantly better EFS and OS compared to delayed ASCT.11,32 In our study, the impact of early or late transplantation on OS and EFS was not statistically significant (OS: p = 0.141, HR 0.284, 95% CI HR 0.015–1.82; EFS: p = 0.393, HR 0.497, 95% CI HR 0.057–3.080). That could be due to our relatively small study group and a small proportion of transplantations to consolidate the second or further remissions. Most of our patients were namely transplanted after first line treatment. TBI conditioning The TBI regimen has been linked to secondary ma­lignancies, mostly hematological - myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML).17,26,33-35 The numbers vary among different studies - they range from a 5-year estimated risk of 3.8%17 to a 7-year cumulative probability of 8.9%34 and a 6-year incidence of 12%.33 The therapy-relat-ed-MDS/therapy-related-AML (t-MDS/t-AML) al­so depended on amount of radiation used for TBI. In Metayer et al. multicenter case-control study, there was a nonsignificant excess risk of tMDS/ tAML with use of conditioning regimens with TBI, compared with no TBI (relative risk [RR] = 2.0; 95% CI 0.95–4.18; p. = .0.07). The dose-response analysis, however, revealed that the excess risk was limited to patients receiving TBI doses of 13.2 Gy (RR =.6.6; p = 0..003). Those with lower TBI doses (5–12 Gy) had no evidence of elevated risk of tMDS/tAML.34 Hypotheses on cellular level are that HDT and TBI cause similar cellular and ge­netic damages as the pre-transplant therapy to the hematopoietic progenitor cells and therefore pro­vide opportunity for causing damage to the stem cells. Reinfusion of peripheral blood progenitor cells with previously damaged DNA can result in clonal abnormal hematopoiesis.35 In this study, we had no hematological malignancies in either of our groups. Even though the number of our patients is relatively small we may assume that TBI with high doses of cyclophosphamide is a safe method of conditioning prior to transplantation in regard of the secondary hematological malignancies. Conclusions Follicular lymphoma and mantle cell lymphoma remain incurable diseases even in modern era. Treatment modalities are improving and trans­plantation of stem cells is gaining a more and more important role each year. We present good long term survivals after autologous stem cell trans­plantation following conditioning with TBI and high dose cyclophosphamide with minimal toxici­ties and low incidence of secondary malignancies. Our study also demonstrates a low relapse rate and therefore we conclude that the autologous stem cell transplantation represents a reliable option for treatment of follicular and mantle cell lymphoma with good long term results. Acknowledgments This study was partially financed by Ministry of Science and Technology of Slovenia – grant P3­0321. References 1. Montoto S, Corradini P, Dreyling M, Ghielmini M, Kimby E, López-Guillermo A, et al. Indications for hematopoietic stem cell transplantation in patients with follicular lymphoma: a consensus project of the EBMT-Lymphoma Working Party. Hematologica 2013; 98: 1014-21. 2. Fisher RI, LeBlanc M, Press OW, Maloney DG, Unger JM, Miller TP. New treatment options have changed the survival of patients with follicular lymphoma. 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Long-term progression-free survival of mantle cell lymphoma after inten­sive front-line immunochemotherapy with in vivo–purged stem cell rescue: a nonrandomized phase 2 multicenter study by the Nordic Lymphoma Group. Blood 2008; 112: 2687-93. 31. Oinonen R, Jantunen E, Itälä M, Lehtinen T, Kuittinen O, Franssila K, et al. Autologous stem cell transplantation in patients with mantle cell lym­phoma. Leuk Lymphoma 2002; 43: 1229-37. 32. Dreger P, Martin S, Kuse R, Sonnen R, Glass B, Kröger N, et al. The impact of autologous stem cell transplantation on the prognosis of mantle cell lymphoma: a joint analysis of two prospective studies with 46 patients. Hematol J 2000; 1: 87-94. 33. Micallef IN, Lillington DM, Apostolidis J, Amess JA, Neat M, Matthews J, et al. Therapy-related myelodysplasia and secondary acute myelogenous leuke­mia after high-dose therapy with autologous hematopoietic progenitor-cell support for lymphoid malignancies. J Clin Oncol 2000; 18: 947-55. 34. Metayer C, Curtis RE, Vose J, Sobocinski KA, Horowitz MM, Bhatia S, et al. Myelodysplastic syndrome and acute myeloid leukemia after autotrans­plantation for lymphoma: a multicentre case-control study. Blood 2003; 101: 2015-23. 35. Akhtari M, Bhatt VR, Tandra PK, Krishnamurthy J, Horstman H, Dreessen A, et al. Therapy-related myeloid neoplasms after autologous hematopoietic stem cell transplantation in lymphoma patients. Cancer Biol Ther 2013; 14: 1077-88. research article Incidence of positive peritoneal cytology in patients with endometrial carcinoma after hysteroscopy vs. dilatation and curettage Andraz Dovnik1, Bojana Crnobrnja1, Branka Zegura1, Iztok Takac1,2, Maja Pakiz1 1 University Clinic for Gynaecology and Perinatology, University Medical Centre Maribor, Maribor, Slovenia 2 Faculty of Medicine, University of Maribor, Maribor, Slovenia Radiol Oncol 2017; 51(1): 88-93. Received 30 January 2016 Accepted 28 April 2016 Correspondence to: Andraž Dovnik, M.D., University Clinic for Gynaecology and Perinatology, University Medical Centre Maribor, Ljubljanska 5, SI-2000 Maribor, Slovenia. Phone: +386 2 321 2178; +386 31 807 874; E-mail: andrazdovnik@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to compare the frequency of positive peritoneal washings in endometrial cancer patients after either hysteroscopy (HSC) or dilatation and curettage (D&C). Patients and methods. We performed a retrospective analysis of 227 patients who underwent either HSC (N = 144) or D&C (N = 83) and were diagnosed with endometrial carcinoma at the University Medical Centre Maribor between January 2008 and December 2014. The incidence of positive peritoneal cytology was evaluated in each group. Results. There was no overall difference in the incidence of positive peritoneal washings after HSC or D&C (HSC = 13.2%; D&C = 12.0%; p = 0.803). However, a detailed analysis of stage I disease revealed significantly higher rates of positive peritoneal washings in the HSC group (HSC = 12.8%; D&C = 3.4%; p = 0.046). Among these patients, there was no difference between both groups considering histologic type (chi-square = 0.059; p = 0.807), tumour differentiation (chi-square = 3.709; p = 0.156), the time between diagnosis and operation (t = 0.930; p = 0.357), and myometrial inva­sion (chi-square = 5.073; p = 0.079). Conclusions. Although the diagnostic procedure did not influence the overall incidence of positive peritoneal washings, HSC was associated with a significantly higher rate of positive peritoneal cytology in stage I endometrial carcinoma compared to D&C. Key words: endometrial carcinoma; peritoneal cytology; FIGO staging; hysteroscopy; dilatation and curettage Introduction The diagnosis of endometrial cancer can be made preoperatively by obtaining a sample of endome­trial tissue either with office endometrial biopsy, most commonly done with a Pipelle aspiration catheter, hysteroscopy (HSC), or dilatation and curettage (D&C).1 The latter two procedures are most commonly used in Slovenia. HSC has been shown to be highly accurate in diagnosing endo­metrial cancer2,3 and is considered a gold standard.1 Conflicting evidence has been published in the past regarding the risk of intraperitoneal spread of ma­lignant cells after HSC with the use of distension media.4-7 In 2007, a retrospective study from our in­stitution reported a significantly higher incidence of positive peritoneal washings after HSC com­pared to D&C.8 However, only 24 patients in this study had undergone HSC compared to 122 who were diagnosed with D&C.8 In recent years, HSC has become an established diagnostic tool at our institution and is now performed more frequently than D&C. The aim of our present study was to find out whether the difference in the incidence of positive peritoneal washings between HSC and D&C persists after including a higher number of patients with hysteroscopy. Patients and methods This retrospective study included all consecu­tive patients who had endometrial carcinoma di­agnosed preoperatively with either D&C or HSC between January 2008 and December 2014 at the University Medical Centre Maribor, Slovenia. The study included patients who had more than one D&C or more than one HSC. Patients who had un­dergone both D&C and HSC were excluded from the study. The study was approved by our insti­tution’s ethics committee (Approval No. 13-03/15, November 26, 2015). All patients signed a written informed consent that their medical records can be used for research matters retrospectively. HSC was performed in the office setting or un­der general anesthesia. Saline solution warmed to body temperature was used as the distension me­dium. In the office setting, the distension medium was installed into the pressure cuff and the intra­uterine pressure was set between 80–150 mmHg. Intrauterine pressure was controlled with the Vario Flow device (Pelta, Slovenia) in operative HSC.9 D&C was performed under general anesthesia. Curettage of the cervical canal and the uterine cav­ity was performed separately. Tissue samples for histologic examination were obtained during both procedures. During the final surgery for endometrial car­cinoma, samples of peritoneal washings from the pouch of Douglas were obtained for cytologic ex­amination. Irrigation of the peritoneal cavity with saline solution was performed to obtain samples in cases with no free fluid. The samples were inspect­ed by an expert cytopathologist. In cases of sus­picious peritoneal cytology additional calretinin, MOC 31, HBME 1 and Ber-EP4 immunostaining was performed during the clarification process. In cases of small numbers of positive cells after im­munostaining peritoneal cytology was described as suspicious. We therefore included suspicious re­sults in the analysis of positive peritoneal cytology. The primary statistical outcome was the inci­dence of positive peritoneal washings after HSC and after D&C. A detailed analysis of tumour histopathologic characteristics was performed including histopathologic type, tumour differen­tiation, depth of myometrial invasion, lympho­vascular invasion and FIGO stage. Different types of endometrial carcinomas were identified in the study population with endometrioid carcinoma representing the majority of cases (N = 211; 93.0%). Other carcinomas (serous adenocarcinoma: N = 8; TABLE 1. Histologic characteristics of the patients with endometrioid endometrial carcinoma (N = 211) and non-endometrioid endometrial carcinoma (N = 16) Tumour differentiation < 0.001 G1 125 (59.2%) 2 (13.3%) G2 63 (29.9%) 4 (26.6%) G3 23 (10.9%) 9 (60.0%) Myometrial invasion none 8 (3.8%) 1 (6.3%) 0.888 less than 1 123 (58.3%) 9 (56.2%) more than 1 80 (37.9%) 6 (37.5%) 2009 FIGO stage IA 114 (54.0%) 7 (43.8%) 0.025 IB 54 (25.6%) 1 (6.3%) II 14 (6.6%) 2 (12.5%) IIIA 7 (3.3%) 1 (6.3%) IIIB 5 (2.4%) 0 (0.0%) IIIC1 11 (4.7%) 2 (12.5%) IIIC2 0 (0.0%) 0 (0.0%) IVA 1 (0.5%) 1 (6.3%) IVB 5 (2.4%) 2 (12.5%) *chi-square test clear cell adenocarcinoma: N = 8) were assigned into non-endometriod group for the purpose of the study. Tumour differentiation was reported as good, moderate or poor. The depth of myometrial invasion was reported as no invasion, less than half of myometrium or more than half of myometrium. Patients treated before 2009 who had been staged according to the 1988 FIGO classification were re­staged according to the new 2009 FIGO classifica­tion for statistical analysis. The time interval from diagnosis to final surgery was also analyzed. Statistical analysis was performed with SPSS software version 22.0 (IBM, Armonk, NY, USA). Descriptive analysis, chi-square test and t-test of independent samples were performed as applica­ble. A p value of less than 0.05 was considered sta­tistically significant. Results Between January 2008 and December 2014, 266 patients had uterine cancer diagnosed with D&C and/or HSC. 227 patients who had either HSC (N = TABLE 2. Histologic characteristics of the patients with endometrial carcinoma (N = 227) who were diagnosed with hysteroscopy (HSC) or dilatation and curettage (D&C) Histologic type endometrioid adenocarcinoma 137 (95.1%) 74 (89.2%) 0.090* non-endometrioid adenocarcinoma 7 (4.9%) 9 (10.8%) Tumour differentiation G1 90 (62.5%) 37 (45.1%) 0.012* G2 40 (27.8%) 27 (32.9%) G3 14 (9.7%) 18 (22.0%) Myometrial invasion none 5 (3.5%) 4 (4.8%) 0.726* less than 1 82 (56.9%) 50 (60.2%) more than 1 57 (39.6%) 29 (34.9%) Lymphovascular invasion present 20 (13.9%) 14 (16.9%) 0.545* absent 124 (86.1%) 69 (83.1%) 2009 FIGO stage IA IB II IIIA IIIB IIIC1 IIIC2 IVA IVB Peritoneal cytology positive or suspicious negative Time from diagnosis to operation (days) 75 (52.1%) 42 (29.2%) 10 (6.9%) 1 (0.7%) 3 (2.1%) 9 (6.3%) 0 (0.0%) 1 (0.7%) 3 (2.1%) 19 (13.2%) 125 (86.8%) 35.8 ± 13.8 46 (55.4%) 13 (15.7%) 6 (7.2%) 7 (8.4%) 2 (2.4%) 4 (4.8%) 0 (0.0%) 1 (1.2%) 4 (4.8%) 10 (12.0%) 73 (88.0%) 32.8 ± 15.4 0.040* 0.803* 0.140** *chi-square test ** independent sample t-test 144) or D&C (N = 83) as well as available informa­tion on peritoneal washings were included in the statistical analysis. Two hundred and eleven (93.0%) patients had endometrioid endometrial carcinoma and 16 (7.0%) had non-endometrioid endometrial carcino­ma. The differences between both groups regard­ing the differentiation, myometrial invasion and tumour stage are shown in Table 1. Significantly higher rate of poorly differentiated tumours (chi-square = 29.114; p < 0.001) and higher stages (chi-square = 16.019; p = 0.025) have been noted in the non-endometrioid group. Overall, there was no significant difference in the incidence of positive or suspicious peritoneal washings regarding the procedure performed dur­ing the diagnostic evaluation (13.2% after HSC, 12.0% after D&C; chi-square = 0.062; p = 0.803). The groups (HSC vs. D&C) did not differ in the prevalence of histologic types of the tumour, depth of myometrial invasion and lymphovascular inva­sion (Table 2). However, there were significant dif­ferences in tumour differentiation as well as FIGO stage, with more patients having FIGO stage I dis­ease in the hysteroscopy group (Table 2). Due to this difference we conducted analysis only in the subgroup of patients with stage I disease. The HSC and D&C groups of stage I patients did not differ in tumour differentiation, the prevalence of histolog­ic types of the tumour, the time from diagnosis to operation and in myometrial invasion. A separate evaluation of patients with stage I tumours showed that 12.8% in the HSC group and only 3.4% in the D&C group had positive peritoneal washings. This difference was statistically significant (chi-square = 2.422; p = 0.046) (Table 3). One out of 15 FIGO stage I patients with posi­tive peritoneal washings after hysteroscopy had disease recurrence by the end of April 2015 (mean follow-up 40.2 months). Neither of the two FIGO stage I patients with positive peritoneal washings after D&C had disease recurrence in the same pe­riod (mean follow-up 39.5 months). Discussion The possibility of microscopic intraperitoneal spread of endometrial cancer cells after hysteros­copy has been a subject of debate for more than a decade. In our study, we did not find an increased incidence of positive peritoneal washings after hysteroscopy in comparison to D&C in the over­all study population of patients with endometrial carcinoma. Several other studies similarly found no association between hysteroscopy and an in­creased rate of positive peritoneal cytology.5,6 On the other hand, Bradley et al.7 reported a higher frequency of positive or suspicious peritoneal cy­tology after hysteroscopy compared to blind en­dometrial sampling using logistic regression con­trolling for confounders of grade and stage. They also reported a higher rate of disease upstaging (according to the 1988 FIGO staging system) after hysteroscopy attributed solely to the positive cytol­ogy. Similar results have been reported by Zerbe et al.10 and Obermair et al.11 In a study conducted at our institution in 20078, positive peritoneal cytol­ogy was present in 12.5% of patients after hyster­oscopy and only in 1.6% after D&C. The difference was statistically significant (chi-square = 4.2455; p < 0.005). In a meta-analysis of nine trials including 1015 patients with confirmed endometrial carcinoma, Polyzos et al.12 evaluated the rate of positive perito­neal washings after hysteroscopy in comparison to other diagnostic procedures or no diagnostic pro­cedures. They concluded that the frequency of pos­itive peritoneal washings was significantly higher after hysteroscopy. The analysis also revealed a higher rate of disease upstaging based only on the positive peritoneal cytology. A detailed literature search performed by Guralp and Kushner13 re­vealed 0–83% of positive peritoneal cytology after hysteroscopy and 0–13.6% after D&C. However, the authors emphasized a number of unanswered questions regarding the type and volume of dis­tension medium, intrauterine pressure during the procedure, time interval between hysteroscopy and definitive surgery, stage, grade of the disease and duration of the procedure.13 Another meta­analysis by Chang et al.14 also reported on higher rates of positive peritoneal cytology after hysteros­copy. Nevertheless, a detailed analysis of patients with stages I or II failed to show significantly high­er rates of positive peritoneal cytology in patients who had hysteroscopy. Interestingly, our results showed a significantly higher incidence of positive or suspicious perito­neal cytology in patients with stage I disease who were diagnosed with hysteroscopy compared to those diagnosed with D&C. This is an unexpected finding because the disease at this stage is confined to the uterus. For example, only 3.3% of patients with stage I and II endometrial cancer in a large retrospective analysis by Garg et al.15 had positive peritoneal cytology. In our study, the rate of posi­tive or suspicious peritoneal cytology in stage I dis­ease was 3.3% in the D&C group but as much as 12.1% in the hysteroscopy group. Positive or sus­picious peritoneal cytology was shown to be more frequent after hysteroscopy in endometrial carci­noma patients who would be staged as FIGO IA in the new staging system by Obermair et al.11 On the other hand, Biewenga et al.6 showed no associa­tion between hysteroscopy and the rate of positive peritoneal washings in stage I disease. Saline solution was used as the distension me­dium in all our patients in the hysteroscopy group. Hysteroscopy with saline solution was specifically linked to a higher rate of positive peritoneal cytol­ogy in a meta-analysis by Polyzos et al.12 In a meta­analysis by Chang et al. 14, the distension medium was either saline solution or 5% glucose solution. TABLE 3. Histologic characteristics of the patients with stage 1 endometrial carcinoma (N = 187) who were diagnosed with hysteroscopy (HSC) or dilatation and curettage (D&C) Histologic type endometrioid adenocarcinoma 112 (95.7%) 56 (94.9%) 0.807* non-endometrioid adenocarcinoma 5 (4.3%) 3 (5.1%) Tumour differentiation G1 75 (64.1%) 29 (49.2%) 0.156* G2 32 (27.4%) 22 (37.3%) G3 10 (8.5%) 8 (13.5%) Myometrial invasion none 5 (4.3%) 3 (5.1%) 0.079* less than 1 71 (60.7%) 45 (76.2%) more than 1 41 (35.0%) 11 (18.6%) Peritoneal cytology positive or suspicious negative 15 (12.8%) 102 (87.2%) 2 (3.4%) 57 (96.6%) 0.046* Time from diagnosis to operation (days) 35.6 ± 14.0 33.1 ± 12.7 0.255** *chi-square test ** independent sample t-test Neither of these two meta-analyses found a con­nection between intrauterine pressure during hys­teroscopy higher than 100 mmHg and a higher in­cidence of positive peritoneal cytology.12,14 In our study, the exact intrauterine pressure during hyst­eroscopy was not known for each patient individu­ally due to the retrospective nature of the analysis. The time interval between the diagnostic proce­dure and definitive surgery was similar in patients with positive and negative peritoneal cytology in our study. This is in line with evidence from an­other retrospective study in 196 patients with en­dometrial cancer diagnosed with hysteroscopy.16 Based on the data from our retrospective study, we cannot give a definite reason for the signifi­cantly higher incidence of positive peritoneal washings after HSC compared to D&C in stage I disease. We used saline solution as the distension medium, which has been previously associated with higher rates of positive peritoneal cytology.12 Unfortunately, we do not have the exact informa­tion on the intrauterine pressure during HSC and the duration of the diagnostic procedure for each patient and therefore we cannot draw conclusions about the influence of these factors on peritoneal cytology. Another important limitation of our study is the inclusion of suspicious peritoneal cytology in the positive peritoneal cytology group. Even after im­munostaining most of the cases without evident malignant cells remained cytologically suspicious because positive immune reaction was seen in only a small fraction of cells. However, it is not easy to differentiate positive from suspicious cytology because severe atypia of reactive mesothelial cells may be interpreted as suspicious. We are aware of this methodological limitation and should aim to lower the incidence of suspicious peritoneal cy­tology in the future firstly by obtaining sufficient amount of fluid for cytological analysis during fi­nal surgery and secondly with accurate cytological diagnosis. Some published research on this subject also included positive and suspicious cytology in the same group.7,8,11 Our data show that among patients with positive or suspicious peritoneal washings after hysterosco­py, in FIGO stage I patients, one out of 15 had local disease recurrence during follow-up of approxi­mately 40 months, whereas neither of the two with positive washings after D&C had the recurrence. As these numbers are small, further research is neces­sary to draw relevant conclusions. Conflicting re­sults exist in the literature regarding the prognostic significance of positive peritoneal cytology.17,18 The updated FIGO staging system from 2009 excluded positive peritoneal cytology as a stage defining variable. Previously, all patients with positive peri­toneal cytology were upstaged to stage IIIA.19 In an analysis of 14,704 patients, Garg et al.15 reported peritoneal cytology to be associated with survival in univariate analysis along with race, age, histol­ogy, grade and the number of removed lymph nodes. In multivariate analysis, positive peritoneal cytology remained an independent prognostic fac­tor in stages I and II. Shiozaki et al.20 studied the influence of positive peritoneal washings on the prognosis of 265 patients with stage I endometrial cancer. Progression-free survival was significantly lower in the group with positive peritoneal cytol­ogy. Other factors associated with progression-free survival in univariate analysis were lymph node dissection and vessel permeation, but positive peri­toneal cytology was the most influential factor.20 Disease-free survival has been shown to be 91% in FIGO stage I patients and 52.5% in those with FIGO stage II, III and IV.21 In the study by Garg et al., sur­vival in patients with stage I endometrioid adeno­carcinoma with positive peritoneal washings was significantly poorer than in patients with negative peritoneal washings (88.2% vs. 98.6%).15 In conclusion, the diagnostic procedure did not influence the overall incidence of positive peritone­al washings in our study. However, hysteroscopy was associated with a significantly higher rate of positive peritoneal cytology in stage I endome­trial carcinoma. Although statistically significant, this finding must be interpreted with caution be­cause of the small sample size of this subgroup. In addition, it is still not known whether iatrogenic dissemination of malignant cells bears the same influence on disease prognosis as spontaneous dis­semination. Despite being excluded as the stage defining variable, peritoneal cytology should still be reported separately as requested by FIGO.15 We believe that additional trials are needed to further clarify the prognostic value of positive peritoneal cytology after hysteroscopy, particularly in the early stages of endometrial cancer. References 1. Burke WM, Orr J, Leitao M, Salom E, Gehrig P, Olawaiye AB, et al. Society of Gynecologic Oncology Clinical Practice Committee. Endometrial cancer: a review and current management strategies: part I. Gynecol Oncol 2014; 134: 385-92. 2. Clark TJ, Voit D, Gupta JK, Hyde C, Song F, Khan KS. Accuracy of hysteroscopy in the diagnosis of endometrial cancer and hyperplasia: a systematic quan­titative review. JAMA 2002; 288: 1610-21. 3. Gkrozou F, Dimakopoulos G, Vrekoussis T, Lavasidis L, Koutlas A, Navrazoglou I, et al. Hysteroscopy in women with abnormal uterine bleeding: a meta­analysis on four major endometrial pathologies. Arch Gynecol Obstet 2015; 291: 1347-54. 4. Gu M, Shi W, Huang J, Barakat RR, Thaler HT, Saigo PE. Association between initial diagnostic procedure and hysteroscopy and abnormal peritoneal washings in patients with endometrial carcinoma. Cancer 2000; 90: 143-7. 5. Selvaggi L, Cormio G, Ceci O, Loverro G, Cazzolla A, Bettocchi S. Hysteroscopy does not increase the risk of microscopic extrauterine spread in endome­trial carcinoma. Int J Gynecol Cancer 2003; 13: 223-7. 6. Biewenga P, de Blok S, Birnie E. Does diagnostic hysteroscopy in patients with stage I endometrial carcinoma cause positive peritoneal washings? Gynecol Oncol 2004; 93: 194-8. 7. Bradley WH, Boente MP, Brooker D, Argenta PA, Downs LS, Judson PL, et al. Hysteroscopy and cytology in endometrial cancer. Obstet Gynecol 2004; 104: 1030-3. 8. Takac I, Zegura B. Office hysteroscopy and the risk of microscopic extrauter­ine spread in endometrial cancer. Gynecol Oncol 2007; 107: 94-8. 9. Tomaževič T. Operative hysteroscopy. In: Ribič-Pucelj M, editor. Endoscopic surgery in gynaecology. Radovljica: Didakta; 2007. p. 251-7. 10. Zerbe MJ, Zhang J, Bristow RE, Grumbine FC, Abularach S, Montz FJ. Retrograde seeding of malignant cells during hysteroscopy in presumed early endometrial cancer. Gynecol Oncol 2000; 79: 55-8. 11. Obermair A, Geramou M, Gucer F, Denison U, Graf AH, Kapshammer E, et al. Does hysteroscopy facilitate tumor cell dissemination? Incidence of peritoneal cytology from patients with early stage endometrial carcinoma following dilatation and curettage (D & C) versus hysteroscopy and D & C. Cancer 2000; 88: 139-43. 12. Polyzos NP, Mauri D, Tsioras S, Messini CI, Valachis A, Messinis IE. Intraperitoneal dissemination of endometrial cancer cells after hysteros­copy: a systematic review and meta-analysis. Int J Gynecol Cancer 2010; 20: 261-7. 13. Guralp O, Kushner DM. Iatrogenic transtubal spill of endometrial cancer: risk or myth. Arch Gynecol Obstet 2011; 284: 1209-21. 14. Chang YN, Zhang Y, Wang YJ, Wang LP, Duan H. Effect of hysteroscopy on the peritoneal dissemination of endometrial cancer cells: a meta-analysis. Fertil Steril 2011; 96: 957-61. 15. Garg G, Gao F, Wright JD, Hagemann AR, Mutch DG, Powell MA. Positive peritoneal cytology is an independent risk-factor in early stage endometrial cancer. Gynecol Oncol 2013; 128: 77-82. 16. Juhasz-Böss I, Fehm T, Nauth A, Becker S, Rothmund R, Gardanis K, et al. Number of hysteroscopies and the time interval between hysteroscopy and surgery: influence on peritoneal cytology in patients with endometrial cancer. Anticancer Res 2010; 30: 2425-30. 17. Saga Y, Imai M, Jobo T, Kuramoto H, Takahashi K, Konno R, et al. Is peritoneal cytology a prognostic factor of endometrial cancer confined to the uterus? Gynecol Oncol 2006; 103: 277-80. 18. Fadare O, Mariappan MR, Hileeto D, Wang S, McAlpine JN, Rimm DL. Upstaging based solely on positive peritoneal washing does not affect out­come in endometrial cancer. Mod Pathol 2005; 18: 673-80. 19. Pecorelli S. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J Gynecol Obstet 2009; 105: 103-4. 20. Shiozaki T, Tabata T, Yamada T, Yamamoto Y, Yamawaki T, Ikeda T. Does positive peritoneal cytology not affect the prognosis for stage I uterine en­dometrial cancer? The remaining controversy and review of the literature. Int J Gynecol Cancer 2014; 24: 549-55. 21. Tejerizo-García A, Jiménez-López JS, Munoz-González JL, Bartolomé-Sotillos S, Marqueta-Marqués L, López-González G, et al. Overall survival and dis-ease-free survival in endometrial cancer: prognostic factors in 276 patients. Onco Targets Ther 2013; 9: 1305-13. research article Implant-prosthetic rehabilitation after radiation treatment in head and neck cancer patients: a case-series report of outcome Jasna Cotic1, Jure Jamsek1, Milan Kuhar1,2, Natasa Ihan Hren3,4, Andrej Kansky3,4, Mutlu Özcan5, Peter Jevnikar1,2 1 Department of Prosthodontics, Faculty of Medicine, University of Ljubljana, Slovenia 2 Department of Prosthodontics, University Medical Centre Ljubljana, Slovenia 3 Department of Maxillofacial and Oral Surgery, Faculty of Medicine, University of Ljubljana, Slovenia 4 Department of Maxillofacial and Oral Surgery, University Medical Centre Ljubljana, Slovenia 5 University of Zürich, Dental Materials Unit, Center for Dental and Oral Medicine, Clinic for Fixed and Removable Prosthodontics and Dental Materials Science, Switzerland Radiol Oncol 2017; 51(1): 94-100. Received 27 November 2015 Accepted 6 January 2016 Correspondence to: Prof. Peter Jevnikar, Department of Prosthodontics, Faculty of Medicine, University of Ljubljana, Hrvatski trg 6, 1000 Ljubljana, Slovenia. Phone: +386 1 522 42 42; E-mail: peter.jevnikar@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Slovenia has a high burden of head and neck cancer. Patients are mostly treated with surgery fol­lowed by radiation therapy. Advanced surgical and prosthodontic techniques have expanded the rehabilitation options. The aim of the study was to review the outcome of implant-prosthetic treatment after radiation therapy. Patients and methods. Twenty irradiated head and neck cancer patients who received a removable implant-supported denture at the University Medical Centre Ljubljana were included in the study. Kaplan-Meier survival analy­sis, Cox proportional hazard models and logistic regression were used to assess the implant survival and success rate. Results. Twenty patients had 100 implants inserted. The estimated implant survival rate was 96% after 1 year and 87% after 5 years. Failures were mostly observed before loading (91.2%). Implants inserted in the transplanted bone were significantly more likely to fail. Out of 89 implants supporting the dentures, 79 implants (88.7%) were successful, meaning that they were functionally loaded and exhibited no pain, radiolucency or progressive bone loss. Prosthetic treatment was significantly less successful in older patients. The attachment system and the number of implants did not have a statistically significant influence on the success rate. Conclusions. Implant-supported dentures have been shown to be a reliable treatment modality after head and neck cancer surgery and radiation therapy. Possible early failures should be communicated with the patients. Key words: head and neck cancer; radiation therapy; dental implants; implant-supported dentures Introduction Slovenia is among the countries with the highest incidence of oral and oropharyngeal cancer.1,2 In a population of 2 million people, approximately 450 cases are diagnosed per year.3 Most tumours are regionally advanced and the patients are treated with radical resection and reconstruction followed by radiation therapy.3 Afterwards, patients ex­perience profound changes in the oral anatomy, function and facial appearance. Radiation therapy causes irreversible damage to both hard and soft tissues by creating a hypoxic, hypocellular and hy­povascular environment which impedes wound healing and creates a risk for osteoradionecrosis.4 A protocol involving hyperbaric oxygen treatment (HBO) has been proposed to enhance wound heal­ing by increasing the tissue oxygenation.5 Following the radical procedures to eradicate cancer, the greatest problems perceived by irradi­ated patients are swallowing, mouth opening, xeros­tomia and compromised aeshetics.6 Prosthodontic treatment is advocated to regain lost oral functions, enhance the physical appearance and enable the patient to take part in normal daily activities with greater confidence. However, comprehensive pros­thetic treatment after head and neck cancer is chal­lenging, time-consuming and costly. Therefore, only 40% of such patients are treated following the postsurgical prosthetic protocol. Among them, 70% receive dentures supported by the residual teeth and bone and 30% receive implant-supported dentures.7 Edentulous patients with head and neck cancer after radiation therapy are an especially vulnerable sub­group. Radiation treatment sequelae persist through­out the patient’s lifetime and in the past, radiation therapy used to be an absolute contraindication to placing of dental implants.8 Treatment options have therefore been limited to conventional complete den­tures or no prosthetic rehabilitation at all. Advanced surgical and prosthodontic tech­niques, such as 3D planning and guided implant surgery, have expanded the treatment options.9 In addition to conventional ball- and bar-retained dentures, new attachments have been introduced to clinical practice. Locator attachment is com­monly used in removable implant prosthodontics. It is a self-aligning system with relatively simple maintenance requirements.10 Locator is, however, a non-rigid type of attachment and does not com­pletely relieve the stress from the underlying mu­cosa. The nylon matrices and male parts are sub­jected to wear, which diminishes retention. Their replacement is one of the most frequent reasons for maintenance visits.11-13 To address these issues, a technically more ad­vanced system of prefabricated double crowns on implants has been introduced.14 The SynCone system (Dentsply, Germany) is indicated in unfa­vourable resection areas, where completely rigid constructions are necessary due to the anatomical constraints. Among the possible complications, debonding of the secondary crowns and the abut­ment screw loosening are reported.15 Both Locator and Syncone systems are common­ly used at the University Medical Centre Ljubljana, Slovenia. These attachment systems have expand­ed the treatment possibilities for the rehabilitation of head and neck cancer patients, which is always a collaborative work between oral surgeons and prosthodontists. The aim of the study was to re­view the outcome of the implant-prosthetic reha­bilitation of irradiated patients performed at the University Medical Centre Ljubljana. Patients and methods A retrospective chart review was performed for the patients who were treated jointly by the Department of oral and maxillofacial surgery and the Department of prosthodontics at the University Medical Centre Ljubljana in the time period from 2008 to 2014. Only patients who underwent resec­tion of malignant tumours with a subsequent ra­diation treatment and received removable implant-supported dentures were included in the study. The recorded data included patient gender and date of birth, smoking status at the time of pros­thetic rehabilitation, cancer type, surgical man­agement, date and dose of radiation treatment, administration of hyperbaric oxygen treatment (HBO), timing to implant surgery and to functional loading, treated jaw, type of implant bed, number of implants supporting the dentures, implant sys­tem and the denture attachment system used. At University Medical Centre Ljubljana, HBO is pro­vided according to the protocol suggested by Marx and Larsen.4,5 Patients are scheduled for 20 sessions before and 10 sessions after the implant insertion, respectively. Each 90-minute session consists of ex­posure to 100% oxygen on 2.5 ATA (1.5 bar ) with three breaks during which patients breathe nor­mal air. The study was conducted according to the Helsinki Declaration. Each patient’s informed con­sent was obtained and Institutional Review board approval was granted. The implants were assessed for survival and success using the guidelines proposed by van Steenberghe et al.16 The survival criteria included osseointegration and presence in the mouth. To be considered successful, the implant had to be functionally loaded, immobile, without persistent pain or inflammation of the periimplant tissue and without progressive bone loss evident from radio­graphs and probing depth at the recall. The survival time was measured from the date of the implant insertion to the date of the implant failure or the last control of the implant. The present series consisted of 20 patients (11 men and 9 women) with a median age of 57.6 years (range 46.7 to 77.2 years) at the time of the implant insertion. Seventeen patients (85%) had a history of squamous cell carcinoma. Mucoepidermoid, ad­enoid cystic and origo ignota metastatic cancer were diagnosed in 1 patient each. The most common sites of primary cancer were the tongue and the floor of the mouth (6 patients each), followed by pharynx and maxilla (2 patients each). Mandible, tonsilla, larynx and origo ignota metastases in lymph nodes FIGURE 1. A patient after segmental resection of the left mandible body due to cancer, with two implants as seen on the radiograph (A). The patient received an implant-supported lower denture (B), where the retention was based on the Locator attachments (C). TABLE 1. Summary of the implant survival according to the factors of interest factors was analysed by fitting univariate Cox pro­portional hazards models. The association between the potential prognostic factors and implant suc­cess was analysed with univariate logistic regres­ Patient’s median age at 58.3 61.5 57.9 sion models. The Holm-Bonferroni method was implant insertion in years (range) (46.7–77.2) (46.7–77.2) (46.7–77.2) used to account for multiple comparisons and the Median time interval between level of significance was set to . = 0.05. Statistical radiation therapy and implant 3.8 3.2 5.2 analyses were conducted with the statistical soft- insertion in years (1.1–38.1) (1.1–38.1) (2.4–12.8) (range) ware package R.17 Patient’s gender Female, n (%) 40 (40%) 32 (36%) 8 (67%) Male, n (%) 60 (60%) 56 (64%) 4 (33%) Smoking Results No, n (%) 66 (66%) 57 (65%) 9 (75%) Yes, n (%) 34 (34%) 31 (35%) 3 (25%) As presented in Table 1, 100 implants of 3 differ- Implant system Astra, n (%) 18 (18%) 18 (20%) 0 (0%) ent implant systems were included in the study: Straumann, n (%) 22 (22%) 17 (20%) 5 (42%) 18 Astra Tech implants (Dentsply, Mannheim, Ankylos, n (%) 60 (60%) 53 (60%) 7 (58%) Jaw Germany), 22 Straumann implants (Institut Lower, n (%) 72 (72%) 63 (72%) 9 (75%) Straumann AG, Basel, Switzerland) and 60 Ankylos Upper, n (%) 28 (28%) 25 (28%) 3 (25%) implants (Dentsply, Mannheim, Germany). 28 im-Bone plants were inserted in the maxilla and 72 in the Native, n (%) 92 (92%) 85 (97%) 7 (58%) Transplanted, n (%) 8 (8%) 3 (3%) 5 (42%) mandible. 92 implants were inserted in native bone HBO administered and 8 in transplanted bone. The median time be- No, n (%) 19 (19%) 14 (16%) 5 (42%) Yes, n (%) 81 (81%) 74 (84%) 7 (58%) tween the end of the radiation therapy and the im­plant surgery was 3.8 years (range 1.1 to 38.1 years). Prophylactic antibiotic therapy was prescribed to all patients and HBO was administered to 16 were encountered in one patient each. A segmental patients. Osteoradionecrosis was not observed in resection of the mandible was performed in 8 pa-this study. All implants were inserted in edentu­tients. In one patient, the tumour resection in the lous jaws, with 5 patients receiving implants in maxilla resulted in an oronasal communication. both jaws, 14 only in the mandible and 1 only in After surgery, all patients were subjected to the ra-the maxilla. Twelve patients were non-smokers diation therapy, with reported doses ranging from and 8 patients were smokers at the time of the 54 to 66 Gy. prosthetic rehabilitation. A two-stage implant in-Reconstruction with bone and soft tissue grafts sertion protocol was used in all cases. The median was accomplished in 3 patients. In two cases the healing period between the implant insertion and fibular graft was used to reconstruct the mandible. functional loading was 15.1 months (range 4.3 to In one patient the maxilla was reconstructed with 54.3 months). Three different attachment systems the iliac crest bone graft. for implant-supported dentures were used: 39 The Kaplan-Meier method was utilized to es-Locator attachments (Zest Anchors, Escondido, timate the implant survival rate. The association USA) (Figure 1), 40 prefabricated conical crowns between the survival and the potential prognostic (SynCone, Dentsply Friadent, Mannheim, FIGURE 2. A patient after surgical treatment of oropharyngeal carcinoma. Segmental resection of the left mandible body is visible on the radiograph (A). Patient received implant-supported lower and upper dentures (B) with retention based on the SynCone double crowns (C). FIGURE 3. The Kaplan-Meier curve for the survival of the implants. 95% confidence intervals and censored data are included on the plot. TABLE 2. Predictors of the implant failure Patient’s age at implant 1.05 1.0000 insertion in years (0.99–1.12) Time interval between the 0.99 radiation therapy and the 1.0000 (0.92–1.07) implant insertion in years 2.74 Female gender 1.0000 (0.82–9.10) 0.72 Smoker 1.0000 (0.19–2.66) 0.84 Lower jaw 1.0000 (0.23–3.09) 12.37 Transplanted bone 0.0003 (3.87–39.56) 0.31 HBO administered 0.4753 (0.10–0.98) Germany) (Figure 2), and 10 custom designed bar-clip systems. Median follow up after implant inser­tion was 61.9 months (range 1.4 to 90.2 months). The Kaplan-Meier estimated 1- and 5-year cu­mulative implant survival rates were 96% (95% confidence interval: 92.2%–99.9%) and 87.0% (95% confidence interval: 80.4–94.2%). The survival curve is shown on Figure 3. During the examina­tion period, three patients died. Time of these 14 implants’ service in the mouth was, as with other implants, registered from the date of the implant insertion to the date of the last follow-up examina­tion. The median time of failure was 19.1 months (range 1.4 to 48.5 months) after implantation. The crude survival rate in our sample was 88%, as 12 implants in 5 patients failed. Data for all the implants and for the subgroups of survived and failed implants are described in Table 1. Primary implant failure during the healing period before functional loading was recorded in 11 implants (91.2%). The causes of implant removal in our sample were incomplete osseointegration (4), per­sistent pain (4), and periimplantitis with recurrent soft tissue hyperplasia (3). The only implant that was lost after functional loading (secondary im­plant failure) was included in a bar-supported den­ture and had to be removed because of periimplan­titis. Results of the analysis with the Cox regression models is presented in Table 2. The survived and failed implants were com­parable considering the patient’s gender, age and smoking status, the time elapsed between the ra­diation therapy and the implant surgery, the jaw of the implant insertion and the administration of HBO. The results for the bone type indicated that the implants inserted in the transplanted bone were statistically significantly more likely to fail than those inserted in the native bone. The influ­ence of the implant system on the survival could not be analysed because of the insufficient number of failure events among the three groups. As pre­sented in Table 3, 89 implants were observed after functional loading. Seventy-nine of those implants (88.7%) were suc­cessful, meaning that they were functionally loaded and exhibited no pain, radiolucency or progressive TABLE 3. Summary of the loaded implants’ success according to the factors of favourable outcome. Two patients with 4 implants interest experienced difficulties in adapting to dentures and did not wear them on regular basis. In addition, two further implants were considered unsuccess­ful because of persistent soft tissue discomfort re­ported by the patients. As shown in Table 4, patient age was a statistically significant predictor for the success. Gender, smoking status, healing time after Median healing time after the implant insertion, the number of implants support­ 15.1 15.2 13.0 implant insertion in months (4.3–54.4) (4.3–54.4) (4.3–24.6) ing the denture, the prosthetic system, jaw, bone (range) type and administered HBO did not have a statisti­ Patient’s gender Female, n (%) 33 (37%) 25 (32%) 8 (80%) cally significant effect on the success rate of loadedMale, n (%) 56 (63%) 54 (68%) 2 (20%) implants in this sample of irradiated patients. Smoking 57 (64%) 49 (62%) 8 (80%)No, n (%) 32 (36%) 30 (38%) 2 (20%)Yes, n (%) Median number of the Discussion implants supporting the 4 4 denture 4 (2–5) (2–5)(range) It has been shown in this study that the implant- Implant denture system supported denture is a reliable treatment modality Bar, n (%) 10 (11%) 6 (8%) 4 (40%) Locator, n (%) 39 (44%) 37 (47%) 2 (20%) for the head and neck cancer patients that undergo SynCone, n (%) 40 (45%) 36 (45%) 4 (40%) radiation therapy. When surgical and prosthodon-Jaw tic protocols are conducted appropriately, dental Upper, n (%) 25 (28%) 21 (27%) 4 (40%) Lower, n (%) 64 (72%) 58 (73%) 6 (60%) implants greatly enhance the stability of the den- Bone tures and improve the facial contours. According Native, n (%) 86 (97%) 76 (96%) 10 (100%) Transplanted, n (%) 3 (3%) 3 (4%) 0 (0%) to the current guidelines, very few absolute con- HBO administered traindications exist for using dental implants in Yes 75 (84%) 65 (82%) 10 (100%) medically compromised patients.18 Radiation No 14 (16%) 14 (18%) 0 (0%) therapy in the head and neck region is no longer a contraindication, as there is a growing number of reports that a high osseointegration rate and a predictable treatment outcome can be expected.19,20 When considering prosthetic treatment options, the socio-economic status of patients should care­fully be evaluated. Head and neck cancer is com­ (0.49–0.80) monly associated with smoking and alcohol abuse. Healing time after the implant insertion 1.09 1.0000 Both tobacco and alcohol are known as strong risk (0.53–2.73) factors and when combined, their carcinogenic Male gender 8.64 0.1456 (1.99–60.09) potential has been shown to be even more pro- Smoker 2.44 1.0000 nounced.21,22 Many patients do not give up smok­ (0.57–16.95) ing and drinking after the initial cancer treatment, Number of the implants supporting the 0.78 1.0000 denture (0.24–1.94) which puts them at risk for cancer recurrence and Denture attachment system might also jeopardize the implant-prosthetic reha-Locator vs bar 12.33 0.1456 bilitation outcome. (1.98–104.98) SynCone vs bar 6.00 0.4416 In this case series of irradiated patients, the im­(1.15–32.68) plant failures were rare and mostly confined to the SynCone vs Locator 0.48 1.0000 (0.06–2.65) healing period. The predominantly early implant Upper jaw 0.54 1.0000 loss is in accordance with the findings of Linsen et (0.14–2.30) al.23 Extended healing time should therefore be al­lowed after implantation and immediate loading protocols are not advised.18 bone loss evident from radiographs and probing The Kaplan-Meier estimated 1- and 5-year cu­depth at yearly recall. Regardless of the appropriate mulative implant survival rates were 96% and 87%. osseointegration, prosthetic rehabilitation of 10 im-This is in accordance with Buddula et al.24 report-plants (11.2%) in four patients (20%) did not have a ing implant survival rates of 98.9% and 89.9% af-ter 1 and 5 years and Yerit et al.25 reporting a 95% and 91% survival after 2 and 5 years, respectively. Due to the small number of failed implants, de­tailed statistical analysis of prognostic factors for implant failure is often not possible or lacks power. It is therefore difficult to draw meaningful conclu­sions from the results of single studies alone. In this study, some limited insight could be obtained regarding the survival of implants. There was a sta­tistically significant higher failure rate in the trans­planted bone. The reduced survival of implants in the transplanted bone may be explained by differ­ences in bone quality, bone volume, and revascu­larization compared to the native bone.9 Our find­ings are in agreement with Yerit et al.25, where low­er survival was also reported for the transplanted bone. In contrast, Buddula et al.24 reported no difference between implant survival in the native and transplanted bone. They also reported no dif­ference in survival between genders and consider­ing the time span between radiation treatment and implant insertion, which is in accordance with this study. Their finding of the statistically significant higher hazard ratio for implants in the upper jaw could not be confirmed in this study. The risk for implant failure is generally higher in smoking patients, as shown in the systematic re­view papers by Chambrone et al. and Moraschini and Barboza.26,27 The smoking status was not a sta­tistically significant predictor for the implant sur­vival in this study, but the effect might have been detected with a larger sample size and more pre­cise smoking classification. It is also notable that the risk for implant failure in smokers was sug­gested to be significantly elevated only for a lim­ited time after surgery, presumably when tobacco smoke components impede bone healing.27 HBO, which is commonly used at the University Medical Centre Ljubljana for the head and neck cancer patients requiring surgery, was also not a statistically significant predictor for the implant survival in this study. Generally, there is no agree­ment on the HBO efficacy and value.28 Implant osseointegration and survival data pro­vide valuable information about the success of the implant therapy. Nevertheless, the final judgement of the implant-prosthetic therapy should be made according the denture performance in the oral cav­ity. Successful implants enable patients to use the dentures and do not cause any persistent discom­fort. To achieve a favourable clinical outcome, it is crucial to design a viable prosthetic plan early in the rehabilitation process. Head and neck cancer patients present severely altered and unfavour­able tissue conditions, making it challenging to model rehabilitation using the optimal top-down approach. The fragile mucosa, xerostomia, limited mouth opening and jaw deviations are additional factors to consider. The possible locations, angula­tions and implant dimensions might not be ideal and should be discussed thoroughly between the oral surgeon and the prosthodontist. The optimal number of implants should be care­fully planned. There is a tendency to insert as few implants as possible in oncological patients, to fa­cilitate bone healing. On the other hand increased number is often required to design rigid, implant-borne prosthetic constructions.18 More implants also allow more flexibility in prosthetic treatment planning. Moreover, if some implants are lost, im­plant dentures can be successfully repaired and worn by the patients. It is currently thought that the number of implants is not critical for the suc­cess of the prosthetic treatment29, but long-term clinical studies are lacking. In the present study the number of implants supporting the dentures was not a detectable factor in the success rate of the prosthodontic rehabilitation. Similarly, neither was the treated jaw or the healing time, which exceeded 4 months in all implants. The loaded implants also exhibited similar suc­cess rates regardless of the denture attachment sys­tem used. While the bar-supporting implants expe­rienced less success, the differences in comparison to the Locator attachments and SynCone systems were not significant. Additional systemic and patient-related factors might play an important role in implant prosthetic rehabilitation. In this case series of irradiated pa­tients, advanced age showed a negative prognos­tic value for the rehabilitation success, but not for implant survival. One of the possible limitations of this study was that the data on systemic diseases and alcohol consumption which might negatively affect implant performance were not included. After the implant-prosthetic rehabilitation, it is of utmost importance to enrol the patient in an appro­priate supportive program, with regular recalls, cancer screening and maintenance of the peri-im­plant conditions. The recall program should meet the individual needs of the patients according to the overall risk profile. Some patients should be re­called every 3 months, while others may need to be checked once per year.30 This study has shown that favourable rehabili­tation results can be obtained with implant-pros­thetic treatment in irradiated patients. With proper collaboration between experienced surgeons and 100 prosthodontists, this treatment modality can be re­garded a viable option for oral rehabilitation after head and neck cancer. References 1. De Camargo Cancela M, Voti L, Guerra-Yi M, Chapuis F, Mazuir M, Curado MP. Oral cavity cancer in developed and in developing countries: popula­tion-based incidence. Head Neck 2010; 32: 357-67. 2. De Camargo Cancela M, de Souza DL, Curado MP. International incidence of oropharyngeal cancer: a population-based study. Oral Oncol 2012; 48: 484-90. 3. Cancer in Slovenia 2011. Ljubljana: Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Cancer Registry of Republic of Slovenia; 2015. 4. Marx RE. Osteoradionecrosis: a new concept of its pathophysiology. J Oral Maxillofac Surg 1983; 41: 283-8. 5. Larsen PE. Placement of dental implants in the irradiated mandible: a protocol involving adjunctive hyperbaric oxygen. J Oral Maxillofac Surg 1997; 55: 967-71. 6. Fierz J, Bürgin W, Mericske-Stern R. Patients with oral tumors. Part 2: Quality of life after treatment with resection prostheses. Resection prosthetics: evaluation of quality of life. Schweiz Monatsschr Zahnmed 2013; 123: 180-91. 7. Brauner E, Cassoni A, Battisti A, Bartoli D, Valentini V. Prosthetic rehabilita­tion of post-oncological patients: Report of two cases. Ann Stomatol 2010; 1: 19-25. 8. Carini F, Pisapia V, Monai D, Barbano L, Porcaro G. Implant rehabilitation in patients irradiated for head and neck cancer: role of Intensity-Moduled Radiotherapy (IMRT) in planning the insertion site. Ann Stomatol 2012; 3: 8-20. 9. Schiegnitz E, Al-Nawas B, Kämmerer PW, Grötz KA. Oral rehabilitation with dental implants in irradiated patients: a meta-analysis on implant survival. Clin Oral Investig 2014; 18: 687-98. 10. Andreiotelli M, Att W, Strub JR. Prosthodontic complications with implant overdentures: a systematic literature review. Int J Prosthodont 2010; 23: 195-203. 11. Kleis WK, Kämmerer PW, Hartmann S, Al-Nawas B, Wagner W. A compari­son of three different attachment systems for mandibular two-implant over-dentures: one-year report. Clin Implant Dent Relat Res 2009; 13: 209-218. 12. Mackie A, Lyons K, Thomson WM, Payne AG. Mandibular two-implant overdentures: three-year prosthodontic maintenance using the locator at­tachment system. Int J Prosthodont 2011; 24: 328-31. 13. Troeltzsch M, Troeltzsch V, Brodine AH, Frankenberger R, Messlinger K, Troeltzsch M. Clinical performance and peri-implant parameters of 132 implants supporting locator-retained overdentures: a case series of 33 patients. Int J Oral Maxillofac Implants 2013; 28: 1132-9. 14. May D, Romanos GE. Immediate implant-supported mandibular overden­tures retained by conical crowns: A new treatment concept. Quintessence Int 2002; 33: 5-12. 15. Bernhart G, Koob A, Schmitter M, Gabbert O, Stober T, Rammelsberg P. Clinical success of implant-supported and tooth-implant-supported double crown-retained dentures. Clin Oral Investig 2012; 16: 1031-7. 16. Van Steenberghe D. Outcomes and their measurement in clinical trials of endosseous oral implants. Ann Periodontol 1997; 2: 291-8. 17. R Development Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2014. [Citated 15 Nov 2015]. Available at http://www.gbif.org/resource/81287 18. Diz P, Scully C, Sanz M. Dental implants in the medically compromised pa­tient. J Dent 2013; 41: 195-206. 19. Ihde S, Kopp S, Gundlach K, Konstantinović VS. Effects of radiation therapy on craniofacial and dental implants: a review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 107: 56-65. 20. Tanaka TI, Chan HL, Tindle DI, Maceachern M, Oh TJ. Updated clinical con­siderations for dental implant therapy in irradiated head and neck cancer patients. J Prosthodont 2013; 22: 432-8. 21. Tobias JS. Cancer of the head and neck. BMJ 1994; 308: 961-6. 22. Cognetti DM, Weber RS, Lai SY. Head and neck cancer: an evolving treat­ment paradigm. Cancer 2008; 113: 1911-32. 23. Linsen SS, Martini M, Stark H. Long-term results of endosteal implants following radical oral cancer surgery with and without adjuvant radiation therapy. Clin Implant Dent Relat Res 2012; 14: 250-8. 24. Buddula A, Assad DA, Salinas TJ, Garces YI, Volz JE, Weaver AL. Survival of dental implants in irradiated head and neck cancer patients: a retrospective analysis. Clin Implant Dent Relat Res 2012; 14: 716-22. 25. Yerit KC, Posch M, Seemann M, Hainich S, Dörtbudak O, Turhani D, et al. Implant survival in mandibles of irradiated oral cancer patients. Clin Oral Implants Res 2006; 17: 337-44. 26. Chambrone L, Preshaw PM, Ferreira JD, Rodrigues JA, Cassoni A, Shibli JA. Effects of tobacco smoking on the survival rates of dental implants placed in areas of maxillary sinus floor augmentation: a systematic review. Clin Oral Implants Res 2014; 25: 408-16. 27. Moraschini V, Barboza ED. Success of dental implants in smokers and non­smokers: a systematic review and meta-analysis. Int J Oral Maxillofac Surg 2015; doi: 10.1016/j.ijom.2015.08.996 28. Spiegelberg L, Djasim UM, van Neck HW, Wolvius EB, van der Wal KG. Hyperbaric oxygen therapy in the management of radiation-induced injury in the head and neck region: a review of the literature. J Oral Maxillofac Surg 2010; 68: 1732-9. 29. Roccuzzo M, Bonino F, Gaudioso L, Zwahlen M, Meijer HJ. What is the optimal number of implants for removable constructions? A systematic review on implant-supported overdentures. Clin Oral Implants Res 2012; 23: 229-37. 30. Lang NP, Lindhe J. Clinical Periodontology and Implant Dentistry. Chichester: John Wiley & Sons; 2015. 101 research article Determination of dosimetric parameters for shielded 153Gd source in prostate cancer brachytherapy Mahdi Ghorbani1, Benyamin Khajetash2, Najmeh Ghatei3, Mohammad Mehrpouyan4, Ali S. Meigooni5, Ramin Shahraini4 1 Biomedical Engineering and Medical Physics Department, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 Medical Physics Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 3 Radiotherapy Department, Namazi Hospital, Shiraz, Iran 4 Radiology and Radiotherapy Department, Faculty of Medicine, Sabzevar University of Medical Sciences, Sabzevar, Iran 5 Comprehensive Cancer Centers of Nevada, Las Vegas, Nevada, USA Radiol Oncol 2017; 51(1): 101-112. Received 22 June 2016 Accepted 27 December 2016 Correspondence to: Behyamin Khajetash, Medical Physics Department, School of Medicine, Iran University of Medical Sciences, Hemmat Highway, Tehran, Iran.E-mail: benyamin.khajetash@gmail.com and Mohammad Mehrpouyan, Radiology and Radiotherapy Department, Faculty of Medicine, Sabzevar University of Medical Sciences, Sabzevar, Iran. E-mail: mehrpouyan.mohammad@gmail.com Disclosure: No potential conflicts of interest were disclosed. Both Corresponding authors have equal benefits Background. Interstitial rotating shield brachytherapy (I-RSBT) is a recently developed method for treatment of prostate cancer. In the present study TG-43 dosimetric parameters of a 153Gd source were obtained for use in I-RSBT. Materials and methods. A 153Gd source located inside a needle including a Pt shield and an aluminum window was simulated using MCNPX Monte Carlo code. Dosimetric parameters of this source model, including air kerma strength, dose rate constant, radial dose function and 2D anisotropy function, with and without the shields were cal­culated according to the TG-43 report. Results. The air kerma strength was found to be 6.71 U for the non-shielded source with 1 GBq activity. This value was found to be 0.04 U and 6.19 U for the Pt shield and Al window cases, respectively. Dose rate constant for the non-shielded source was found to be 1.20 cGy/(hU). However, for a shielded source with Pt and aluminum window, dose rate constants were found to be 0.07 cGy/(hU) and 0.96 cGy/(hU), on the shielded and window sides, respectively. The values of radial dose function and anisotropy function were tabulated for these sources. Additionally, isodose curves were drawn for sources with and without shield, in order to evaluate the effect of shield on dose distribution. Conclusions. Existence of the Pt shield may greatly reduce the dose to organs at risk and normal tissues which are located toward the shielded side. The calculated air kerma strength, dose rate constant, radial dose function and 2D anisotropy function data for the 153Gd source for the non-shielded and the shielded sources can be used in the treatment planning system (TPS). Key words: interstitial rotating shield brachytherapy; 153Gd; TG-43 dosimetric parameters; Monte Carlo simulation Introduction Prostate cancer is the most common cancer in men. In 2013, it was reported that the number of 2,850,139 men were living with prostate cancer in the United States.1 The number of new cases of prostate cancer from the year 2009 to 2013, was 129.4 per 100,000 men per year and the number of deaths caused by prostate cancer was 20.7 per 100,000 men per year.1 This cancer is observed with a higher prevalence in African-American men than in white men.2 Brachytherapy is one of the com­ 102 mon therapeutic methods for treatment of prostate cancer. This approach has shown successful out­comes in treatment of this cancer. The success of brachytherapy is due to advantages such as sim­ple performance and reduced side effects of treat­ment compared to external radiation therapy and surgical removal of the tumor.3-6 Brachytherapy is a treatment in which one or a number of covered radioactive sources are inserted at a short distance from the target. Brachytherapy is performed either as intracavitary procedure, in which the brachy­therapy source is placed inside the natural body cavity, adjacent to the tumor. The second method is interstitial brachytherapy, in which brachyther­apy seeds are implanted directly inside the tumor mass. With this treatment method, the required amounts of radiation dose can be delivered to the tumor with the rapid dose fall off to the healthy tis­sues around the tumor. Originally, brachytherapy treatments were per­formed with 226Ra sources. Nowadays, the use of artificial radionuclides such as 125I, 131Cs, and 103Pd is rapidly increasing.7-9 Presently, brachytherapy sources are widely used for treatment of various types of cancer patients. 153Gd is a medium ener­gy brachytherapy source emitting photons in the range of 40 to 100 keV. This isotope has a low dose rate and its half-life is 242 days. Interstitial rotating shield brachytherapy (I-RSBT) is a type of brachy­therapy in which a shield is used to spare normal tissues from radiation damage. The rotating capa­bility enables the user to select an emission angle to direct the radiation to a specified tumor. It can also be used in sequencing the rotating shield in dynamic rotational shield brachytherapy. With I-RSBT it is possible to have a significant dose re­duction in urethra while delivering higher prostate organ dose. The main reason for the use of 153Gd in I-RSBT method is that it requires less shielding within the source than other higher energy iso­topes such as iridium. The main goal of any radiation therapy treat­ment is to maximize the dose to the tumor while limiting the dose to adjacent normal tissue and organs. Therefore, shielding the sensitive organs around the target volume has always been one of the important issues. In brachytherapy of prostate and cervix, the sensitive organs such as the rectum and bladder should be protected to receive the ra­diation dose lower than their tolerance level. For the purpose (I-RSBT), the use of special cylindri­cally shielded source can reach the aim. The shield there is a platinum sleeve with an aluminium gap leaking the narrow source beam. The difficulty of manufacturing of a shielded source is in its limit­ed size. Namely, the cylindrical source should be capable to enter the hollow brachytherapy needle with the diameter e.g. 1.6 gauge. Energetic sources (Ir-192) need more shielding thickness which pre­vents them entering the small diameter brachy­therapy needles. The report by task group No. 43 (TG-43) from American Association of Physicists in Medicine is known as the most common formalism for calcula­tion of the dose distribution around brachytherapy sources. According to this protocol, dosimetric parameters around brachytherapy sources are ob­tained by experimental measurement or simulation techniques using the Monte Carlo codes in a uni­form water phantom.10-11 Monte Carlo is a compu­tational technique for solving problems in various fields including physical and mathematical scienc­es. This method is based on random sampling to achieve the required results. Different Monte Carlo codes are currently used in medical radiation phys­ics: MCNP, EGSnrc, GEANT4, SIMIND, etc. Monte Carlo N-Particle (MCNP) is specially designed for nuclear application of radiation transport but it also can be used for other fields, for example in medical physics applications. Monte Carlo codes are of high capabilities in particle transport physics lacking applications in practical medical physics dosimetric problems.12-14 These methods can have desired results to assess dosimetric parameters such as air kerma strength, dose rate constant, radial dose function and anisotropy function of brachytherapy sources.15,16 In a number of previ­ous studies, dosimetric parameters of hypotheti­cal brachytherapy sources were calculated prior to their fabrications in order to assess the feasibility of its fabrication. One of these sources is 153Gd that has been introduced by Enger et al.17 Additionally, 153Gd radionuclide has been recently used in nu­clear medicine imaging systems e.g. linear scanners and quality assurance procedures such as phantom calibrations. In radiotherapy the radiation dose destroys the tumor cells causing the healing of cancer and improving the patient’s quality of life. There is a need in prostate brachytherapy to reduce the dose to radiosensitive organs such as rectum and blad­der reducing the dose side effects to these sensitive organs. Therefore, in a proper patient treatment plan the amount of absorbed dose to critical organs should be exactly specified and controlled.18 Interstitial rotating shield brachytherapy is a new type of high dose rate brachytherapy in which the radiation dose is delivered by shielded TABLE 1. The energy spectrum of 153Gd radionuclide.18 In these data, the energy values were rounded to two decimal places 5.18 0.374 5.82 0.976 5.82 0.1341 5.85 8.86 6.44 0.559 6.46 5.64 6.57 0.9214 6.62 0.0845 6.84 1.8512 7.48 0.947 7.77 0.173 7.79 0.244 14.06 0.0183 21.20 0.0224 40.47 0.00953 40.90 35.29 41.54 63.516 46.90 6.2516 47.04 12.13 47.37 0.1848 48.25 4.001 48.39 1.597 54.19 40.01622 69.67 2.41923 75.42 0.078323 83.37 0.1964 89.49 0.0694 96.88 0.0022 97.43 29 103.18 21.1123 118.11 0.000121 166.56 0.00033 172.30 0.00022 172.85 0.036017 with 62 GBq activity and by dividing a quantity of this source to 62 GBq it is feasible to calculate that quantity per GBq. The active core is surrounded by 75 µm titanium capsule with a density of 4.506 g/ cm3. At one side of the titanium capsule there is a window of pure aluminum with 2.70 g/cm3 density and the thickness of 75 µm. On the other side of the source construction, there is a Pt shield which is an alloy of 90% Pt and 10% Ir expressed in weight percentages. The density of this shield is 21.45 g/ cm3. The radioactive source is within Nitnol cath­eter and a Nitinol needle (Figure 1). These are composed of Ni and Ti with 55.6% and 44.4% per­centage weight fractions, respectively. The density of the catheter and needle was considered as 6.54 g/cm3. The outer diameter of the needle is 1.651 mm. Figure 1 illustrates transverse and longitu­dinal views of the 153Gd source, catheter, and the needle including the Pt shield and Al windows. It should be noticed that some of the dimensions in this Figure are based on the study by Adams et al.19, while the others are based on assumptions. In other words, we assumed all the dimensions which were not directly reported in the study by Adams et al. The photon spectrum of the 153Gd radionuclide is listed in Table 1.21 Calculation of dosimetric parameters According to TG-43U1 report10, dose distribution around a brachytherapy source can be obtained us­ing the following equation: [1] In this equation the SK, ., G(r, .), g(r) and F(r, .), are air kerma strength, dose rate constant, geom­etry function, radial dose function and anisotropy function. 105 The TG-43U1 quantities agenda are as follows: TABLE 2. Radial dose function values for the non-shield and shield modes. The shield MCNPX (version 2.6.0) Monte Carlo simulation mode is related to the Pt and Al window sides code was used to calculate the TG-43 dosimetric parameters of the 153Gd source. The parameters including air kerma strength, dose rate constant, radial dose function and anisotropy function were calculated for the source with the shield and with-0.5 0.90 0.52 0.93 out it. 1 1.00 1.00 1.00 To calculate the air kerma strength in the non­ 1.5 1.06 1.39 1.04 shielded mode, the source was assumed to be 2 1.11 1.73 1.04 inside a vacuum sphere with a radius of 100 cm. 2.5 1.13 2.03 1.07 Then, the spherical tally cells were placed at dis­ tances ranging from 1 to 40 cm from the source 3 1.18 2.22 1.07 center at 1 cm intervals. These spheres were made 3.5 1.14 2.44 1.07 of air and their radii were defined according to a 4 1.19 2.72 1.05 joint report from AAPM and European Society for 5 1.17 2.65 1.02 Therapeutic Radiology and Oncology (ESTRO).22 8 1.01 3.01 0.91 The amount of air kerma was scored by F6 tally 10 0.89 2.63 0.80 (in terms of MeV/g). Throughout this project the energy cut off for photons and electrons was de-12 0.77 2.44 0.71 fined as 1 keV. Each simulation was performed for 1.4 × 109 particles and the maximum type A uncer­tainty of Monte Carlo simulation in these calcula­tions was 3.08%. The uncertainty quantity in tally calculation is normally listed in the output file of a Monte Carlo program. For the shielded source, the implementation of the program and the calculation of air kerma strength was similar to the non-shield­ed source. In calculation of air kerma strength for the Pt shield case, the scoring spheres were defined on the Pt shield side. In calculation for the Al win­dow case, the scoring spheres were defined on the Al window side. The maximum type A uncertain­ties of Monte Carlo simulation calculations in the calculation of air kerma strength for the Pt shield and Al window cases were 32.78% and 3.24%, re­spectively. Calculation of dose rate constant was performed based on the formalism presented in the TG-43 re­port. In the non-shield mode, the 153Gd source was defined in a water sphere with 100 cm radius and a spherical water tally cell with 0.005 cm radius was defined at 1 cm distance from the source. Then, *F4 tally was calculated in combination with mass en­ergy absorption coefficient in order to score the en­ergy deposition per mass. Mass energy absorption coefficients of water listed in Table 4 of National Institute of Standards and Technology (NIST) web-page were used.23 This program was run for 1.2 × 109 particles and the type A Monte Carlo uncertain­ty was obtained as 0.34% that is related to statistical fluctuation of the simulation data. By dividing the absorbed dose rate to air kerma strength, the dose rate constant was achieved. In the shielded source FIGURE 3. (A) Radial dose function for the 153Gd source without the shield and with the shield on Al window side (B) radial dose function for the shielded source at Pt shield side. 106 cases the calculations were performed for both sides of source. In the calculation of dose rate con­stant in each case, the dose rate at 1 cm was divided by the air kerma strength for the non-shielded case. The type A uncertainties in Monte Carlo simula­tions were 0.37% and 1.50% for the calculation at Al window and Pt shield sides, respectively. The other details of the simulations for the shielded source were the same as the simulation details for the non-shielded source. Radial dose function for the non-shielded source was calculated by selecting a spherical water phan­tom with 100 cm radius and placing spherical wa­ter tally cells at distances ranging from 0.5 cm to 12 cm with 0.5 cm intervals, relative to the source center. The radii of these tally cells were defined according to the report by AAPM and ESTRO22, in other words, for distances less than 1 cm the radius of the spheres was considered as 0.005 cm, and for distances 1 cm . r<5 cm the radius was considered as 0.025 cm and for distances 5 cm . r< 10 cm the radius was considered 0.05 cm and finally for dis­tances more than 10 cm the radius was considered to be 0.1 cm. In this program *F4 tally was scored and the tally outputs in various energy bins were multiplied by mass energy absorption coefficients using DE and DF cards in MCNP code. Based on the MCNP manual, “DE” and “DF” stand for dose energy and dose function, respectively. In DE cards, energy bins are defined, while in DF card mass energy absorption coefficients are intro­duced. The program was run for 1.2 × 109 particles and the maximum type A Monte Carlo uncertainty was 1.81%. A similar program was defined for the shielded case so that the source was defined in a water sphere with 100 cm radius and also the same intervals and water spheres were defined. Unlike the previous case in this program, spheres were de­fined on both sides of the source. In this program the output data were obtained using *F4 tally and “DE” and “DF” cards. This program was run for 1.2 × 109 particles. The maximum Monte Carlo un­certainty in these investigations in the non-shield­ed case was observed as 2.07% and in the case of the Pt shield it was observed as 4.49%. It can be considered that the values of these uncertainties are different at different parts of the simulation, and it is because the uncertainty depends on the complexity of geometry, tally type used, number of particle histories, etc. To calculate the anisotropy function in the case of non-shielded source, a water sphere with 100 cm radius was defined as the phantom. In order to ob­tain anisotropy function, spheres were defined at different radial distances and angles. These spheres were defined at distances of 0.5, 1, 2, 3, 5, 10 and 12 cm. The angles were ranging from 0 to 180 degrees with 10 degrees intervals. The energy flux was scored in these spheres using *F4 tally. In the cal­culation of this program mass energy absorption coefficient was utilized. The radius of each sphere was considered according to the report by AAPM and ESTRO. This program had been run for 2 × 109 photons and the maximum uncertainty in the Monte Carlo calculation was observed as 1%. The calculations defined for the shielded source were performed similar to the non-shielded source. The difference was that the spheres were on both sides (Pt shield side and Al window side). The uncertain­ty in the calculation in this case was observed less than 5%. Polar angles that were used to calculate the anisotropy function of the Pt shield case are il­lustrated in Figure 2. Based on this Figure zero angle begins from the tip of the source for the Pt shield case. For the rest of the cases (nonshielded source and the Al window case) the polar angles are likewise. Results Air kerma strength per source activity (per GBq) for the non-shielded mode was obtained equal to 6.71 cGyh-1cm2GBq-1. Based on this value for the nonshielded 153Gd source, 1 U is equal to 4.03 mCi. This value was found to be 0.04 cGyh-1cm2GBq-1 107 TABLE 3. Anisotropy function values for the 153Gd source at different distances for the non-shielded mode 0 0.71 0.59 0.48 0.41 0.29 - 10 0.75 0.63 0.58 0.51 0.44 0.75 20 0.82 0.76 0.69 0.64 0.63 0.81 30 0.86 0.83 0.77 0.75 0.76 0.86 40 0.90 0.85 0.85 0.84 0.87 0.91 50 0.95 0.91 0.90 0.90 0.93 0.94 60 0.95 0.98 0.94 0.94 0.96 0.96 70 1.00 0.96 0.97 0.98 0.98 0.97 80 0.99 0.99 0.97 0.98 1.01 1.00 90 1.00 1.00 1.00 1.00 1.00 1.00 100 0.97 0.97 0.99 1.00 1.01 1.00 110 1.01 0.95 0.97 0.97 0.96 0.97 120 0.97 0.95 0.92 0.94 0.96 0.97 130 0.95 0.93 0.89 0.90 0.93 0.94 140 0.92 0.87 0.85 0.84 0.87 0.90 150 0.88 0.80 0.78 0.76 0.79 0.87 160 0.83 0.74 0.68 0.65 0.63 0.81 170 0.76 0.65 0.56 0.50 0.45 0.75 180 0.69 - - - - - and 6.19 cGyh-1cm2GBq-1 for the Pt shield and Al window cases, respectively. The values of dose rate constant for the non-shielded mode was obtained as 1.20 cGyh-1U-1 and for the shielded mode for the Pt shield and Al window sides were achieved as 0.07 cGyh-1U-1 and 0.96 cGyh-1U-1, respectively. It should be noted that in calculation of dose rate constant for these three cases the dose rate at the reference point (at 1 cm and 90 degrees angle) in each case was divided by the air kerma strength value for the non-shielded source. It can be noted that in the shielded case the dose rate constant is reduced. Radial dose function values for 153Gd source are listed in Table 2. The first column there refers to the source without shield, the second column re­fers to the Pt shield side of the source and the third column refers to the Al window side of the source. The radial dose function values are plotted in the Figure 3B for the Pt shield side. Figure 4 shows the relative dose for the non-shielded mode of the source, the Pt shield and Al window sides of the shielded source. In this graph, for all three cases, the dose values have been normalized to the dose at 1 cm distance of the non-shielded source. Anisotropy function values for the 153Gd source were calculated for 0.5, 1, 2, 3, 5, 10 and 12 cm dis­tances from the source at 19 different angles (rang­ing from 0 to 180 degrees) with angular intervals of 10 degrees. The results of anisotropy function are presented in Table 3 and Table 4 for the non-shield­ed and shielded cases, respectively. Figure 5A also shows anisotropy function values at different dis­tances from the source for non-shielded mode. In 108 TABLE 4. Anisotropy function values for the 153Gd source at different distances for the shielded mode at the Pt and Al windows sides 0 0.42 0.39 0.30 - - - - 1.80 1.78 1.56 - - - - 10 0.51 0.52 0.42 0.35 0.31 0.29 0.76 1.54 1.57 1.38 1.32 1.22 0.35 - 20 0.62 0.61 0.58 0.51 0.50 0.55 0.81 1.41 1.36 1.23 1.21 1.12 0.88 0.38 30 0.72 0.71 0.72 0.66 0.66 0.70 0.85 1.24 1.31 1.18 1.17 1.10 0.87 0.56 40 0.81 0.81 0.79 0.77 0.78 0.81 0.90 1.15 1.23 1.14 1.07 1.07 1.05 0.77 50 0.88 0.88 0.87 0.86 0.87 0.90 0.93 1.09 1.14 1.07 1.08 0.98 0.86 0.84 60 0.93 0.91 0.95 0.92 0.94 0.97 0.96 1.05 1.07 0.98 1.06 1.05 0.91 0.94 70 0.98 0.96 0.99 0.97 0.98 1.01 0.98 1.02 1.07 0.91 1.04 0.99 1.03 1.00 80 0.99 0.99 1.00 1.00 1.00 1.06 0.99 1.02 1.01 0.98 0.99 0.96 1.00 1.00 90 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 100 0.98 0.99 1.00 0.98 1.00 1.02 1.00 0.96 1.01 1.01 1.04 0.99 1.03 1.00 110 0.96 0.97 0.98 0.97 0.99 0.97 0.98 0.96 1.07 0.96 1.01 1.04 0.99 0.95 120 0.93 0.93 0.95 0.92 0.94 0.96 0.97 1.08 1.09 1.02 1.07 1.01 1.08 0.82 130 0.88 0.89 0.90 0.88 0.88 0.93 0.93 1.13 1.24 1.03 1.07 1.09 0.95 0.84 140 0.82 0.83 0.81 0.77 0.78 0.84 0.89 1.18 1.21 1.14 1.12 1.07 1.04 0.74 150 0.73 0.70 0.69 0.67 0.66 0.71 0.86 1.27 1.29 1.16 1.15 1.13 0.87 0.63 160 0.63 0.62 0.56 0.52 0.50 0.54 0.81 1.45 1.53 1.23 1.33 1.21 0.89 0.37 170 0.53 0.49 0.43 0.36 0.33 0.30 0.75 1.59 1.57 1.47 1.51 1.38 1.06 - 180 0.43 0.41 - - - - - 1.83 1.89 - - - - - TABLE 5. Mass attenuation coefficient (µ/.) of Pt, Al and Ti in the 40 keV-100 keV energy range 21 40 12.45 0.57 2.21 50 6.95 0.37 1.21 60 4.34 0.28 0.77 80 8.73 0.20 0.41 100 4.99 0.17 0.27 ed. In Figure 6 anisotropy function for three modes is presented. There are four graphs for distances of the evaluation point of the anisotropy function from the source: (A) 0.5 cm, (B) 2 cm, (C) 5 cm and (D) 12 cm distance. Isodose curves (%) for the non-shielded source and the shielded source (Pt shield side) are plot­ted in Figure 7. In these plots the dose distributions were normalized to the dose at 1 cm distance from the source at transverse plane. Figure 5A anisotropy function for the non-shielded source is presented, in Figure 5B anisotropy func­tion for the shielded source at Pt shield side and in Figure 5C anisotropy function for the shielded source at Al window side of the source is present- Discussion In this study TG-43 dosimetric parameters for a 153Gd source with Pt shield were calculated for use in I-RSBT of prostate brachytherapy. Air kerma strength value for the non-shielded 153Gd source was found to be 6.71 U per 1 GBq activity. 110 in terms of r×Pt density and the data be normal­ized to the dose rate at 1 cm physical distance from source center for the density corrected value. That way the trend of radial dose function may not be increasing. In the calculation of radial dose function in Figure 3, each case was normalized to the dose value at 1 cm of itself. Based on Figure 3A the ra­dial dose function in the Al window side is lower. This is due to higher photon attenuation in Al than in the water. Figure 3B, which corresponds to the Pt shield side, the radial dose function at the Pt shield side has increasing trend up to the distance of about 8 cm. In Figure 4 all the obtained dose val­ues in three cases (non-shielded, Pt shield, Al win­dow) were normalized to dose at 1 cm distance of the non-shield mode. With this calculation, there is evidence to have more accurate comparison. The graph in Figure 4 reveals the dependence of the source distance to the point of absorbed dose in the phantom. By increasing that distance the dose be­comes lower for all three cases. Secondly, the dose in the case of Pt shield side is lower than at the Al window side. Additionally the dose at the Al wid­ow source is lower than at the non-shielded source. These two effects are related to different absorp­tion coefficients in Pt, Al and phantom. Pt has high­er absorption coefficient than Al and Al has higher absorption coefficient than phantom (i.e water ). Figure 5A is related to the non-shielded source. Figure 5B is related to the Pt window source. In case of Pt window that side of the source receive lower dose. It indicates that Pt side should be turned to the side of organs at risk which means that central areas of the organs will receive low­er dose. According to Figure 5A and C it can be mentioned that anisotropy at Al window mode is lower than at the non-shielded mode of the I-RSBT source. This is due to the attenuation of radiation in Al. This effect is similar for the comparison of anisotropy of Pt shield side versus non-shielded case. In directions other than 90o, beam traverses through longer path inside the needle and source, therefore the beam attenuation is more pro­nounced. The higher beam attenuation results to lower doses at these angles. Calculating the anisot­ropy function we normalized it to the dose at 90o. Thus the lower isotropy at these angles can be ex­plained. The lower isotropy in I-RSBT source is like a disadvantage, because it causes non-uniformity of tumor dose. The peripheral parts of the tumor receive systematically lower doses than the central ones. In the Figure 6, anisotropy function is further analyzed. We compared different shielding effects of the I-RSBT source at four different distances in the phantom. We compared the source without shield, Pt shielded source and the source with Al window. They were compared at each of the four distances (0.5 cm, 2 cm, 5 cm, and 12 cm). As it can be concluded from the charts, at 0.5 cm from the source the anisotropy functions for the non-shield­ed mode and Al Window mode are relatively the same. This is because of the similar magnitude of self-absorption in both modes. Increasing the dis­tance from the source there is a change of energy spectrum of photons in the phantom. The differ­ences become greater by increasing the distance 111 from the source. The reason for anisotropy func­tion equality at a distance of 0.5 cm for these two modes is their almost equal self-absorption. In Figure 7, isodose curves are plotted for both non-shielded and shielded cases. As it can be seen in the shielded source considerable dose reductions occur at the Pt shield side. Thus the organs at risk located near the shield side of the source receive significantly lower dose. Organs that are located at 1 cm distance from the source (Pt shield side) re­ceive 5% dose. This relative dose value is reduced further to 1% when the distance is approaching 4 cm. It should be noted that the shield should be turned to the side of organs at risk, such as the rec­tum and bladder. In prostate cancer the total organ volume is tumor which is covered with many ir­radiation source dwell positions. Thus the optimi­zation of treatment plan can compensate eventual cold spots from the individual source dwell posi­tions. As it was mentioned in the study of Adams et al.19, by adjusting the number of needles and dwell positions as well as the number of rotations per dwell position, prostate dose can be controlled. It seems that in I-RSBT treatment of prostate cancer of Adams et al.19 the radiation sensitive organs at risk are protected receiving the relative lower dose compared to tumor dose. As a consequence some parts of the tumor can receive lower dose which has to be compensated extending the affected ir­radiation dwell times in the treatment plan calcu­lations. Overall radiation time is thus prolonged using this irradiation technique. The I-RSBT could be promising also for some other sites in brachy­therapy like gynecology or breast brachytherapy. It is interesting as a subject of further research. Additionally, the dose distribution around the source in shielded mode is less symmetrical com­pared to unshielded source which could be ac­counted as a disadvantage (Figure 7). However this can be compensated in treatment planning. This leads to “Intensity Modulated Brachytherapy” which is considered as an innovation at this time. Only the future will show us the real possibilities of these new techniques. As it was aforementioned in the text, Adams et al.19 have introduced the inter­stitial rotating shield brachytherapy using a 153Gd source for brachytherapy of prostate. In the study they used Computed Tomography imaging to of­fer clinical data for the shielded source positioning and rotation in the treatment planning process. They used 19 needles and 16 rotational angles per dwell position of the 153Gd source rotating shield for annonimized prostate patient. The dose-volume histograms from conventional brachytherapy with 192Ir source were compared with those from I-RSBT with 153Gd source. Our study continues the scientif­ic efforts adding TG-43 simulated dose parameters which enable Treatment Planning Systems to cal­culate the I-RSBT doses in phantoms and tissues. Conclusions The calculated TG-43 dosimetric parameters in this study for a combination of the 153Gd source and the shielded needle could be used in treatment plan­ning system. Dose rate constant and radial dose function with the shielded sources are considerably different than the non-shielded sources. Further studies will be required to justify our Monte Carlo simulation parameters as dose calculation tools on a real human RANDO phantom. From clinical point of view there are some issues relating the tu­mor dose uniformity which is related to dose vol­ume histograms. The I-RSBT source has lower dose isotropy which can lead to lack of uniformity distri­bution in the tumor. This can be compensated with new Treatment Planning Systems based on the live Monte Carlo calculations. 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Available at: http://physics.nist.gov/PhysRefData/XrayMassCoef/tab3.html 113 research article Optimal planning strategy among various arc arrangements for prostate stereotactic body radiotherapy with volumetric modulated arc therapy technique Sang Won Kang1,2, Jin Beom Chung3, Jae Sung Kim3, In Ah Kim3, Keun Yong Eom3, Changhoon Song3, Jeong Woo Lee4, Jin Young Kim5, Tae Suk Suh1,2 1 Research Institute of Biomedical Engineering, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Biomedical Engineering, College of Medicine, The Catholic University of Korea, Seoul, Korea 3 Department of Radiation Oncology, Seoul National University Bundang Hospital, Seongnam, Korea 4 Department of Radiation Oncology, Konkuk University Medical center, Seoul, Korea 5 Department of Radiation Oncology, Haeundae Paik Hospital, Inje University, Busan, Korea Radiol Oncol 2017; 51(1): 112-120. Received 19 July 2016 Accepted 25 October 2016 Correspondence to: Jin-Beom Chung, Department of Radiation Oncology, Seoul National University Bundang Hospital, Seongnam, 13620, Korea. E-mail: jbchung1213@gmail.com and Tae Suk Suh, Department of Biomedical Engineering, College of Medicine, The Catholic University of Korea, Seoul, 137701, Korea. E-mail: suhsanta@catholic.ac.kr Disclosure: No potential conflicts of interest were disclosed. Jin Beom Chung and Tae Suk Suh contribution equally to this work Backgorund. The aim of this study was to determine the optimal strategy among various arc arrangements in pros­tate plans of stereotactic body radiotherapy with volumetric modulated arc therapy (SBRT-VMAT). Patients and Methods. To investigate how arc arrangements affect dosimetric and biological metrics, SBRT­VMAT plans for eighteen patients were generated with arrangements of single-full arc (1FA), single-partial arc (1PA), double-full arc (2FA), and double-partial arc (2PA). All plans were calculated by the Acuros XB calculation algorithm. Dosimetric and radiobiological metrics for target volumes and organs at risk (OARs) were evaluated from dose-volume histograms. Results. All plans were highly conformal (CI<1.05, CN=0.91) and homogeneous (HI=0.09-0.12) for target volumes. For OARs, there was no difference in the bladder dose, while there was a significant difference in the rectum and both femoral head doses. Plans using 1PA and 2PA showed a strong reduction to the mean rectum dose compared to plans using 1FA and 2FA. Contrastively, the D2% and mean dose in both femoral heads were always lower in plans us­ing 1FA and 2FA. The average tumor control probability and normal tissue complication probability were comparable in plans using all arc arrangements. Conclusions. The use of 1PA had a more effective delivery time and produced equivalent target coverage with better rectal sparing, although all plans using four arc arrangements showed generally similar for dosimetric and biological metrics. However, the D2% and mean dose in femoral heads increased slightly and remained within the tolerance. Therefore, this study suggests that the use of 1PA is an attractive choice for delivering prostate SBRT-VMAT. Key words: stereotactic body radiotherapy; volumetric modulated arc therapy; prostate cancer; arc arrangement; dosimetric and biological metrics Introduction noses, with the number of prostate cancer patients rapidly increasing. Various radiotherapy tech-Prostate cancer is the most common cancer in men, niques for treating prostate cancer have been con-accounting for over one fifth of male cancer diag-sidered effective noninvasive treatment options, 114 especially for elderly patients and those unfit for surgery.1-3 Radiation therapy options for prostate cancer in­clude external beam radiation therapy (EBRT) and brachytherapy. Volumetric modulated arc therapy (VMAT) and intensity-modulated radiation thera­py (IMRT) for EBRT is widely used as the stand­ard treatment for prostate cancer. VMAT and IMRT permit dose optimization, in which the dose to the prostate can be increased while reducing toxicities and doses to the rectum and bladder, resulting in improved local control and reduced complications when compared to three-dimensional conformal radiation therapy (3DCRT).4-7 In addition, VMAT can produce equivalent or even better target cover­age and normal tissue sparing compared to fixed-field IMRT while taking advantage of more efficient monitor unit (MU) and reducing the delivery times. The use of SBRT to irradiate primary or meta­static tumors in several anatomical sites is becom­ing the standard treatment.8–13 SBRT with VMAT (SBRT-VMAT) is a radiotherapy method, which very precisely delivers a high dose of radiation to the target area in either a single dose or a small number of fractions. It is an attractive approach to dose escalation. Recently, researchers using SBRT, including the linear accelerator (Linac) and CyberKnife (CK), have achieved promising re­sults in the treatment of prostate cancer.14–17 To our knowledge, there is very little information regard­ing the optimal planning for various arc arrange­ments of prostate SBRT-VMAT.18,19 Moreover, there is no study, which investigates the radiobiological effect of prostate SBRT-VMAT plans. The aim of this study is to determine the optimal treatment planning approach under the different arc arrangements by analyzing the dosimetric and radiobiological impact in plans for prostate SBRT­VMAT. Patients and methods Patient selection and contouring For this retrospective study, we chose 18 patients diagnosed with prostate cancer that had previous­ly been treated in our department from September 2013 to October 2015. All prostate cancer patients were enrolled in our SBRT planning study, which was approved by the institutional review board of Seoul National University Bundang Hospital. (IRB No. B-1501/284-107). A computer tomography (CT; The Brilliance CT Big Bore, Philips, Eindhoven, Netherlands) simu­lation was performed with the patients, who were placed in a supine position on a flat bench and stabilized with Knee-fix™ and Feet-fix™ (CIVCO Medical Solutions, Coralville, IA, USA). Prior to the CT simulation, the patients were asked to drink 300 ml of water 1 h before the simulation to ensure that the bladder was completely filled. An endorec­tal balloon (ERB) was inserted into the rectum and filled with 70 cc of air. After 1 min, the ERB catheter was placed at the pre-marked position and the in­flated ERB was immobilized above the anal sphinc­ter. A detailed description of the patient setup was given in our previous study.20,21 The CT scans were acquired with a 3 mm slice thickness. The prostatic bed was delineated as the clinical target volume (CTV), and the planning target volume (PTV) was defined as the CTV plus a treatment margin of 7 mm posteriorly and 10 mm in all other directions. The relevant normal tissue including rectum, blad­der, and femoral head were delineated as OARs. The rectum was defined as extending from the sig­moid flexure to the bottom of the ischium. Planning strategy for SBRT Prostate SBRT using VMAT plans were created by the Eclipse™ Treatment Planning System (ver. 11.0.34, Varian Medical Systems). The SABR­VMAT planning system was commissioned for a TruebeamTM Linac (Varian Medical Systems) with a high definition multileaf collimator (HD MLC). Dose distributions were calculated using a 10-MV flattening-filter-free (FFF) beam and the Acuros XB (AXB) dose calculation algorithm with inhomoge­neity correction. The calculation grid size was 2.5­mm. The SBRT-VMAT plans were set up with four arc arrangements such as single-full arc (181° to 179°; 1FA), single-partial arc (240° to 120°; 1PA), double-full arcs (181° to 179° and 179° to 181°; 2FA), and double-partial arcs (240° to 120° and 120° to 240°; 2PA). The arc arrangements for SBRT-VMAT plans are presented in Figure 1 A to D. The collimator rotation angles were 30°. The prescription dose was 42.7 Gy and was administered in seven fractions. Compared to 78 Gy in 39 fractions, which is the standard prostate fractionation, this delivers a higher biologically effective dose (BED) to the prostate (216.3 Gy vs. 182.0 Gy; ./ß 1.2 Gy) but an equivalent dose to late responding tissues (129.5 Gy vs. 130.0 Gy; ./ß 3.0 Gy). For all cases, a minimum of 95% of the pre­scription dose (40.6 Gy) was assigned to cover 95% of the PTV (V95% . 95%). No OAR constraints for 115 prostate SBRT have yet been reported. Therefore, we used the modified constraints for the OARs de­rived from those reported by Murray et al.22 that were suitable for our planning study. The OAR constraints for this planning study are listed in Table 1. Evaluation of dosimetric and biological parameters The mean, maximum, and minimum doses for PTV were measured from cumulative dose-volume histograms (DVH) of plans using four arc arrange­ments for all patients. In order to investigate the target coverage, V100% for CTV and PTV were evalu­ated. The conformity index (CI) was defined as fol­lows: The ideal conformity is defined as CI = 1. A value of CI > 1 indicated that healthy tissue has been irradiated.23 The conformation number (CN) takes into consideration the irradiation of healthy tissue. It is the product of two fractions, TVRI/TV and TVRI/VRI, where TV is the volume of the PTV, TVRI is the volume of the PTV covered by the refer­ence isodose line, and VRI is the volume enclosed by the reference isodose line. TVRI/TV is the quality of the target coverage and TVRI/VRI is the volume of healthy tissue irradiated with the reference isodose (95% of prescribed dose) or more.24 The dose homogeneity index (HI) was deter­mined as follows: where D5% is the maximum dose received by 5% of PTV, D95% is the minimum dose received by 95% of PTV, and D50% is the dose received by 50% of PTV. A lower HI represents a more homogeneous plan, because D5% and D95% were surrogate markers of maximum dose and minimum dose in the PTV, respectively. The near-to-maximum dose (D2%) and mean dose for the OARs were evaluated. In addition, we conducted a detailed analysis of the rectum and bladder volumes that received at least 95% (V95%), 80% (V80%), 50% (V50%), and 20% (V20%) of the pre­scription dose; these values represent very high, high, intermediate, and low doses, respectively. In addition, total monitor units (MUs) were com­pared in each plan between single arc and double arc arrangements. TABLE 1. Dose volume constraints adopted for planning study V42.7 Gy (100%) < 5% V42.7 Gy(100%) < 10% V29.9 Gy (70%) < 50% V38.4 Gy (90%) < 15% V34.7 Gy (81%) < 25% Dmax <29.9 Gy Constraints V32.0 Gy (75%) < 35% V29.9 Gy(70%) < 50% V28.0 Gy (65%) < 45% V24.8 Gy (58%) < 70% V20.0 Gy (47%) < 80% D= the maximum dose; Vxx Gy = the volume receiving dose of xx Gy (x% of the prescription dose) max For the radiobiological model evaluation, we utilized the MATLAB program to calculate the Niemierko’s equivalent uniform dose (EUD)-based tumor control probability (TCP) and normal tissue complication probability (NTCP).25,26 According to Niemierko’s phenomenological model, the EUD is defined as follows: where . is a tissue-specific parameter describ­ing the volume effect, and is the partial tumor volume, which receives dose D in Gy. For tumors, . takes negative values; for serial-like structures, . takes large positive values; and for parallel-like structures, . takes values close to 1. The prostate TCP was calculated with Niemierko’s EUD-based TCP. The equation is de­fined as follows: where is the tumor dose to control 50% of the tumors when the tumor is homogeneously irradiated, and the is the slope of dose response at a TCP of 50%. NTCP for OARs were calculated using Niemierko’s EUD-based NTCP with the fol­lowing equation: where is the tolerance dose for a 50% com­plication rate at a specific time interval (e.g., 5 years in the Emami et al. normal tissue tolerance data27) when the whole organ of interest is homogenously irradiated. The is specific to the normal struc­ture of interest and describes the slope of the dose-response curve. Table 2 lists parameters used to calculate Niemierko’s EUD-based TCP and NTCP. These 116 TABLE 2. Parameters used to calculated Niemierko’s EUD-based TCP and NTCP Prostate 6.1 7 -10 1.0 28.34 2 1.20 Rectum 6.1 7 8.33 4 80 2 3.90 Bladder 6.1 7 2 4 80 2 8.00 Femur 6.1 7 4 4 65 2 0.85 Alpha-beta ratio; 100%dpf = 100% dose per fraction; dpf = Parameters’ source data’s dose per fraction FIGURE 1. An arrangement of (A) one full arc (1FA), (B) one partial arc (1PA), (C) two full arcs (2FA), and (D) two partial arcs (2PA) in prostate SBRT-VMAT for the same patient. parameters come from data reported in previous study28 and were suitably modified for our study. Results Figure 2A to 2D show the dose distributions achieved with four arc arrangements for the same patient. There were small differences in the dose distributions and corresponding DVHs among each arc arrangement. Average cumulated DVHs of the PTV, rectum, bladder, and left and right fem­oral head are presented in Figure 3A to 3E for each of the four arc arrangements. Tables 3 and 4 show the average (range) values for the dosimetric results of target volumes and several OARs (i.e., bladder, rectum, and left and right femoral head). The resulting PTV and CTV of the four arc arrangements of all patients were very similar as shown in Table 3 and Figure 3A. In four arc arrangements, the volume of the CTVs that received 100% of prescription dose was great­er than 97.6% (range 96.7–98.4%), which indicated very good coverage the CTV in the VMAT plan. All plans were highly conformal with CI < 1.05 and CN = 0.91, and the doses were homogeneous (HI = 0.09±0.12). The doses to bladder tissue showed no differ­ence among all arc arrangements. However, there were significant differences in the doses of the rec­tum, left and right femoral head (Table 4, Figure 3 A, C, D, E). Compared to 1FA and 2FA, 1PA and 2PA arrangements resulted in a significant reduc­tion of the mean dose (V50%) of the rectum. These arc arrangements resulted in a significant increase in the near-to-maximum dose D2% and mean dose of the left and right femoral head. Table 5 summarizes the MUs and delivery time for the prostate SBRT plans using the four arc ar­rangements. The average MU was 1575 ± 63, 1607 ± 56, 1646 ± 97, and 1660 ± 98 for plan using 1FA, 1PA, 2FA, and 2PA, respectively. Plans using 1FA required lower MUs than those using other arc ar­rangements. The average delivery time was 1.01 ± 0.02, 0.69 ± 0.01, 2.00 ± 0.01, and 1.36 ± 0.02 for 1FA, 1PA, 2FA, and 2PA, respectively. The ratio of deliv­ery time was 1.46, 2.90, and 1.97 for 1FA, 2FA, and 2PA compared to 1PA. The average TCP of prostate tumors and the av­erage NTCP of OARs in the four arc arrangement plans are shown in Table 6. TCP and NTCP values were comparable for all arc arrangements. Discussion Much has been published regarding the use of VMAT in prostate cancer, but little regarding the use of SBRT using VMAT for prostate cancer. Most of the previous studies for prostate SBRT em­ployed the CK technique. The CK technique is an 117 accurate image guided method for delivering ra-TABLE 3. Dosimetric results for target volumes in prostate SABR-VMAT plans using four arc arrangements diation to a precisely targeted area using multiple nonisocentric beams with steep surrounding dose gradient.16,29 In the previous study reported by Chow and CTV coverage Jiang30, the dosimetry and radiobiological model 44.9±0.4 44.6±0.5 44.5±0.5 44.4±0.3 variation was investigated in prostate VMAT plans D50% (Gy) (44.4-45.4) (44.1-45.1) (44.0-45.3) (44.0-44.7) using the single- and double-full arc technique. 46.0±0.1 45.9±0.1 45.9±0.1 45.8±0.2D5% (Gy) (45.9-46.1) (45.7-46.0) (45.8-46.0) (45.7-45.9) The authors reported that the double-arc technique 43.5±0.5 43.1±0.4 43.1±0.6 42.8±0.2could lower the dose-volume criteria of the rectum D95% (Gy) (43.0-44.2) (42.8-43.5) (42.6-43.9) (42.7-43.1) and bladder but increase the rectal NTCP. 98.4±1.8 97.9±0.0 97.8±3.8 97.6±2.0 (%) V100% (95.5-100.0) (95.0-99.8) (95.2-100.0) (94.2-99.9) In addition to the study above, we included the PTV coverage single and double-partial arc (1PA and 2PA), which 44.3±0.2 44.2±0.1 44.1±0.3 44.3±0.1 meanavoided irradiation of the rectum for the optimal D(44.2-44.6) (44.0-44.4) (44.0-44.6) (44.2-44.5) prostate SBRT-VMAT plan. This study focused on 48.8±0.8 49.5±0.9 48.5±1.1 49.0±1.4 D (47.8-49.8) (48.4-50.5) (47.2-50.0) (47.4-50.5) arc arrangements as a key preparatory step in fa-max 33.0±0.4 32.3±0.0 32.8±0.8 33.7±0.3 cilitating clinical planning studies. Dmin (32.4-33.2) (31.9-33.7) (32.1-33.9) (33.4-34.1) The prostate SBRT-VMAT plans for all arc ar-(Gy) 44.6±0.3 44.5±0.2 44.4±0.4 44.5±0.2 D50% (44.2-45.0) (44.3-44.8) (44.1-45.0) (44.3-44.8) rangements generated conformal dose distribu­ 46.1±0.2 46.1±0.1 46.1±0.2 46.0±0.1 (Gy) D5% tions for target volumes (PTV and CTV). We found (45.9-46.3) (45.9-46.2) (45.9-46.3) (45.9-46.2) 41.5±0.3 41.4±0.2 41.3±0.3 41.7±0.1 that the dose distributions in the anterior and pos-(Gy) D95% (41.1-41.8) (41.2-41.8) (41.0-41.7) (41.6-41.9)terior direction were lower when the partial arc 87.5±1.9 86.6±1.2 87.7±1.5 88.1±1.0 V100% (%) (85.1-90.2) (85.0-87.8) (85.8-89.3) (87.1-89.9) arrangements (1PA and 2PA) were used instead of Conformity 1.05±0.00 1.04±0.01 1.03±0.01 1.05±0.01the full arc (1FA and 2FA). In contrast, the dose dis-index (1.02-1.03) (1.02-1.04) (1.02-1.04) (1.02-1.03) tributions in the left and right direction for full arc Conformation 0.91±0.01 0.91±0.01 0.91±0.01 0.91±0.01 number (0.90-0.92) (0.09-0.92) (0.91-0.92) (0.90-0.93) arrangements were lower than those of the partial Homogeneity 0.10±0.01 0.11±0.00 0.11±0.01 0.10±0.00arc (Figure 2). This was due to the application of index (0.09-0.12) (0.10-0.11) (0.10-0.12) (0.09-0.10) arc angles, which avoided the direct irradiation of Avg. = average; D5% = the dose received at least 5% volume; D= the mean dose; D = the mean maxthe rectum. maximum dose; Dmin = the minimum dose; D50% = the dose received at least 50% volume; D95% = the dose received at least 95% volume; SD = the standard deviation; V100% = the volume received With respect to the dose to the OARs, the effect of 100% of prescription dose. the dose difference on bladder tissue was negligible for the different arc arrangements. The differences between the partial and full arc arrangements were observed in several dose-volume criteria (e.g., V20% and V50%) and were slightly lower in plans using the partial arc arrangements than the full arc arrange­ments. For the rectum, the partial arc arrangements showed relatively strong reductions of the mean dose compared to the full arc arrangements while all plans using four arc arrangements showed no dramatic differences in the high and low doses. For the left and right femoral head in Table 3, Figures 3D,E, we found that the D2% and mean dose were always lower when the full arc arrangements were used compared to the partial arc arrangements. We also observed better sparing in the right femoral head compared to the left femoral head. The clinical delivery time of prostate SBRT­VMAT plans using the one arc arrangements (1FA and 1PA) was approximately half compared to that of the two arc arrangements (2FA and 2PA). The reduced delivery time by using one arc arrange- FIGURE 2. An example of the dose distributions achieved with (A) one full arc (1FA), ments has the potential to decrease the effects of (B) one partial arc (1PA), (C) two full arcs (2FA), and (D) two partial arcs (2PA) intrafractional motion because prostate motion is arrangement in prostate SBRT-VMAT for the same patient. 118 TABLE 4. Dosimetric results for organs at risk in prostate SABR-VMAT plans using four time dependent. The delivery time for partial arc arc arrangements and full arc in single or double arc arrangements was similar because the delivery duration is lim­ited by the gantry rotation speed and leaf speed, not the dose rate. Organs at risk The MUs were observed to significantly increase Rectum D(Gy) 25.2±1.7 23.3±2.0 23.6±1.8 23.4±1.9 with the number of arcs used in an SBRT-VMAT mean (23.1-27.2) (21.4-25.8) (21.3-26.0) (21.3-26.4) plan. The MU value of the 2FA and 2PA was up to 44.2±0.3 44.6±0.4 44.1±0.4 44.5±0.2 Rectum D2% (Gy) (43.7-44.5) (44.0-45.1) (43.8-44.7) (44.3-44.9) 5% greater than that of the 1FA. This was in line Rectum V95% (%) 9.0±1.4 10.1±1.3 8.3±1.3 9.8±1.5 with the previous study20, which reported the in­(6.6-10.3) (8.1-11.5) (7.0-10.2) (7.6-11.6) censement of MUs in plans using double arcs com­ 19.1±3.0 19.7±3.2 18.0±2.8 19.4±3.4 Rectum V80% (%) (15.1-23.2) (15.2-23.8) (15.2-21.9) (14.8-24.3) pared to those with single arcs. However, the plan 71.0±8.8 53.9±9.4 60.0±11.1 54.1±9.0 using 1FA for this study required lower MUs than Rectum V50% (%) (61.1-80.1) (41.9-63.7) (46.9-77.4) (42.5-66.7) the plan using 1PA. The ratio of delivery time for 90.2±2.5 90.0±2.7 89.6±3.0 90.0±2.8 Rectum V20% (%) (87.7-93.4) (87.1-94.1) (85.6-93.5) (87.3-94.0) 1FA, 2FA, and 2PA compared to 1PA was between 19.0±6.9 18.7±7.1 18.5±6.1 18.3±6.9 1.5 and 3. The use of 1PA appeared to reduce the Bladder Dmean (Gy) (13.4-31.1) (13.2-31.0) (15.7-29.0) (12.9-30.4) treatment delivery time, which has obvious ben­ 45.6±0.2 45.6±0.2 45.5±0.3 45.6±0.4 Bladder D2% (Gy) efits for SBRT. (45.5-45.8) (45.3-45.9) (45.0-45.8) (45.3-46.4) 21.2±11.1 21.4±11.1 20.9±10.5 21.4±11.3 In this study, we compared the radiobiological Bladder V95% (%) (10.5-39.3) (10.6-39.4) (10.3-37.7) (10.7-39.9) impact among four arc arrangements within SBRT­ 25.6±11.9 26.2±11.9 25.7±11.1 26.0±12.0 Bladder V80% (%) VMAT plans for prostate cancer. There were no (16.6-46.4) (16.5-46.3) (16.6-44.6) (16.3-46.2) 40.4±13.6 38.6±12.6 39.5±12.4 38.2±12.9 obvious differences in the TPC and the NTCP for Bladder V50% (%) (29.4-64.1) (28.5-60.5) (29.2-60.7) (27.5-60.6) plans using four arc arrangements, excluding the 54.4±17.0 52.5±16.1 54.1±17.4 52.1±16.1 Bladder V20% (%) NTCP value of the rectum. The NTCP difference of (37.2-79.1) (36.4-76.5) (37.0-79.4) (36.2-76.1) Left femoral head 12.8±1.8 18.7±2.3 12.7±2.4 13.9±1.4 rectum was also small (within 0.2%) among the four D(Gy) (10.5-15.1) (16.9-22.7) (9.3-14.9) (11.7-15.3) arc arrangements as shown in Table 6. These mean mean Left femoral head 19.7±2.2 23.5±4.4 18.3±3.0 20.1±2.0 that radiobiological outcomes have no difference (Gy) (16.9-21.8) (15.6-26.1) (13.4-20.7) (16.7-21.8) Right femoral 10.8±0.9 13.3±2.5 12.4±1.7 13.7±2.1 relative to four arc arrangements. Furthermore, the D2% head D(Gy) (9.2-11.3) (9.2-15.5) (10.5-14.2) (10.7-16.5) TCP increase/decrease was correlated with D, mean mean Right femoral 16.4±1.3 20.6±3.7 18.1±1.9 19.7±2.3 which is related to the mean dose. The mean dose head D2% (Gy) (14.3-18.0) (14.3-23.6) (15.8-20.1) (16.2-22.5) of PTV for four arc arrangements showed no dif- Avg. = average; D5% = the dose received at least 5% volume; Dmean = the mean dose; Dmax = the ference (Table 3). The NTCP of the rectum for the maximum dose; Dmin = the minimum dose; D50% = the dose received at least 50% volume; D95% = the dose received at least 95% volume; SD = the standard deviation; V100% = the volume received partial arc arrangements (1PA and 2PA) was higher 100% of prescription dose. than that of the full arc arrangements (1FA and TABLE 5. Average monitor unit and delivery time for prostate SBRT-VMAT plans using 2FA). The reason for this is that the rectum includes four arc arrangements the high-dose region with higher mean and maxi­ mum doses in plans using partial arc arrangements. Nevertheless, such an increased NTCP of the rec­tum is still within the acceptable range. MU 1575±63 1627±56 1646±97 1660±98 Limitation of this study is that there are no de- Delivery time (min) 1.01±0.02 0.69±0.01 2.00±0.01 1.36±0.02 finitive clinical data on short- and long-term out­comes. This study focused mainly on the investi- TABLE 6. TCP of prostate tumor and NTCP of OARs for four arc arrangement plans gation of optimal treatment planning in prostate SBRT-VMAT. Therefore, future follow-up studies are required to evaluate the clinical outcome and toxicity for practice application of this technique. TCP (%) Prostate 93.35±0.08 99.28±0.06 93.32±0.08 93.36±0.08 NTCP (%) Conclusions Rectum 0.44±0.23 0.61±0.21 0.40±0.23 0.56±0.22 Prostate SBRT-VMAT plans using four arc (1FA, Bladder 0.01±0.02 0.01±0.03 0.00±0.01 0.00±0.02 1PA, 2FA, and 2PA) arrangements offered high LT Femur 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 conformity for target volumes. This study demon- RT Femur 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 strated that prostate SBRT-VMAT using 1PA (240° to 120°) showed reasonably fast delivery time and produced equivalent target coverage and better rec­tum sparing, although the near-to-maximum dose and mean dose of the left and right femoral heads increased slightly. However, the doses in both fem­oral heads remained well within the clinical normal tissue tolerance. For evaluating the radiobiologi­cal metrics, all plans using four arc arrangements produced comparable TCP for prostate tumors and NTCP for OARs. Therefore, it was concluded that the use of 1PA was an attractive choice for treating prostate cancer using SBRT-VMAT. Author Contribution Conception and design of the study: JBC TSS. Coordination of the study: SWK JSK JWL. Case se­lection, radiation treatment planning, and analysis: KYE CHS JSK JYK JBC. Manuscript preparation: JBC SWK. Manuscript revision and editing: JBC JSK IAK TSS. Manuscript approval: all authors. Acknowledgments This research was supported by a grant of the Korea Health technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea. 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J App Clin Med Phy 2014; 14: 3-12. doi:10.1120/jacmp. v14i3.4053 Radiol Oncol 2017; 51(1): 1-7. doi:10.1515/raon-2016-0038 Primarni pljučni horiokarcinom Snoj Ž, Kocijančič I, Škof E Izhodišča. Primarni pljučni horiokarcinom je redek in visoko maligen pljučni tumor s slabo napovedjo poteka bolezni. Namen raziskave je bil ugotoviti, ali obstajajo različne klinične oblike primarnega pljučnega horiokarcinoma, ki zahtevajo različen diagnostični pristop in primernejšo izbiro zdravljenja. Bolniki in metode. S pomočjo iskalnika PubMed smo naredili pregled literature predhodno objavljenih primerov primar­nega pljučnega horiokarcinoma. Vključitveni dejavniki za raziskavo so bili bolniki, ki so imeli histološko potrjeno diagnozo pljuč­nega horiokarcinoma in temeljito preiskavo reproduktivnih organov za izključitev morebitnega horiokarcinoma v gonadah. Predstavili smo tudi bolnika zdravljenega v naši ustanovi. Rezultati. Vključili smo 55 primerov (17 moških) s srednjo starostjo 34 let. Ženske z anamnezo gestacijskega dogodka so imele daljšo dobo preživetja kot ženske brez gestacijskega dogodka. Bolniki, zdravljeni s kombinacijo kirurgije in kemoterapije, so imeli daljšo dobo preživetja kot bolniki, ki niso bili zdravljeni s kombinacijo kirurgije in kemoterapije. Z multivariatno analizo smo prikazali, da je zdravljenje s kombinacijo kirurgije in kemoterapije neodvisni napovedni dejavnik. Z univariatno in multivariatno analizo pa smo prikazali, da ima velikost tumorja statistično pomemben vpliv na izid bolezni. Zaključki. Primarni pljučni horiokarcinom je izjemno redka bolezen z različnimi kliničnimi oblikami in izidom. Pomembno je, da bolnike zajamemo in začnemo zdraviti v zgodnjih fazah bolezni. Ženske z anamnezo gestacijskega dogodka imajo daljšo dobo preživetja, kar nam lahko s pomočjo genetske analize pomaga oceniti napoved poteka bolezni pri bolnicah. Kirurško zdravljenje z adjuvantno kemoterapijo predstavlja najboljšo izbiro zdravljenja primarnega pljučnega horiokarcinoma. Radiol Oncol 2017; 51(1): 8-14. doi:10.1515/raon-2017-0001 PET/CT slikovna tehnika pri revmatični polimialgiji: predsramnično kopičenje z 18F označene fluorodeoksiglukoze je povezano z entezitisi in tenosinovitisi mišic grebenke in dolge pritezalke stegna Rehak Z, Sprlakova-Pukova A, Bortlicek Z, Fojtik Z, Kazda T, Joukal M, Koukalova R, Vasina J , Eremiasova J, Nemec P Izhodišča. V diagnostiki revmatične polimialgije (RPM), ki velja za eno najpogostejših revmatičnih bolezni, ima PET/CT s 18F označeno fluorodeoksiglukozo (18F-FDG PET/CT) vse večjo vlogo. Poleg kopičenj 18F-FDG na drugih lokacijah se pri nekaterih bolnikijh povečano kopiči 18F-FDG tudi v predsramničnem področju. Pri tem pa povečano predsramnično kopičenje 18F-FDG še vedno ni natančno opisano predvsem pa ne patofiziološko razloženo. Namen naše raziskave je bil dokazati zmanjšano predsramnično kopičenje 18F-FDG kot odgovor na zdravljenje pri bolnikih z RP ter opisati potencialne povezave z drugimi 18F-FDG PET/CT značilnostmi. Poleg tega pa smo želeli osvetliti patološki vidik predsramničnega kopičenja 18F-FDG. Bolniki in metode. V retrospektivno študiji smo pregledali bolnike s RPM obravnavane med feburajem 2010 in marcem 2016. Tiste bolnike, pri katerih smo ugotovili predsramnično kopičenje 18F-FDG smo natančno analizirali vizualno in semi-kvanti­tativno z določanjem razmerja med predsramničnim kopičenjem in kopičenjem v jetrih. Pri teh bolnikih smo sistematično smo opisali tudi kopičenje 18F-FDG v ostalih področjih (ramenskem, kolčnem in sternoklavikularnem sklepu, sedničnozadnjični burzi in medtrnastimi prostori). Rezultati. Pri triindvajsetih od 89 pregledanih bolnikov (26 %) je bilo vidno predsramnično kopičenje 18F-FDG. Pri petnajstih od 23 bolnikov smo naredili tudi dodatno 18F-FDG PET/CT preiskavo med samim spremljanjem poteka bolezni. Pri petih od petnajstih bolnikov je bilo vidno kopičenje 18F-FDG tudi v velikih arterijah, kot znak velikoceličnega arteritisa. Med 18F-FDG PET/ CT preiskavo ob spremeljanju petnajstih bolnikov smo, kot posledica zdravljenja, ugotovili zmanjšano kopičenje 18F-FDG v vseh prvotno pozitivnih področjih. Poleg tega nismo našli novih kopičenj ob sklepih ali zunaj njih ali v tarčnih velikih žilah. Pri vseh petnajstih biolnikih se je po zdravljanju zmanjšalo tudi predsramnično kopičenje 18F-FDG. Zaključki. Povečano predsramnično kopičenje 18F-FDG pri bolnikih s RPM je pogosto in ga je potrebno med diagnostičnim postopkom revmatičnih ali malignih obolenj sistematično proučiti. Predsramnična vnetja so verjetno v povezana z entesitisom in tenosinovitisom grebenske mišice in dolge pritezalke stegna pred samo sramno zrastjo. Radiol Oncol 2017; 51(1): 15-22. doi:10.1515/raon-2016-0037 Ehokardiografija in srčni biološki označevalci pri bolnikih z nedrobnoceličnim pljučnim rakom zdravljenih s kemoterapijo na osnovi platine Omersa D, Čufer T, Marčun R, Lainščak M Izhodišča. Nedrobnocelični pljučni rak (NSCLC) je v svetu eden izmed glavnih razlogov za smrt. Namen raziskave je bil oceniti kardiotoksičnost pri bolnikih z NSCLC zdravljenih s kemoterapijo na osnovi platine ter ugotoviti, kateri bolniki imajo večje tveganje za nastanek kardiotoksičnosti. Bolniki in metode. V prospektivno, opazovalno raziskavo smo vključili bolnike z začetnim in napredovalim NSCLC, ki smo jih zdravili s kemoterapijo na osnovi platine. Pri bolnikih smo ugotavljali kardiotoksičnost, ki smo jo opredelili kot zvišanje ultra­senzitivnega troponina T, N-terminalnega natriuretičnega propeptida tipa B ali zmanjšanje iztisnega deleža levega prekata (LVEF) pred in po zaključku kemoterapije ter 6 mesecev po zaključku kemoterapije. Rezultati. Vključili smo 41 bolnikov (povprečna starost 61 let ± 9 let, 54 % moških, 68 % z napredovalo boleznijo) z NSCLC zdravljenih s kemoterapijo na osnovi platine (v povprečju s 4 krogi). Med raziskavo so umrli 3 bolniki, pri nobenem od njih nismo ugotovili srčnega popuščanja. Povprečne vrednosti bioloških označevalcev in LVEF se niso razlikovale (p > 0,2). Kljub temu pa je 10 bolnikov (25 %) imelo kardiotoksičnost, ki je bila neodvisno povezana z znano ishemično boleznijo srca (p = 0.026). Zaključki. Pri bolnikih z NSCLC zdravljenih s kemoterapijo na osnovi platine, ki imajo ishemično bolezen srca in pri tistih z dolgo pričakovano življenjsko dobo, bi bilo smiselno razmisliti o kardiološki obravnavi in modifikaciji življenjskega sloga. Radiol Oncol 2017; 51(1): 23-29. doi:10.1515/raon-2017-0002 Diagnostika in staging tumorjev želodca – doprinos difuzijsko obremenjenega slikanja z magnetno resonanco in primerjava z računalniško tomografsko preiskavo Arslan H, Özbay MF, Çalli I, Dogan E, Çelik S, Batur A, Bora A, Yavuz A, Bulut MD,Özgökçe M, Kotan MC Izhodišča. Primerjava diagnostične učinkovitosti magnetnoresonančnega difuzijskega slikanja (angl. Diffusion-Weighted Imaging – DWI) in računalniške tomografije (angl. CT) pri določanju TNM stadija raka želodca. Bolniki in metode. Pri 51 bolnikih z rakom želodca smo predoperativno uporabili T2 obtežene slike in DWI (b-0,400 in b­-800s/mm2). Opravili smo tudi CT slikanje. Iskali smo zvišanje signala na sekvencah DWI. Ocenjevali smo globino vraščanja tumorja (T), prizadetost regionalnih bezgavk (N) in prisotnost ali odsotnost oddaljenih metastaz (M) na sekvencah DWI in CT preiskavi. Izračunali smo občutljivost, specifičnost, pozitivno in negativno napovedno vrednost DWI in CT preiskave v primerjavi s patološkim izvidom kirurgije, ki velja za zlati standard. Ujemanje smo ocenili s koeficientom ujemanja Kapa. Rezultati. Senzitivnost in specifičnost preiskavav DWI in CT pri ocenjevanju prizadetosti regionalnih bezgavk sta bili sledeči: N1: DWI: 75 %, 84,6 %; CT: 66,7 %, 82 %; N2: DWI: 79,3 %, 77,3 %; CT 69%, 68,2 %; N3: DWI: 60%, 97,6 %; CT 50 %, 90.2 %. DWI sli­kanje se je pri ocenjevanju regionalnih bezgavk izkazalo za bolj primerljivo z zlatim standardom (kirurškim patološkim izvidom) kot CT preiskava. Pri ocenjevanju globine vraščanja tumorja (T) so bili rezultati preiskav DWI in CT boljši kot zlati standard pri višjem stadiju T, vendar se DWI ni izkazal za boljšega od CT preiskave. Pri določanju oddaljenih metastaz (M) sta senzitivnost in specifičnost obeh preiskovalnih metod bili 100 %. Zaključki. Diagnostična natančnost slikanja DWI pri TNM klasifikaciji raka želodca je primerljiva s CT slikanjem in dodatna uporaba DWI pri rutinskem protokolu lahko izboljša diagnostično natančnost kadar ocenjujemo metastatsko spremenjene bezgavke. Radiol Oncol 2017; 51(1): 30-39. doi:10.1515/raon-2017-0011 Elektroprenos plazmidne DNA radiosenzibilzira tumorje B16F10 preko aktivacije imunskega odgovora Savarin M, Kamenšek U, Čemažar M, Heller R, Serša G Izhodišča. Obsevanje tumorjev v kombinaciji z različnimi žilno ciljanimi ali imunskimi dodatnimi zdravili je predmet številnih sodobnih raziskav. Genski elektroprenos terapevtskih plazmidov je eden izmed primernih pristopov. V predstavljeni raziskavi nas je zanimalo, ali lahko z genskim elektroprenosom plazmidne DNA z zapisom za shRNA za utišanje endoglina radiosenzibi­liziramo tumorje melanoma B16F10. Materiali in metode. Poskuse smo izvedli na mišjem melanomskem tumorskem modelu B16F10. Tumorje smo inducirali na hrbtu miši C57Bl/6 in jih zdravili s trikratnim genskim elektroprenosom terapevtskih plazmidov in obsevanjem. Protitumorsko učinkovitost smo ovrednotili z določanjem zaostanka v rasti tumorjev in števila popolnih odgovorov. Dodatno smo za dolo­čanje mehanizmov delovanja izvedli še histološko analizo tumorjev (določili smo nekrozo, apoptozo, proliferacijo, ožiljenje, prisotnost hipoksije in infiltracijo imunskih celic). Rezultati. Rezultati raziskave so pokazali, da ima genski elektroprenos plazmida za utišanje endoglina predvsem žilno ciljan učinek; zdravljenje je povzročilo značilen zaostanek v rasti tumorjev in 44 % pozdravljenih miši. Obsevanje je imelo manjši učinek na ta radiorezistenten melanomski model, saj smo zabeležili le 11% ozdravitev. Kombinirana terapija pa je bila zelo uspešna; zabeležili smo kar 88 % ozdravljenih miši, podoben odziv pa smo opazili tudi po kombinirani terapiji s plazmidom brez terapevtskega gena. Histološka analiza tumorjev po kombinirani terapiji je pokazala podoben mehanizem delovanja plazmi­da za utišanje endoglina in plazmida brez terapevtskega gena, namreč preko indukcije imunskega odziva. Zaključki. Rezultati naše raziskave nakazujejo, da lahko obsevanje pri sicer radiorezistentem melanomskem tumorskem mo­delu deluje kot aktivator imunskega odziva proti tumorskim antigenom sproščenim iz obsevanih celic. Ta aktiviran protitumorski imunski odgovor lahko dodatno pospešimo z genskim elektroprenosom plazmidov z ali celo brez terapevtskega gena, kar smo potrdili z visoko radisenzibilizacijo, ki se je kazala v 88 % popolnih ozdravitev in zaostanku v rasti tumorjev. Poleg tega ima genski elektroprenos terapevtskega plazmida za utišanje endoglina tudi direkten učinek na tumorsko žilje in tumorske celice, ki pa po kombinirani terapiji z obsevanjem ni bil viden, saj je bil zakrit z močnim imunskim odzivom. Radiol Oncol 2017; 51(1): 40-46. doi:10.1515/raon-2017-0003 Primerjava krioablacije in ireverzibilne elektroporacije jeter v zajcih neposredno ob žolčniku Zeng J, Qin Z, Zhou L, Fang G, Chen J, Li J, Niu L, Liang B, Xu K Izhodišča. Ablacija jetrnih tumorjev v bližini žolčnika lahko povzroči komplikacije. Namen študije je bil primerjati učinkovitost in varnost ablacije jeter v bližini žolčnika s krioablacijo in ireverzibilno elektroporacijo. Materiali in metode. Krioablacijo in ireverzibilno elektroporacijo smo naredili na 12 novozelandskih zajcih, na jetrih v bližini žolčnika, tako da je bil zajet tudi ta organ. Spremljali smo serumske aminotrasferaze, pred in po terapiji. Tkivne poškodbe smo spremljali histopatološko v ablativni coni, 7. dan po terapiji. Rezultati. Po 7. dneh so bile vse živali žive. Ireverzibilna elektroporacija ni povzročila perforacije žolčnika, le nekrozo mukoze in edem. Nasprotno je krioablacija povzročila perforacijo žolčnika pri 4. zajcih. Zabeležili smo povišane serumske aminotrans­ferase 3. dan po terapiji tako pri ireverzibilni elektroporaciji, kot pri krioablaciji, vendar so za tem upadle. Povišanje aminotra­snferaz in bilirubina je bilo večje po krioablaciji kot po ireverzibilni elektroporaciji. Obe metodi ablacije sta povzročili nekrozo jetrnega parenhima od ablacijskega centra proti žolčniku. Kriobalacije je povzročila nekrozo stene žolčnika, medtem ko ireverzibilna elektroporacije tega ni povzročila, le granulacijo in hiperplazijo smo opazili. Zaključki. Za ablacijo jetrnega tkiva v bližini žolčnika je primernejša ireverzibilna elektroporacija kot krioablacija, saj je var­nejša, ker ne povzroča perforacije žolčnika in je učinkovitejša, sa povzroči popolno nekrozo s hitro regeneracijo tkiva. Radiol Oncol 2017; 51(1): 47-55. doi:10.1515/raon-2017-0008 Breme raka v Sloveniji z oceno časovnih trendov Zadnik V, Primic Žakelj M, Lokar K, Jarm K, Ivanuš U, Žagar T Izhodišče. Z raziskavo smo želeli opisati breme raka v Sloveniji in oceniti časovne trende za vse rake skupaj, kot tudi za po­ samezne pogoste lokacije in nekatere redke rake. Bolniki in metode. Osnovni vir podatkov je bil populacijski Register raka Republike Slovenije. Breme raka je predstavljeno z incidenco in prevalenco za obdobje 1950–2013 ter z umrljivostjo za obdobje 1985–2013. Pri časovnih trendih smo ocenje­vali delež letne spremembe z Jointpoint regresijsko analizo. Za oceno incidence leta 2016 smo uporabili metodo po Dybi in Hakulinenu, za projekcijo števila novo zbolelih v letu 2025 pa program Globocan. Rezultati. V zadnjem obdobju v Sloveniji letno za rakom zboli okrog 14.000 ljudi, nekaj več kot 6.000 jih umre, med nami pa živi 94.000 ljudi, ki so bili že obravnavani zaradi rakave bolezni. Pet najpogostejši rakov pri nas – nemelanomski kožni rak, raki debelega črevesa in danke, pljučni rak, rak dojke in rak prostate – obsega skoraj 60 % vseh novih primerov rakavih bolezni. Incidenca pogostih rakov se veča povprečno letno za 3,0 %, medtem ko je incidenca redkih rakov stabilna. Zaključek. Rak je bolezen starejših in ker se številčna povojna generacija hitro stara, lahko pričakujemo, da bo breme raka v Sloveniji raslo še naprej, kljub pričakovanim uspehom primarne in sekundarne preventive. Radiol Oncol 2017; 51(1): 56-64. doi:10.1515/raon-2016-0051 Leiomiosarkom ledvične vene. Analiza preživetja in napovednih dejavnikov do sedaj objavljenih 67 primerov bolnikov Novak M, Perhavec A, E. Maturen K, Pavlović Djokić S, Jereb S, Eržen D Izhodišča. Leiomiosarkom je redek maligen mezenhimski tumor. V svetovni literaturi so opisali več primerov leiomiosarkoma ledvične vene, analiza preživetja in napovednih dejavnikov pa še ni bila opravljena. Namen raziskave je bil opisati leiomi­osarkom ledvične vene ter glede na dosedanje objave primerov opredeliti celokupno preživetje, preživetje brez lokalne ponovitve bolezni, preživetje brez oddaljene ponovitve bolezni in napovedne dejavnike zanje. Bolniki in metode. Objavljenim primerom leiomiosarkoma ledvične vene iz literature smo dodali naš primer. Rezultati. Zbrali smo 67 primerov bolnikov, srednja starost je bila 56,6 let, 76,1 % je bilo žensk. Srednja velikost tumorja je bila 8,9 cm, pri 68,7 % bolnikov je bil tumor na levi strani. Tumorski trombus se je širil v lumen spodnje votle vene pri 13,4 %. Operirani so bili vsi bolniki razen enega (98,5 %). Celokupno preživetje je bilo 79,5 %, srednji čas sledenja 24 mesecev. Preživetje brez lokalne ponovitve bolezni je bilo 83,5 %, srednji čas sledenja 21,5 meseca, preživetje brez oddaljene ponovitve bolezni pa je bilo 76,1 %, srednji čas sledenja 22 mesecev. V univariatni analizi je bil napovedni dejavnik za celokupno preživetje status kirur­ških robov, za preživetje brez lokalne ponovitve bolezni pa širjenje tumorja v lumen spodnje votle vene in gradus. Napovednih dejavnikov za preživetje brez oddaljene ponovitve bolezni nismo uspeli opredeliti. V multivariatni analizi nismo uspeli opredeliti napovednih dejavnikov za nobenega od parametrov. Zaključki. Leiomiosarkom ledvične vene zraste običajno v hilusu ledvice. Nanj naj bi pomislili v diferencialni diagnozi ledvičnih in retroperitonealnih tumorjev, še posebaj pri ženskah starejših od 40 let, če je tumor na levi strani in ob odsotnosti hematurije. Opraviti moramo histološko biopsijo tumorja. Bolniki naj bodo napoteni v obravnavo k multidisciplinarnemu timu za sarkome. Tumor naj bo odstranjen z negativnimi kirurškimi robovi. Radiol Oncol 2017; 51(1): 65-73. doi:10.1515/raon-2016-0024 Napovedni pomen vrednosti uPA/PAI-1, statusa HER2 in tradicionalnih histoloških dejavnikov za preživetje pri bolnicah z rakom dojk brez zasevkov v bezgavkah Fokter Dovnik N, Takač I Izhodišča. Urokinazni aktivator plazminogena (uPA) in inhibitor aktivatorja plazminogena 1 (PAI-1) sta povezana s statusom HER2, zaradi česar se postavlja vprašanje, ali nosi vrednost uPA/PAI-1 dodatno klinično pomembno napovedno informacijo neodvisno od statusa HER2. Namen naše raziskave je bil primerjati napovedno moč vrednosti uPA/PAI-1, statusa HER2 in tra­dicionalnih napovednih dejavnikov za preživetje pri bolnicah z rakom dojk brez zasevkov v bezgavkah. Bolniki in metode. Opravili smo retrospektivno analizo 858 bolnic z rakom dojk brez zasevkov v bezgavkah, zdravljenih v Univerzitetnem kliničnem centru Maribor v letih 2000-2009. Podatke smo pridobili iz zdravstvene dokumentacije bolnic. Srednji čas sledenja je bil 100 mesecev. Univariatne in multivariatne analize preživetja brez ponovitve bolezni in celokupnega preži­vetja smo opravili s pomočjo Coxove regresije in Coxovega modela sorazmernih tveganj. Rezultati. V univariatnih analizah so bili s preživetjem brez ponovitve bolezni povezani starost, velikost tumorja, diferencia­cija, limfovaskularna invazija, status HER2 in vrednost uPA/PAI-1, s celokupnim preživetjem pa starost, velikost tumorja, diferen­ciacija in vrednost uPA/PAI-1. V multivariatnem modelu so bili s preživetjem brez ponovitve bolezni najbolj povezani dejavniki starost, status estrogenskih receptorjev in vrednost uPA/PAI-1, s celokupnim preživetjem pa starost bolnic in diferenciacija tumorja. V primerjavi z bolnicami z nizkim uPA in PAI-1 so imele bolnice z visoko eno ali obema vrednostma razmerje tveganj za smrt iz kateregakoli razloga v multivariatnem modelu 1,98 (95 % interval zaupanja 0,83–4,76). Zaključki. Vrednost uPA/PAI-1 ima pri bolnicah z rakom dojk brez zasevkov v bezgavkah jasno napovedno vrednost ne glede na status HER2, zato bi jo pri teh bolnicah lahko uporabljali kot dodaten napovedni dejavnik pri odločanju o dopolnil­nem sistemskem zdravljenju. Radiol Oncol 2017; 51(1): 74-80. doi:10.1515/raon-2017-0007 Varnost in učinkovitost, superabsorbentnih polimernih kroglic eluiranih z doksirubicinom za zdravljenje jetrnih zasevkov nevroendokrinih tumorjev. Preliminarni rezultati Bonne L, Verslype C, Laenen A, Cornelissen S, Deroose C, Prenen H, Vandecaveye V, Van Cutsem E, Maleux G Izhodišča. Namen retrospektivne raziskave je bil oceniti nadzor nad simptomi, tumorski odgovor na zdravljenje in stopnjo zapletov pri bolnikih z metastatskimi nevroendokrinimi tumorji jeter. Bolnike smo zdravili s transarterijsko kemoembolizacijo in uporabo superabsorbentnih polimernih (SAP) mikrokroglic, eluiranih z doksorubicinom. Bolniki in metode. V raziskavo smo vključili bolnike z jetrnimi metastazami nevroendokrinih tumorjev, ki smo jih zdravljeni s transarterijsko kemoembolizacijo in uporabo mikrokroglic SAP (delci 50–100 um Hepasphere / Quadrasphere Microsphere®, Merit Medical, South Jordan, Utah, ZDA), eludiranih z doksirubicinom. Bolnike smo slikali pred in po postopku zdravljenja, tako smo ocenjevali kratkotrajni in dolgoročni odgovor tumorjev z uporabo kriterijev RECIST. Ovrednotili smo stopnjo zmanjšanja simptomov in zaplete povezane z zdravljenjem. Rezultati. Skupno 27 postopkov embolizacije smo izvedli pri 17 bolnikih. Dvanajst od 17 bolnikov (70 %) je bilo simptomatskih, vključno s karcinoidnim sindromom (n = 8) in hudo, neobvladljivo hiperglikemijo (n = 4). Pri 8 od 12 bolnikov (67 %) smo ugotovili popolno izginotje simptomov, pri ostalih 4 (33 %) pa delno izboljšanje. En bolnik (6 %) je razvil ishemični holecistitis, sicer pa nismo zaznali nobenih drugih hepatobiliarnih zapletov. Kratkoročno sledenje bolnikov (> 3 mesece) s pomočjo slikanja je bilo na voljo pri 15 bolnikih in je pokazalo delni odgovor pri 14 bolnikih, pri enem bolniku pa napredovanje bolezni. Srednjeročno sledenje bolnikom (< 3 mesece) je bilo na voljo pri 12 bolnikih. Pri 7 bolnikoh (58 %) smo našli delen odgovor na zdravljenje, pri 3 (25 %) stabilno stanje bolezni in pri 2 (17 %) napredovanje bolezni. Zaključki. Kemoembolizacija s kroglicami SAP, eluiranimi z doksorubicinom, je varna in učinkovita metoda za zdravljenje jetrnih metastaz neuroendokrinih tumorjev in ima malo zapletov. Pomembno je, da klinično ni bilo zaslediti zapletov, kot so nekroza jeter ali žočnih vodov. Radiol Oncol 2017; 51(1): 81-87. doi:10.1515/raon-2016-0040 Dolgoročni izidi visokodoznega zdravljenja in transplantacije krvotvornih matičnih celic pri zdravljenju folikularnega limfoma in limfoma plaščnih celic. Izkušnje posamičnega centra Boltežar L, Pintarić K, Pretnar J, Pohar Perme M, Jezeršek Novaković B Izhodišča. Napredovali folikularni limfom in limfom plaščnih celic sta neozdravljivi bolezni, če jih zdravimo konvencionalno. Visokodozno zdravljenje in transplantacija krvotvornih matičnih celic predstavljata možnost podaljšanja časa brez napre­dovanja bolezni ter celokupnega preživetja. V raziskavi smo ugotavljali preživetje brez dogodka in celokupno preživetje pri bolnikih s folikularnim limfomom in limfomom plaščnih celic, zdravljenih s transplantacijo. Bolniki in metode. Analizirali smo 17 bolnikov s folikularnim limfomom in 29 bolnikov z limfomom plaščnih celic, ki smo jih zdravili s transplantacijo. 15 bolnikov je bilo v remisiji po zdravljenju drugega reda in 24 v remisiji po zdravljenju prvega reda. Vse bolnike smo zdravili z obsevanjem celega telesa in z visokim odmerkom ciklofosfamida med letoma 2006 in 2014. Vsi so s transplantacijo prejeli matične celice, ki smo jih pridobili iz periferne krvi. Rezultati. Ocenjeno 5-letno celokupno preživetje je bilo v skupini folikularnega limfoma 87,8 % (95 % interval zaupanja [CI] 59,5–96,8 %) in v skupini limfoma plaščnih celic 79,3 % (95 % CI 56,1–91,1 %). Ocenjeno 5-letno preživetje brez dogodka je bilo v skupini folikularnega limfoma 76,0 % (95 % CI 48,0–90,3 %) in v skupini limfoma plaščnih celic 69,8 % (95 % CI 45,5–84,8 %). V času analize podatkov nismo beležili nobenega sekundarnega hematološkega obolenja. Zaključki. Avtologna transplantacija krvotvornih matičnih celic je dobra terapevtska možnost za dolgoročna preživetja bolnikov s folikularnim limfomom in limfomom plaščnih celic z relativno nizko stopnjo poznih zapletov in sekundarnih hemato­loških malignomov. Radiol Oncol 2017; 51(1): 88-93. doi:10.1515/raon-2016-0035 Pogostnost pozitivnih peritonealnih izpirkov pri bolnicah z endometrijskim rakom po histeroskopiji ali abraziji maternične votline Dovnik A, Crnobrnja B, Žegura B, Takač I, Pakiž M Izhodišča. Namen raziskave je bil primerjati pogostnost pozitivnih peritonealnih izpirkov pri bolnicah z rakom endometrija, ki smo jim v diagnostičnem postopku naredili histeroskopijo ali abrazijo maternične votline. Metode. Opravili smo retrospektivno analizo 227 bolnic z rakom endometrija, ki smo jim naredili histeroskopijo (N = 144) ali abrazijo maternične votline (N = 83) v Univerzitetnem kliničnem centru Maribor med januarjem 2008 in decembrom 2014. V vsaki skupini smo določili pogostnost pozitivnih peritonealnih izpirkov. Rezultati. Med obema skupinama nismo ugotavili razlik v pogostnosti pozitivnih peritonealnih izpirkov (histeroskopija 13,2 %; abrazija maternične votline 12,0 %; p = 0,803). Natančnejša analiza bolnic s stadijem I pa je razkrila statistično značilno več pozitivnih izpirkov pri bolnicah po histeroskopiji (histeroskopija 12,8 %; abrazija maternične votline 3,4 %; p = 0.046). Med temi bolnicami ni bilo razlik v histološkem tipu tumorja (hi-kvadrat = 0,059; p = 0,807), diferenciaciji tumorja (hi-kvadrat = 3,709; p = 0,156), času med diagnozo in operacijo (t = 0,930; p = 0,357) in miometrijski invaziji (hi-kvadrat = 5,073; p = 0,079). Zaključki. Kljub temu, da v celotni populaciji ni bilo razlik v pogostnosti pozitivnih peritonealnih izpirkov po histeroskopiji ali po abraziji, smo pri bolnicah s stadijem I po histeroskopiji ugotovili statistično značilno pogostejše pozitivne peritonealne izpirke v primerjavi z abrazijo. Radiol Oncol 2017; 51(1): 94-100. doi:10.1515/raon-2016-0005 Izid implantatno-protetične rehabilitacije po obsevanju zaradi raka glave in vratu Cotič J, Jamšek J, Kuhar M, Ihan Hren N, Kansky A, Özcan M, Jevnikar P Izhodišča. V Sloveniji je visoka pojavnost raka glave in vratu. Zdravljenje najpogosteje vključuje kombinacijo kirurške obrav­nave in obsevanja. Možnosti kasnejše protetične rehabilitacije smo izboljšali ob uporabi sodobnih kirurških tehnik in zobnih vsadkov. Namen prispevka je prikazati izid implantatno-protetične oskrbe obsevanih bolnikov na Univerzitetnem kliničnem centru Ljubljana. Bolniki in metode. V raziskavi smo preverili izid zdravljenja 20 bolnikov po operaciji in obsevanju zaradi raka glave in vratu, ki smo jih oskrbeli z implantatno podprtimi protezami. Preživetje in uspešnost zobnih vsadkov smo statistično ovrednotili z metodo Kaplan-Meier, Coxovimi modeli sorazmernih tveganj in logistično regresijo. Rezultati. 20 bolnikov je skupno prejelo 100 zobnih vsadkov. Ocenjena stopnja preživetja vsadkov je bila 96 % po 1 letu in 87 % po 5 letih. Do odpovedi vsadkov je večinoma prišlo pred obremenitvijo (91,2 %). Vsadki v presajeni kosti so imeli statistič­no značilno slabše preživetje. Izmed 89 obremenjenih vsadkov jih je bilo 79 (88,7 %) uspešnih, kar je pomenilo funkcionalno uporabo protez in odsotnost bolečin ali napredujoče izgube kostnine. Opazili smo statistično značilno slabšanje uspeha z napredujočo starostjo, medtem ko izbor protetičnega sidra in število vsadkov pod protezo na uspeh rehabilitacije nista imela statistično značilnega vpliva. Zaključki. Implantatno podprte proteze so zanesljiv način protetične oskrbe po obsevanju zaradi raka glave in vratu. Bolnike je potrebno seznaniti z možnostjo zgodnje odpovedi vsadkov. Radiol Oncol 2017; 51(1): 101-112. doi:10.1515/raon-2017-0009 Določitev dozimetričnih parametrov za ščiten vir 153Gd pri brahiterapiji raka prostate Ghorbani M, Khajetash B, Ghatei N, Mehrpouyan M, Meigooni AS, Shahraini R Izhodišča. Intersticijska brahiterapija z rotirajočo zaščito (I-RSBT) je nedavno razvita metoda za zdravljenje raka prostate. V raziskavi smo želeli določiti dozimetrične parametre TG-43 vira 153Gd za uporabo v I-RSBT. Materiali in metode. Simulacijo vira 153Gd, umeščenega znotraj igle in zaščitenega s platino in aluminijevim oknom, smo naredili s kodo Monte Carlo MCNPX. Dozimetrične parametre za ta model vira smo izračunali v skladu s poročilom TG-43: moč zračne kerme, konstanta hitrosti doze, funkcija radialne doze in 2D funkcija anizotropije z in brez ščitenja. Rezultati. Moč zračne kerme je bila 6,71 U za neščiten vir z aktivnostjo 1 GBq. Za primere z zaščito iz platine in z aluminijevim oknom je bila ta vrednost 0,04 U in 6,19 U. Konstanta hitrosti doze za neščiten vir je bila 1,20 cGy/(hU); za vir z zaščito iz platine in z aluminijevim oknom na strani zašite 0,07 cGy/(hU); oziroma in na straneh z okni 0,96 cGy/(hU). Za te vire smo izdelali tabele za vrednosti funkcije radialne doze in anizotropije. Poleg tega smo za oceno učinka zaščite na razporeditev doze za vire z in brez zaščite izrisali izodozne krivulje. Zaključki. Dodatek zaščite iz platine lahko močno zniža dozo na kritične organe in normalna tkiva, ki ležijo na ščiteni strani. Izračunane vrednosti za moč zračne kerme, konstante hitrosti doze, funkcije radialne doze in podatki o funkciji 2D anizotropije za neščiten in ščiten vir 153Gd lahko uporabljamo v sistemih za načrtovanje obsevanj (TPS). Radiol Oncol 2017; 51(1): 113-120. doi:10.1515/raon-2017-0005 Optimalna strategija načrtovanja različnih ureditev lokov pri stereotaktičnem obsevanju prostate z volumsko modulirano ločno tehniko Kang SW, Chung JB, Kim JS, Kim IA, Eom KY, Song C, Lee JW, Kim JY, Suh TS Izhodišča. Cilj te raziskave je bil ugotoviti optimalno strategijo različnih ureditev lokov pri stereotaktičnem obsevanju prosta­ te z volumsko modulirano ločno tehniko (SBRT-VMAT). Bolniki in metode. Da bi raziskali, kako različne ureditve lokov vplivajo na dozimetrične in biološke podatke, smo SBRT­VMAT načrte za osemnajst bolnikov naredili z ureditvijo z enim polnim lokom (1PL), enim delnim lokom (1DL), dvojnim polnim lokom (2PL), in dvema delnima lokoma (2DL). Vse načrte smo izračunali s pomočjo algoritma Acuros XB. Dozimetrične in radiobiološke podatke za tarčne volumne in rizične organe smo ovrednotili iz dozno volumskih histogramov. Rezultati. Vsi načrti so bili zelo konformni (CI <1.05, CN = 0,91) in homogeni (HI = 0,09-0,12) za tarčne volumne. Pri rizičnih organih ni bilo razlike v dozi na mehur, medtem ko je bila velika razlika v dozi na rektum in na glavico stegnenice. Načrti, ki so uporabljali 1DL in 2DL so pokazali močno zmanjšanje srednje doze na rektum v primerjavi z načrti, ki so uporabljali 1PL in 2PL. D2% in srednja doza na obe glavici stegnenice pa so bile vedno nižje v načrtih z uporabo 1PL in 2PL. Povprečna verjetnost nadzora tumorja in verjetnost zapletov normalnih tkiv so bili primerljivi v vseh načrtih z uporabo različnih ločnih ureditev. Zaključki. Uporaba 1DL je omogočala krajši čas obsevanja ob enaki pokritosti tarče z boljšim varovanjem rektuma, čeprav so vsi načrti, ki so uporabljali štiri različne ločne ureditve, v splošnem pokazali podobne dozimetrične in biološke rezultate meritev. D2% in povprečni odmerek na glavico stegnenice se je nekoliko povečal, vendar je ostal v okviru dovoljenega od­stopanja. Ta študija kaže, da je uporaba 1DL privlačna izbira za zdravljenje prostate s SBRT-VMAT. 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 first quarter of 2017 Doc. dr. Josip Cholewa Foundation for cancer research and education continues with its planned activi­ties in the first quarter of 2017 and is commencing to prepare for the activities the whole year. Its pri­mary focus remains the provision of grants and scholarships and other forms of financial assistance for basic, clinical and public health research in the field of oncology. An analysis of the ongoing activities in the last year was made in order to make an assessment of the impact of Foundation's activities, thus providing a basis for developing new strategies and approaches in its scope of fight against cancer. 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 can­cer. The Foundation makes great efforts to provide financial and other kinds of support for the organisation of various forms of meetings to extend and broaden the knowledge about prevention of cancer, early detection of various types of cancer, its treatment and rehabilitation of cancer patients. The advances in knowledge of all aspects of dealing with cancer should be in Foundation's opinion available to all the professionals that treat cancer patients, to the patients themselves and their closest relatives and friends, and finally also to the general public. The problems associated with cancer affect more and more people and their relatives in Slovenia and elsewhere. The Foundation will therefore continue with its activities in the years to come. Treatment of cancer is ever more successful with many patients surviving decades after the start of their treatment and many new problems and challenges have thus come into place. Longer survival of an increasing number of patients with previously incurable cancer conditions adds many new dimensions to their life and to the life of their families. It also confronts cancer specialists, all the other experts and lay pub­lic dealing with cancer with new challenges and new goals to achieve. Borut Štabuc, M.D., Ph.D. Tomaž Benulič, M.D. Andrej Plesničar, M.D., M.Sc. Viljem Kovač M.D., Ph.D. Borut Štabuc, M.D., Ph.D. O VA INDIKACIJA1 Vectibix® + FOLFIRI v 1. liniji zdravljenja bolnikov z mKRR in nemutiranim genom RAS Zdravilo Vectibix® je sedaj indicirano za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (mKRR) in nemutiranim genom RAS: v 1. liniji zdravljenja v kombinaciji s FOLFOX ali FOLFIRI v 2. liniji zdravljenja v kombinaciji s FOLFIRI pri bolnikih, ki so v prvi liniji zdravljenja prejemali kemoterapijo, ki je vkljucevala fluoropirimidin ˇ (vendar ni vkljucevala irinotekana) ˇ kot monoterapija po neuspehu shem kemoterapije, ki so vkljucevale fluoropirimidin, oksaliplatin in irinotekan. ˇ Pred predpisovanjem natančno preberite celoten Povzetek glavnih Zdravila Iclusig se ne sme uporabljati pri bolnikih z miokardnim infarktom, zdravila: Predpisovanje in izdaja zdravila je le na recept. Imetnik dovoljenja za substratih P-glikoproteina (P-gp) ali beljakovine rezistence za raka dojke promet z zdravilom: ARIAD Pharma Ltd., Riverbridge House, Guildford (BCRP). Pri sočasni uporabi ponatiniba z zdravili proti strjevanju krvi pri Road, Leatherhead, Surrey KT22 9AD, Velika Britanija. Zadnja revizija bolnikih, pri katerih obstaja tveganje za krvavitev, je potrebna previdnost. Plodnost, nosečnost in dojenje: Ženskam v rodni dobi je treba svetovati, Podrobnejše informacije o zdravilu Iclusig so na voljo pri predstavniku da naj v času zdravljenja z zdravilom Iclusig ne zanosijo, moškim pa, da naj v imetnika dovoljenja za promet z zdravilom: Angelini Pharma d.o.o., Koprska času zdravljenja ne zaplodijo otroka. Med zdravljenjem je treba uporabljati ulica 108A, 1000 Ljubljana, Tel.: +386 1 544 65 79, E-pošta: info@angelini.si Za to zdravilo se izvaja dodatno spremljanje varnosti. SESTAVA*: Lonsurf 15 mg/6,14 mg: Ena filmsko obložena tableta vsebuje 15 mg trifluridina in 6,14 mg tipiracila (v obliki klorida). Lonsurf 20 mg/8,19 mg: Ena filmsko obložena tableta vsebuje 20 mg trifluridina in 8,19 mg tipiracila (v obliki klorida). TERAPEVTSKE INDIKACIJE*: Zdravilo Lonsurf je indicirano za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom, ki so bili predhodno že zdravljeni ali niso primerni za zdravljenja, ki so na voljo. Ta vključujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF – Vascular Endothelial Growth Factor) in zaviralci receptorjev za epidermalni rastni dejavnik (EGFR – Epidermal Growth Factor Receptor). ODMERJANJE IN NAČIN UPORABE*: Odmerjanje: Priporočeni začetni odmerek zdravila Lonsur f pri odraslih je 35 mg/m2/odmerek peroralno dvakrat dnevno na 1. do 5. dan in 8. do 12. dan vsakega 28-dnevnega cikla zdravljenja, najpozneje 1 uro po zaključku jutranjega in večernega obroka. Odmerjanje, izračunano glede na telesno površino, ne sme preseči 80 mg/ odmerek. Možne prilagoditve odmerka glede na varnost in prenašanje zdravila: dovoljena so največ 3 zmanjšanja odmerka na najmanjši odmerek 20 mg/m2 dvakrat dnevno. Potem ko je bil odmerek zmanjšan, povečanje ni dovoljeno. KONTRAINDIKACIJE*: Preobčutljivost na zdravilni učinkovini ali katero koli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI*: Supresija kostnega mozga: Pred uvedbo zdravljenja, pred vsakim ciklom zdravljenja in po potrebi je treba pregledati celotno krvno sliko. Zdravljenja ne smete začeti, če je absolutno število nevtrofilcev < 1,5 x 109/l, če je število trombocitov < 75 x 109/l ali če se je pri bolniku zaradi predhodnih zdravljenj pojavila klinično pomembna nehematološka toksičnost 3. ali 4. stopnje, ki še traja. Bolnike je treba skrbno spremljati zaradi morebitnih okužb, uvesti je treba ustrezne ukrepe, kot je klinično indicirano. Toksičnost za prebavila: Potrebna je uporaba antiemetikov, antidiaroikov ter drugih ukrepov, kot je klinično indicirano. Če je potrebno, prilagodite odmerke. Ledvična okvara: Zdravilo Lonsurf ni primerno za uporabo pri bolnikih s hudo ledvično okvaro ali končno stopnjo ledvične okvare. Bolnike z zmerno ledvično okvaro je treba zaradi hematološke toksičnosti bolj pogosto spremljati. Jetrna okvara: Uporaba zdravila Lonsur f pri bolnikih z zmerno ali hudo jetrno okvaro ni priporočljiva. Proteinurija: Pred začetkom zdravljenja in med njim je priporočljivo spremljanje proteinurije z urinskimi testnimi lističi. Pomožne snovi: Vsebujejo laktozo. INTERAKCIJE*: Zdravila, ki medsebojno delujejo z nukleozidnimi prenašalci CNT1, ENT1 in ENT2, zaviralci OCT2 ali MATE1, substrati humane timidin-kinaze (npr. zidovudinom), hormonskimi kontraceptivi. PLODNOST*, NOSEČNOST IN DOJENJE*: Ni priporočljivo. KONTRACEPCIJA*: Ženske in moški morajo uporabljati učinkovito metodo kontracepcije med zdravljenjem in do 6 mesecev po zaključku zdravljenja. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVL JANJA S STROJI*: Med zdravljenjem se lahko pojavijo utrujenost, omotica ali splošno slabo počutje. NEŽELENI UČINKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, zmanjšan apetit, diareja, navzea, bruhanje, utrujenost. Pogosti: okužba spodnjih dihal, okužba zgornjih dihal, febrilna nevtropenija, limfopenija, monocitoza, hipoalbuminemija, nespečnost, disgevzija, periferna nevropatija, omotica, glavobol, vročinski oblivi, dispneja, kašelj, bolečina v trebuhu, zapr tje, stomatitis, bolezni ustne votline, hiperbilirubinemija, sindrom palmarne plantarne eritrodisestezije, izpuščaj, alopecija, pruritus, suha koža, proteinurija, pireksija, edem, vnetje sluznice, splošno slabo počutje, zvišanje jetrnih encimov, zvišanje alkalne fosfataze v krvi, zmanjšanje telesne mase. Občasni: septični šok, infekcijski enteritis, pljučnica, okužba žolčevoda, gripa, okužba sečil, vnetje dlesni, herpes zoster, tinea pedis, kandidiaza, bakterijska okužba, okužba, bolečina zaradi raka, pancitopenija, granulocitopenija, monocitopenija, eritropenija, levkocitoza, dehidracija, hiperglikemija, hiperkaliemija, hipokaliemija, hipofosfatemija, hipernatriemija, hiponatriemija, hipokalciemija, protin, anksioznost, nevrotoksičnost, disestezija, hiperestezija, hipoestezija, sinkopa, par­estezija, pekoč občutek, letargija, zmanjšana ostrina vida, zamegljen vid, diplopija, katarakta, konjunktivitis, suho oko, vrtoglavica, neugodje v ušesu, angina pektoris, aritmija, palpitacije, embolija, hipertenzija, hipotenzija, pljučna embolija, plevralni izliv, izcedek iz nosu, disfonija, orofaringealna bolečina, epistaksa, hemoragični enterokolitis, kr vavitev v prebavilih, akutni pankreatitis, ascites, ileus, subileus, kolitis, gastritis, refluksni gastritis, ezofagitis, moteno praznjenje želodca, abdominalna distenzija, analno vnetje, razjede v ustih, dispepsija, gastroezofagealna refluksna bolezen, proktalgija, bukalni polip, krvavitev dlesni, glositis, parodontalna bolezen, bolezen zob, siljenje na bruhanje, flatulenca, slab zadah, hepatotoksičnost, razširitev žolčnih vodov, luščenje kože, ur tikarija, preobčutljivostne reakcije na svetlobo, eritem, akne, hiperhidroza, žulj, bolezni nohtov, otekanje sklepov, artralgija, bolečina v kosteh, mialgija, mišično-skeletna bolečina, mišična oslabelost, mišični krči, bolečina v okončinah, občutek teže, ledvična odpoved, neinfektivni cistitis, motnje mikcije, hematurija, levkociturija, motnje menstruacije, poslabšanje splošnega zdravstvenega stanja, bolečina, občutek spremembe telesne temperature, kseroza, zvišanje kreatinina v krvi, podaljšanje intervala QT na elektrokardiogramu, povečanje mednarodnega umerjenega razmerja (INR), podaljšanje aktiviranega parcialnega tromboplastinskega časa (aPTČ), zvišanje sečnine v krvi, zvišanje laktatne dehidrogenaze v kr vi, znižanje celokupnih proteinov, zvišanje C-reaktivnega proteina, zmanjšan hematokrit. Post-marketinške izkušnje: pri bolnikih, zdravljenih z zdravilom Lonsurf na Japonskem, so poročali o primerih intersticijske bolezni pljuč. PREVELIKO ODMERJANJE*: Neželeni učinki, o katerih so poročali v povezavi s prevelikim odmerjanjem, so bili v skladu z uveljavljenim varnostnim profilom. Glavni pričakovani zaplet prevelikega odmerjanja je supresija kostnega mozga. FARMAKODINAMIČNE LASTNOSTI*: Farmakoterapevtska skupina: zdravila z delovanjem na novotvorbe, antimetaboliti, oznaka ATC: L01BC59. Zdravilo Lonsurf sestavljata antineoplastični timidinski nukleozidni analog, trifluridin, in zaviralec timidin-fosforilaze (TPaze), tipiracilijev klorid. Po privzemu v rakave celice timidin-kinaza fosforilira trifluridin. Ta se v celicah nato presnovi v substrat deoksiribonukleinske kisline (DNA), ki se vgradi neposredno v DNA ter tako preprečuje celično proliferacijo. TPaza hitro razgradi trifluridin in njegova presnova po peroralni uporabi je hitra zaradi učinka prvega prehoda, zato je v zdravilo vključen zaviralec TPaze, tipiracilijev klorid. PAKIRANJE*: 20 filmsko obloženih tablet. NAČIN PREDPISOVANJA IN IZDA JANJA ZDRAVILA: Rp/Spec. Imetnik dovoljenja za promet z zdravilom: Les Laboratoires Servier, 50, rue Carnot, 92284 Suresnes cedex, Francija. Številka dovoljenja za promet z zdravilom: EU/1/16/1096/001 (Lonsurf 15 mg/6,14 mg), EU/ 1/16/1096/004 (Lonsurf 20 mg/8,19 mg). * Pred predpisovanjem preberite celoten povzetek glavnih značilnosti zdravila. Datum zadnje revizije besedila: april 2016. Celoten povzetek glavnih značilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., tel: 01 563 48 11, ww w.servier.si. Datum priprave informacije: januar 2017. LNF16/17C1AD2 Samo za strokovno javnost. DVOJNO ZAVIRANJE. VEČJA UČINKOVITOST. Skrajšan povzetek glavnih značilnosti zdravila COTELLIC: Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Kako poročati o neželenih učinkih, si poglejte skrajšani povzetek glavnih značilnosti zdravila pod ‘’Poročanje o domnevnih neželenih učinkih’’. Ime zdravila: Cotellic 20 mg filmsko obložene tablete. Kakovostna in količinska sestava: Ena filmsko obložena tableta vsebuje kobimetinibijev hemifumarat, kolikor ga ustreza 20 mg kobimetiniba. Pomožna snov z znanim učinkom: Ena filmsko obložena tableta vsebuje 36 mg laktoze monohidrata. Terapevtske indikacije: Zdravilo Cotellic je v kombinaciji z vemurafenibom indicirano za zdravljenje odraslih bolnikov z neoperabilnim ali metastatskim melanomom, ki ima mutacijo BRAF V600. Odmerjanje in način uporabe: Zdravljenje z zdravilom Cotellic v kombinaciji z vemurafenibom sme uvesti in nadzorovati le usposobljen zdravnik, ki ima izkušnje z uporabo zdravil proti raku. Pred začetkom zdravljenja je treba z validirano preiskavo potrditi, da ima bolnik melanom z mutacijo BRAF V600. Odmerjanje: Vsak odmerek obsega tri 20-mg tablete (60 mg) in ga je treba vzeti enkrat na dan 21 dni zapored, temu sledi 7-dnevni premor. Zdravljenje z zdravilom Cotellic je treba nadaljevati, dokler bolniku ne koristi več oziroma do pojava nesprejemljive toksičnosti. Če bolnik izpusti odmerek, ga lahko vzame do 12 ur pred naslednjim odmerkom, da ohrani shemo enkrat na dan. Če bolnik po uporabi zdravila Cotellic bruha, tisti dan ne sme vzeti dodatnega odmerka, temveč mora zdravljenje nadaljevati naslednji dan, kot je predpisano. Splošne prilagoditve odmerka: Odločitev za zmanjšanje odmerka zdravila Cotellic ali vemurafeniba mora temeljiti na zdravnikovi oceni varnosti in prenašanja pri posameznem bolniku. Ko je bil odmerek enkrat zmanjšan, se ga kasneje ne sme več povečati. Nasvet za prilagoditev odmerka v primeru disfunkcije levega prekata: Če so srčni simptomi posledica zdravila Cotellic in se po prehodni prekinitvi njegove uporabe ne izboljšajo, je treba razmisliti o trajnem prenehanju zdravljenja z zdravilom Cotellic. Nasvet za prilagoditev odmerka zdravila Cotellic med uporabo z vemurafenibom: Jetrna laboratorijska odstopanja: Stopnja 3: Zdravilo Cotellic je treba nadaljevati v predpisanem odmerku. Odmerek vemurafeniba je mogoče zmanjšati, kot je klinično primerno. Stopnja 4: Zdravljenje z zdravilom Cotellic in zdravljenje z vemurafenibom je treba prekiniti. Za dodatna navodila glejte celotni povzetek glavnih značilnosti zdravila Cotellic. Zvišanja kreatin-fosfokinaze (CPK): Za obvladovanje nesimptomatskih zvišanj CPK odmerka zdravila Cotellic ni treba prilagoditi ali prekiniti. Fotosenzibilnost: Fotosenzibilnost stopnje . 2 je treba obvladovati s podpornim zdravljenjem. Fotosenzibilnost stopnje 2 ali stopnje . 3: zdravili Cotellic in vemurafenib je treba prekiniti, dokler se ne zmanjša na stopnjo . 1. Zdravljenje je mogoče znova začeti brez spremembe odmerka zdravila Cotellic. Odmerjanje vemurafeniba je treba zmanjšati, kot je klinično primerno; za dodatne informacije glejte povzetek glavnih značilnosti vemurafeniba. Izpuščaj: Izpuščaj se lahko pojavi tako med zdravljenjem z zdravilom Cotellic kot med zdravljenjem z vemurafenibom. Odmerek zdravila Cotellic in/ali vemurafeniba je mogoče začasno prekiniti in/ali zmanjšati, kot je klinično primerno. Za dodatna navodila glejte celotni povzetek glavnih značilnosti zdravila Cotellic. Podaljšanje intervala QT: Če interval QTc med zdravljenjem preseže 500 ms, prosimo, glejte povzetek glavnih značilnosti vemurafeniba. Sprememba odmerkov zdravila Cotellic ni potrebna, kadar se ta uporablja v kombinaciji z vemurafenibom. Posebne populacije bolnikov: Bolnikom, starim . 65 let, odmerka ni treba prilagoditi. Pri bolnikih s hudo okvaro ledvic je treba zdravilo Cotellic uporabljati previdno. Bolnikom z okvaro jeter odmerka ni treba prilagoditi. Bolniki s hudo okvaro jeter imajo lahko v primerjavi z bolniki z normalnim delovanjem jeter zvišane koncentracije prostega kobimetiniba v plazmi. Pri zdravljenju z zdravilom Cotellic se lahko pojavijo z jetri povezane laboratorijske nepravilnosti, zato je treba pri bolnikih z okvaro jeter katere koli stopnje zdravilo uporabljati previdno.Varnost in učinkovitost zdravila Cotellic pri otrocih in mladostnikih, mlajših od 18 let, nista ugotovljeni. Način uporabe: Zdravilo Cotellic je za peroralno uporabo. Tablete je treba zaužiti cele, z vodo. Lahko se jemljejo skupaj s hrano ali brez nje. Kontraindikacije: Preobčutljivost na zdravilno učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Zdravilo Cotellic v kombinaciji z vemurafenibom pri bolnikih, katerih bolezen je napredovala med zdravljenjem z zaviralcem BRAF: Podatkov pri teh bolnikih je malo. Ti kažejo, da je učinkovitost kombinacije pri teh bolnikih manjša. Zato je treba razmisliti o drugih možnostih zdravljenja pred uvedbo kombinacije. Zdravilo Cotellic v kombinaciji z vemurafenibom pri bolnikih z zasevki v možganih: Varnost in učinkovitost kombinacije zdravila Cotellic in vemurafeniba pri teh bolnikih ni bila ocenjena. Intrakranialna aktivnost kobimetiniba trenutno ni poznana. Serozna retinopatija: Pri bolnikih, zdravljenih z zaviralci MEK, vključno z zdravilom Cotellic, so opažali serozno retinopatijo. Večina primerov je bila opisana kot horioretinopatija ali odstop mrežnice. Večina dogodkov, opaženih v kliničnih preskušanjih, je po prekinitvi ali zmanjšanju odmerka izzvenela ali se izboljšala na nesimptomatsko stopnjo 1. Bolnike je treba na vsakem pregledu oceniti glede simptomov novih motenj vida ali poslabšanja obstoječih motenj vida. V primeru simptomov novih motenj vida ali poslabšanju obstoječih je priporočljiv oftalmološki pregled. Serozno retinopatijo je mogoče obvladati s prekinitvijo zdravljenja, zmanjšanjem odmerka ali prenehanjem zdravljenja. Disfunkcija levega prekata: Pri bolnikih, ki so prejemali zdravilo Cotellic, so poročali o zmanjšanju iztisnega deleža levega prekata (LVEF) v primerjavi z izhodiščem. LVEF je treba izmeriti pred začetkom zdravljenja za določitev izhodiščne vrednosti, nato pa ga kontrolirati po prvem mesecu zdravljenja ter vsaj na 3 mesece oziroma kot je klinično indicirano, do prenehanja zdravljenja. Zmanjšanje LVEF od izhodišča je mogoče obvladati s prekinitvijo zdravljenja, zmanjšanjem odmerka ali prenehanjem zdravljenja. Bolnikov z izhodiščnim LVEF pod spodnjo mejo normalne vrednosti (SMN) za ustanovo ali pod 50 % niso proučevali. Jetrna laboratorijska odstopanja: Jetrna laboratorijska odstopanja se lahko pojavijo tako med uporabo zdravila Cotellic v kombinaciji z vemurafenibom kot med samostojnim zdravljenjem z vemurafenibom. Pri bolnikih, zdravljenih s kombinacijo zdravila Cotellic in vemurafeniba, so opažali jetrna laboratorijska odstopanja, zlasti zvišanje ALT, AST in AF. Jetrna laboratorijska odstopanja je treba kontrolirati z laboratorijskimi preiskavami jeter pred začetkom kombiniranega zdravljenja in vsak mesec med zdravljenjem, lahko pa tudi pogosteje, če je klinično indicirano. Laboratorijska odstopanja stopnje 3 je treba obvladati s prekinitvijo uporabe vemurafeniba ali zmanjšanjem odmerka. Jetrna laboratorijska odstopanja stopnje 4 se obvlada s prekinitvijo zdravljenja, zmanjšanjem odmerka ali prenehanjem zdravljenja z zdravilom Cotellic in z vemurafenibom. Driska: Pri bolnikih, zdravljenih z zdravilom Cotellic, so poročali o primerih driske stopnje . 3 in resni driski. Drisko je treba obvladati z antidiaroiki in podpornim zdravljenjem. V primeru driske stopnje . 3, ki se pojavi kljub podpornemu zdravljenju, je treba obe zdravili, Cotellic in vemurafenib, prekiniti, dokler se driska ne izboljša na stopnjo . 1. Če se driska stopnje . 3 ponovi, je treba odmerek zdravila Cotellic in vemurafeniba zmanjšati. Intoleranca za laktozo: Zdravilo vsebuje laktozo. Bolniki z redko dedno intoleranco za galaktozo, laponsko obliko pomanjkanja laktaze ali malabsorpcijo glukoze/galaktoze se morajo posvetovati z zdravnikom in se z njim pogovoriti, ali zanje koristi zdravljenja odtehtajo tveganje. Podaljšanje intervala QT: Če interval QTc med zdravljenjem preseže 500 ms, prosimo, glejte povzetka glavnih značilnosti vemurafeniba. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Učinki drugih zdravil na kobimetinib: Zaviralci CYP3A: Med zdravljenjem s kobimetinibom se izogibajte sočasni uporabi močnih zaviralcev CYP3A. Če se sočasni uporabi močnega zaviralca CYP3A ni mogoče izogniti, je treba bolnike skrbno nadzirati glede varnosti. Previdnost je potrebna, če se kobimetinib uporablja sočasno z zmernimi zaviralci CYP3A. Če se kobimetinib uporablja sočasno z zmernim zaviralcem CYP3A, je treba bolnike skrbno nadzirati glede varnosti. Induktorji CYP3A: Treba se je izogibati sočasni uporabi z zmernimi in močnimi induktorji CYP3A. Razmisliti je treba o uporabi drugih zdravil, ki CYP3A inducirajo le malo ali sploh ne. Ker se med sočasno uporabo zmernih do močnih induktorjev CYP3A koncentracija kobimetiniba verjetno bistveno zniža, se lahko njegova učinkovitost pri bolniku poslabša. Zaviralci P-glikoproteina: Sočasna uporaba zaviralcev P-gp, npr. ciklosporina in verapamila, lahko zviša koncentracijo kobimetiniba v plazmi. Učinki kobimetiniba na druga zdravila: Substrati CYP3A in CYP2D6: Klinična študija medsebojnega delovanja zdravil pri onkoloških bolnikih je pokazala, da se koncentraciji midazolama in dekstrometorfana v plazmi v prisotnosti kobimetiniba nista spremenili. Substrati CYP1A2: In vitro je kobimetinib potencialen induktor CYP1A2 in lahko zato zmanjša izpostavljenost substratom tega encima, npr. teofilinu. Substrati BCRP: In vitro kobimetinib zmerno zavira BCRP. Klinično pomembnega zavrtja BCRP na ravni črevesja ni mogoče izključiti. Druga zdravila proti raku: vemurafenib: Pri bolnikih z neoperabilnim ali metastatskim melanomom ni dokazov o klinično pomembnem medsebojnem delovanju med kobimetinibom in vemurafenibom, zato prilagoditve odmerkov niso potrebne. Vpliv kobimetiniba na transportne sisteme zdravil: Študije in vitro kažejo, da kobimetinib ni substrat jetrnih privzemnih prenašalcev OATP1B1, OATP1B3 in OCT1, vendar pa jih rahlo zavira. Klinični pomen teh izsledkov ni raziskan. Pediatrična populacija: Študije medsebojnega delovanja so izvedli le pri odraslih. Neželeni učinki: Neželeni učinki pri bolnikih, zdravljenih z zdravilom Cotellic v kombinaciji z vemurafenibom: Zelo pogosti: anemija, serozna retinopatija, zamegljen vid, hipertenzija, krvavitev, driska, navzea, bruhanje, fotosenzibilnost, izpuščaj, makulopapulozen izpuščaj, akneiformni dermatitis, hiperkeratoza, zvišana telesna temperatura, mrzlica, zvišanja CPK, ALT, AST, gama-glutamiltransferaze (GGT) in AF v krvi. Pogosti: bazalnocelični karcinom, ploščatocelični karcinom kože, keratoakantom, dehidracija, hipofosfatemija, hiponatriemija, hiperglikemija, okvara vida, pnevmonitis, zmanjšan iztisni delež in zvišanje bilirubina v krvi. Poročanje o domnevnih neželenih učinkih: Poročanje o domnevnih neželenih učinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoča namreč stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da poročajo o katerem koli domnevnem neželenem učinku zdravila na: Univerzitetni klinični center Ljubljana, Interna klinika, Center za zastrupitve, Zaloška cesta 2, SI-1000 Ljubljana, Faks: + 386 (0)1 434 76 46, e-pošta: farmakovigilanca@kclj.si. Režim izdaje zdravila: Rp/Spec. Imetnik dovoljenja za promet: Roche Registration Limited, 6 Falcon Way, Shire Park, Welwyn Garden City, AL7 1TW, Velika Britanija. Verzija: 2.0/16. Informacija pripravljena: januar 2017. Skrajšan povzetek glavnih značilnosti zdravila ZELBORAF: Ime zdravila: Zelboraf 240 mg filmsko obložene tablete. Kakovostna in količinska sestava: Ena tableta vsebuje 240 mg vemurafeniba (v obliki precipitata vemurafeniba in hipromeloze acetat sukcinata). Terapevtske indikacije: Vemurafenib je indiciran za samostojno zdravljenje odraslih bolnikov z neresektabilnim ali metastatskim melanomom, s pozitivno mutacijo BRAF V600. Odmerjanje in način uporabe: Zdravljenje z vemurafenibom mora uvesti in nadzorovati usposobljen zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje raka. Odmerjanje: Priporočeni odmerek vemurafeniba je 960 mg (4 tablete po 240 mg) dvakrat na dan (to ustreza celotnemu dnevnemu odmerku 1920 mg). Vemurafenib lahko vzamemo s hrano ali brez nje, izogibati pa se moramo stalnemu jemanju obeh dnevnih odmerkov na prazen želodec. Zdravljenje z vemurafenibom moramo nadaljevati do napredovanja bolezni ali pojava nesprejemljive toksičnosti. Če bolnik izpusti odmerek, ga lahko vzame do 4 ure pred naslednjim odmerkom za ohranitev sheme dvakrat na dan. Obeh odmerkov pa ne sme vzeti hkrati. Če bolnik po zaužitju vemurafeniba bruha, ne sme vzeti dodatnega odmerka zdravila, ampak mora z zdravljenjem normalno nadaljevati. Prilagoditve odmerjanja: Za obvladovanje neželenih učinkov ali ob podaljšanju intervala QTc je potrebno zmanjšanje odmerka, začasna prekinitev in/ali dokončno prenehanje zdravljenja (za podrobnosti o prilagoditvi odmerka, prosimo glejte SmPC zdravila). Zmanjšanje odmerka pod 480 mg dvakrat na dan ni priporočljivo. Če se pri bolniku pojavi ploščatocelični karcinom kože, priporočamo nadaljevanje zdravljenja brez zmanjšanja odmerka vemurafeniba. Posebne populacije: Za bolnike, starejše od 65 let, prilagajanje odmerka ni potrebno. O bolnikih z okvaro ledvic ali jeter je na voljo malo podatkov. Bolnike s hudo okvaro ledvic ali z zmerno do hudo okvaro jeter je treba pozorno spremljati. Varnost in učinkovitost vemurafeniba pri otrocih in mladostnikih, mlajših od 18 let, še nista bili dokazani. Podatkov ni na voljo. Način uporabe: Vemurafenib je namenjen peroralni uporabi. Tablete je treba zaužiti cele, z vodo. Ne sme se jih žvečiti ali zdrobiti. Kontraindikacije: Preobčutljivost na zdravilno učinkovino ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Pred uporabo vemurafeniba je treba z validirano preiskavo potrditi, da ima bolnik tumor s pozitivno mutacijo BRAF V600. Dokazi o učinkovitosti in varnosti vemurafeniba pri bolnikih s tumorji z izraženo redko BRAF V600 mutacijo, ki ni V600E ali V600K, niso prepričljivi. Vemurafeniba se ne sme uporabljati pri bolnikih z malignim melanomom, ki ima divji tip BRAF. Preobčutljivostne reakcije: V povezavi z vemurafenibom so bile opisane resne preobčutljivostne reakcije, vključno z anafilaksijo. Hude preobčutljivostne reakcije lahko vključujejo Stevens-Johnsonov sindrom, generaliziran izpuščaj, eritem ali hipotenzijo. Pri bolnikih, pri katerih se pojavijo resne preobčutljivostne reakcije, je treba zdravljenje z vemurafenibom dokončno opustiti. Kožne reakcije: Pri bolnikih, ki so prejemali vemurafenib, so v ključnem kliničnem preskušanju poročali o hudih kožnih reakcijah, vključno z redkim Stevens-Johnsonovim sindromom in toksično epidermalno nekrolizo. Po prihodu vemurafeniba na trg so v povezavi z njim poročali o reakciji na zdravilo z eozinofilijo in sistemskimi simptomi. Pri bolnikih, pri katerih se pojavi huda kožna reakcija, je treba zdravljenje z vemurafenibom dokončno opustiti. Povečanje toksičnosti obsevanja: Pri bolnikih, ki so se pred, med ali po zdravljenju z vemurafenibom zdravili z obsevanjem, so poročali o primerih vnetnih reakcij na mestu obsevanja (t.i. radiation recall) in povečane občutljivosti na obsevanje. Večina primerov je bila po naravi kožnih, a nekaj primerov, ki je vključevalo visceralne organe, je imelo smrtni izid. Pri sočasni ali zaporedni uporabi vemurafeniba in obsevanja je potrebna previdnost. Podaljšanje intervala QT: V nekontrolirani, odprti študiji faze II pri predhodno zdravljenih bolnikih z metastatskim melanomom, so opazili podaljšanje intervala QT, odvisnega od izpostavljenosti vemurafenibu. Podaljšanje intervala QT lahko poveča tveganje za ventrikularne aritmije, vključno s t. i. Torsade de Pointes. Z vemurafenibom ni priporočljivo zdraviti bolnikov z elektrolitskimi motnjami (vključno z magnezijem), ki jih ni mogoče odpraviti, bolnikov s sindromom dolgega intervala QT in bolnikov, zdravljenih z zdravili, ki podaljšajo interval QT. Pred zdravljenjem z vemurafenibom, en mesec po zdravljenju in po spremembi odmerka je treba pri vseh bolnikih posneti EKG in kontrolirati elektrolite (vključno z magnezijem). Nadaljnje kontrole so priporočljive predvsem pri bolnikih z zmerno do hudo jetrno okvaro, in sicer mesečno prve 3 mesece zdravljenja, potem pa na 3 mesece oziroma pogosteje, če je to klinično indicirano. Zdravljenja z vemurafenibom ni priporočljivo uvesti pri bolnikih, ki imajo interval QTc > 500 ms. Bolezni oči: Poročali so o resnih neželenih učinkih na očeh, vključno z uveitisom, iritisom in zaporo mrežnične vene. Bolnikom je treba oči redno kontrolirati glede morebitnih neželenih učinkov na očeh. Ploščatocelični karcinom kože: Pri bolnikih, zdravljenih z vemurafenibom, so bili opisani primeri ploščatoceličnega karcinoma kože, vključno s ploščatoceličnim karcinomom, opredeljenim kot keratoakantom ali mešani keratoakantom. Priporočljivo je, da vsi bolniki pred uvedbo zdravljenja opravijo dermatološki pregled in da so med zdravljenjem deležni rednih kontrol. Vsako sumljivo spremembo je treba izrezati, poslati na histopatološko oceno in jo zdraviti v skladu z lokalnimi smernicami. Med zdravljenjem in do šest mesecev po zdravljenju ploščatoceličnega karcinoma mora zdravnik enkrat mesečno pregledati bolnika. Pri bolnikih, ki se jim pojavi ploščatocelični karcinom kože, je priporočljivo nadaljevati zdravljenje brez zmanjšanja odmerka. Nadzor se mora nadaljevati še 6 mesecev po prenehanju zdravljenja z vemurafenibom ali do uvedbe drugega antineoplastičnega zdravljenja. Bolnikom je treba naročiti, naj svojega zdravnika obvestijo o pojavu kakršnih koli sprememb na koži. Ploščatocelični karcinom, ki se ne nahaja na koži: Pri bolnikih, ki so prejemali vemurafenib v kliničnih preskušanjih, so poročali o primerih ploščatoceličnega karcinoma, ki se ne nahaja na koži. Bolnikom je treba pred uvedbo zdravljenja in na 3 mesece med zdravljenjem pregledati glavo in vrat (pregled mora obsegati vsaj ogled ustne sluznice in palpacijo bezgavk). Poleg tega morajo bolniki pred zdravljenjem in na 6 mesecev med zdravljenjem opraviti računalniško tomografijo prsnega koša. Pred in po končanem zdravljenju ali kadar je klinično indicirano, je priporočljivo opraviti pregled zadnjika in ginekološki pregled. Po prenehanju zdravljenja z vemurafenibom se mora nadzor glede ploščatoceličnega karcinoma, ki se ne nahaja na koži, nadaljevati še 6 mesecev ali do uvedbe drugega antineoplastičnega zdravljenja. Nenormalne spremembe je treba obravnavati v skladu s klinično prakso. Novi primarni melanom: V kliničnih preskušanjih so poročali o novih primarnih melanomih. Bolnike s takšnimi primeri so zdravili z ekscizijo, bolniki pa so nadaljevali z zdravljenjem brez prilagoditve odmerka. Nadzor nad pojavom kožnih lezij je treba izvajati, kot je navedeno zgoraj pri ploščatoceličnem karcinomu kože. Druge malignosti: Glede na mehanizem delovanja lahko vemurafenib povzroči napredovanje rakov, povezanih z mutacijo RAS. Pred dajanjem vemurafeniba bolnikom, ki so imeli ali imajo raka, povezanega z mutacijo RAS, skrbno razmislite o koristih in tveganjih. Pankreatitis: Pri bolnikih, zdravljenih z zdravilom Zelboraf, so poročali o pankreatitisu. Nepojasnjeno bolečino v trebuhu je treba nemudoma preiskati. Bolnike je treba skrbno spremljati, ko po epizodi pankreatitisa ponovno uvedemo vemurafenib. Poškodbe jeter: Med uporabo vemurafeniba so poročali o poškodbah jeter, vključno s primeri hudih poškodb. Pred uvedbo zdravljenja je treba izmeriti jetrne encime (transaminaze in alkalno fosfatazo) ter bilirubin in jih spremljati mesečno med zdravljenjem oz. kot je klinično indicirano. Laboratorijske nepravilnosti je treba obvladati z zmanjšanjem odmerka, prekinitvijo zdravljenja ali prenehanjem zdravljenja (za podrobnosti o prilagoditvi odmerka, prosimo glejte SmPC zdravila). Ledvična toksičnost: Med uporabo vemurafeniba so poročali o ledvični toksičnosti v razponu od zvišanega serumskega kreatinina do akutnega intersticijskega nefritisa in akutne tubulne nekroze. Serumski kreatinin je treba izmeriti pred začetkom zdravljenja in ga med zdravljenjem spremljati, kot je klinično indicirano. Jetrna okvara: Bolnikom z jetrno okvaro začetnih odmerkov ni treba prilagajati. Bolnike, ki imajo zaradi metastaz v jetrih blago jetrno okvaro in nimajo hiperbilirubinemije, se lahko nadzoruje v skladu s splošnimi priporočili. Podatkov o bolnikih z zmerno do hudo jetrno okvaro je le malo; pri takih bolnikih je izpostavljenost lahko večja. Tako je posebej po prvih tednih zdravljenja potreben skrben nadzor, saj lahko po daljšem obdobju (več tednih) pride do kopičenja. Ledvična okvara: Bolnikom z blago ali zmerno ledvično okvaro začetnih odmerkov ni treba prilagajati. Pri bolnikih z hudo ledvično okvaro je treba vemurafenib uporabljati previdno ter jih pozorno spremljati. Fotosenzibilnost: Pri bolnikih, ki so v kliničnih študijah prejemali vemurafenib, je bila opisana blaga do huda fotosenzibilnost. Vsem bolnikom je treba naročiti, naj se med jemanjem vemurafeniba ne izpostavljajo soncu. V primeru fotosenzibilnosti stopnje 2 ali več so priporočljive prilagoditve odmerka. Ženske v rodni dobi morajo med zdravljenjem in vsaj še 6 mesecev po zdravljenju uporabljati učinkovito kontracepcijsko zaščito. Vemurafenib lahko zmanjša učinkovitost hormonskih kontraceptivov. Sočasna uporaba ipilimumaba in vemurafeniba ni priporočljiva. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Vplivi vemurafeniba na substrate CYP Vemurafenib lahko poveča izpostavljenost v plazmi tistim snovem, ki se presnavljajo pretežno s CYP1A2 (npr. agomelatinu, duloksetinu, melatoninu, tizanidinu, teofilinu); v takem primeru se lahko razmisli o prilagoditvi odmerka, če je klinično indicirano. Vemurafenib lahko zmanjša plazemsko izpostavljenost zdravilom, ki se presnavljajo pretežno s CYP3A4. Tako je lahko učinkovitost kontracepcijskih tablet, ki se presnavljajo s CYP3A4 in se uporabljajo sočasno z vemurafenibom, zmanjšana. Pri substratih CYP3A4, ki imajo ozko terapevtsko okno, se lahko razmisli o prilagoditvi odmerka, če je klinično indicirano. Zaenkrat še ni znano ali lahko vemurafenib pri koncentraciji v plazmi, ki je bila opažena pri bolnikih v stanju dinamičnega ravnovesja, zmanjša plazemske koncentracije sočasno dajanih substratov CYP2B6, kot je bupropion. Kadar se vemurafenib pri bolnikih z melanomom uporabi hkrati z varfarinom (CYP2C9), je potrebna previdnost. Tveganja za klinično pomemben učinek na sočasno uporabljene učinkovine, ki so substrati CYP2C8, ni mogoče izključiti. Zaradi dolge razpolovne dobe vemurafeniba je mogoče, da popolnega inhibitornega učinka vemurafeniba na sočasno dajano zdravilo ne opazimo, dokler ne mine 8 dni zdravljenja z vemurafenibom. Po končanem zdravljenju z vemurafenibom bo morda potreben 8-dnevni premor, da se izognemo interakcijam z nadaljnjim zdravljenjem. Zdravljenje z obsevanjem: Pri bolnikih, zdravljenih z vemurafenibom, so poročali o povečanju toksičnosti obsevanja. V večini primerov so bolniki prejeli protokole obsevanja z 2 Gy/dan ali več (hipofrakcionirane protokole). Medsebojno delovanje vemurafeniba s transportnimi sistemi zdravil: Ob sočasni uporabi vemurafeniba in substrata P-gp je potrebna previdnost. Pri uporabi zdravil, ki so substrati P-gp in imajo ozko terapevtsko okno (npr. digoksina, dabigatran eteksilata, aliskirena), je treba razmisliti o dodatnem spremljanju koncentracije zdravila. Možnosti, da vemurafenib morda poveča izpostavljenost zdravilom, ki se prenašajo z BCRP, ni mogoče izključiti. Vplivi sočasno uporabljenih zdravil na vemurafenib: Vemurafenib je treba uporabljati previdno v kombinaciji z močnimi inhibitorji CYP3A4, glukuronidacije in/ali prenašalnih beljakovin (npr. ritonavirjem, sakvinavirjem, telitromicinom, ketokonazolom, itrakonazolom, vorikonazolom, posakonazolom, nefazodonom, atazanavirjem). Sočasna uporaba močnih induktorjev P-gp, glukuronidacije in/ali CYP3A4 (npr. rifampicina, rifabutina, karbamazepina, fenitoina ali šentjanževke) lahko vodi v suboptimalno izpostavljenost vemurafenibu in se ji je treba izogibati. Vplivi induktorjev in inhibitorjev P-gp in BCRP na izpostavljenost vemurafenibu niso znani. Ne moremo pa izključiti možnosti, da imajo lahko zdravila, ki vplivajo na P-gp (npr. verapamil, ciklosporin, ritonavir, kinidin, itrakonazol) ali BCRP (npr. ciklosporin, gefitinib), vpliv na farmakokinetiko vemurafeniba. Neželeni učinki: Med najpogostejšimi neželenimi učinki (> 30 %), o katerih so poročali v zvezi z vemurafenibom, so artralgija, utrujenost, kožni izpuščaj, fotosenzibilnostna reakcija, navzea, alopecija in srbenje. Zelo pogosto je bil opisan ploščatocelični karcinom kože. Sledijo drugi zelo pogosti in pogosti neželeni učinki. Zelo pogosti: seboroična keratoza, kožni papilom, zmanjšanje teka, glavobol, disgevzija, kašelj, driska, bruhanje, zaprtost, aktinična keratoza, makulo-papulozen izpuščaj, papulozen izpuščaj, hiperkeratoza, eritem, suha koža, sončne opekline, mialgija, bolečina v okončini, mišično-skeletne bolečine, bolečine v hrbtu, pireksija, periferni edem, astenija, zvišanje GGT. Pogosti: folikulitis, bazalnocelični karcinom, novi primarni melanom, ohromelost sedmega živca, omotica, uveitis, sindrom palmarno-plantarne eritrodisestezije, panikulitis, pilarna keratoza, artritis, zvišanje ALT, alkalne fosfataze, bilirubina in izguba telesne mase, podaljšanje QT in zvišanje kreatinina v krvi. Posebne populacije: Pri starejših bolnikih (. 65 let) je možna večja verjetnost neželenih učinkov, vključno s ploščatoceličnim karcinomom kože, zmanjšanjem teka in motnjami srčnega ritma. Med neželene učinke stopnje 3, ki so bili med kliničnimi preskušanji vemurafeniba pri ženskah opisani pogosteje kot pri moških, spadajo kožni izpuščaj, artralgija in fotosenzibilnost. Poročanje o domnevnih neželenih učinkih: Poročanje o domnevnih neželenih učinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoča namreč stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da poročajo o katerem koli domnevnem neželenem učinku zdravila na: Univerzitetni klinični center Ljubljana, Interna klinika, Center za zastrupitve, Zaloška cesta 2, SI-1000 Ljubljana, Faks: + 386 (0)1 434 76 46, e-pošta: farmakovigilanca@kclj.si. Režim izdaje zdravila: Rp/Spec. Imetnik dovoljenja za promet: Roche Registration Limited, 6 Falcon Way, Shire Park, Welwyn Garden City, AL7 1TW, Velika Britanija. Verzija: 2.0/16. Informacija pripravljena: januar 2017. Samo za strokovno javnost. DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o., Vodovodna cesta 109, 1000 Ljubljana. PG-002-0117-ME2 DVOJNO ZAVIRANJE. 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The statement of disclosure must be in the Cover letter accompany­ing the manuscript or submitted on the form available on www.icmje.org/coi_disclosure.pdf Page proofs Page proofs will be sent by E-mail to the corresponding author. It is their responsibility to check the proofs carefully and return a list of essential corrections to the editorial office within three days of receipt. Only grammatical corrections are ac­ceptable at that time. Open access Papers are published electronically as open access on www.degruyter.com/view/j/raon, also papers accepted for publication as E-ahead of print. XALKORI®- prvi zaviralec ALK, odobren za 1. linijo zdravljenja napredovalega, ALK pozitivnega nedrobnoceliínega pljuínega raka1 ALK = anaplastiína limfomska kinaza BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNACˇILNOSTI ZDRAVILA XALKORI 200 mg, 250 mg trde kapsule Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da porocˇajo o kateremkoli domnevnem neželenem ucˇinku zdravila. Glejte poglavje 4.8 povzetka glavnih znacˇilnosti zdravila, kako porocˇati o neželenih ucˇinkih. Sestava in oblika zdravila: Ena kapsula vsebuje 200 mg ali 250 mg krizotiniba. Indikacije: Prva linija zdravljenja odraslih bolnikov z napredovalim nedrobnocelicˇnim pljucˇnim rakom (NSCLC – Non-Small Cell Lung Cancer), ki je ALK (anaplasticˇna limfomska kinaza) pozitiven. Zdravljenje odraslih bolnikov s predhodno zdravljenim, napredovalim NSCLC, ki je ALK pozitiven. Zdravljenje odraslih bolnikov z napredovalim NSCLC, ki je ROS1 pozitiven. Odmerjanje in nacˇin uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik z izkušnjami z uporabo zdravil za zdravljenje rakavih bolezni. Preverjanje prisotnosti ALK in ROS1: Pri izbiri bolnikov za zdravljenje je treba pred zdravljenjem opraviti tocˇno in validirano preverjanje prisotnosti ALK ali ROS1. Odmerjanje: Priporocˇeni odmerek je 250 mg dvakrat na dan (500 mg na dan), bolniki pa morajo zdravilo jemati brez prekinitev. Cˇe bolnik pozabi vzeti odmerek, ga mora vzeti takoj, ko se spomni, razen cˇe do naslednjega odmerka manjka manj kot 6 ur. V tem primeru bolnik pozabljenega odmerka ne sme vzeti. Prilagajanja odmerkov: Glede na varnost uporabe zdravila pri posameznem bolniku in kako bolnik zdravljenje prenaša, utegne biti potrebna prekinitev in/ali zmanjšanje odmerka zdravila na 200 mg dvakrat na dan; cˇe je potrebno še nadaljnje zmanjšanje, pa znaša odmerek 250 mg enkrat na dan. Za prilagajanje odmerkov pr i hematološki in nehematološki (povecˇanje vrednosti AST, ALT, bilirubina; ILD/pnevmonitis; podaljšanje intervala QTc, bradikardija, bolezni ocˇi) toksicˇnosti glejte preglednici 1 in 2 v povzetku glavnih znacˇilnosti zdravila. Okvara jeter: Pri blagi in zmerni okvari je zdravljenje treba izvajati previdno, pri hudi okvari se zdravila ne sme uporabljati. Okvara ledvic: Pri blagi in zmerni okvari prilagajanje zacˇetnega odmerka ni priporocˇeno. Pri hudi okvari ledvic (ki ne zahteva peritonealne dialize ali hemodialize) je zacˇetni odmerek 250 mg peroralno enkrat na dan; po vsaj 4 tednih zdravljenja se lahko povecˇa na 200 mg dvakrat na dan. Starejši bolniki (. 65 let): Prilagajanje zacˇetnega odmerka ni potrebno. Pediatricˇna populacija: Varnost in ucˇinkovitost nista bili dokazani. Nacˇin uporabe: Kapsule je treba pogoltniti cele, z nekaj vode, s hrano ali brez nje. Ne sme se jih zdrobiti, raztopiti ali odpreti. Izogibati se je treba uživanju grenivk, grenivkinega soka ter uporabi šentjanževke. Kontraindikacije: Preobcˇutljivost na krizotinib ali katerokoli pomožno snov. Huda okvara jeter. Posebna opozorila in previdnostni ukrepi: Dolocˇanje statusa ALK in ROS1: Pomembno je izbrati dobro validirano in robustno metodologijo, da se izognemo lažno negativnim ali lažno pozitivnim rezultatom. Hepatotoksicˇnost: V klinicˇnih študijah so porocˇali o hepatotoksicˇnosti, ki jo je povzrocˇilo zdravilo (vkljucˇno s primeri s smrtnim izidom). Delovanje jeter, vkljucˇno z ALT, AST in skupnim bilirubinom, je treba preveriti enkrat na teden v prvih 2 mesecih zdravljenja, nato pa enkrat na mesec in kot je klinicˇno indicirano. Ponovitve preverjanj morajo biti pogostejše pri povecˇanjih vrednosti stopnje 2, 3 ali 4. Intersticijska bolezen pljucˇ (ILD)/pnevmonitis: Lahko se pojavi huda, življenjsko nevarna ali smrtna ILD/pnevmonitis. Bolnike s simptomi ILD/pnevmonitisa, je treba spremljati, zdravljenje pa prekiniti ob sumu na ILD/pnevmonitis. Podaljšanje intervala QT: Opažali so podaljšanje intervala QTc. Pri bolnikih z obstojecˇo bradikardijo, podaljšanjem intervala QTc v anamnezi ali predispozicijo zanj, pri bolnikih, ki jemljejo antiaritmike ali druga zdravila, ki podaljšujejo interval QT, ter pri bolnikih s pomembno obstojecˇo srcˇno boleznijo in/ali motnjami elektrolitov je treba krizotinib uporabljati previdno; potrebno je redno spremljanje EKG, elektrolitov in delovanja ledvic; preiskavi EKG in elektrolitov je treba opraviti cˇimbližje uporabi prvega odmerka, potem se priporocˇa redno spremljanje. Cˇe se interval QTc podaljša za 60 ms ali vecˇ, je treba zdravljenje s krizotinibom zacˇasno prekiniti in se posvetovati s kardiologom. Bradikardija: Lahko se pojavi simptomatska bradikardija (lahko se razvije vecˇ tednov po zacˇetku zdravljenja); izogibati se je treba uporabi krizotiniba v kombinaciji z drugimi zdravili, ki povzrocˇajo bradikardijo; pri simptomatski bradikardiji je treba prilagoditi odmerek. Srcˇno popušcˇanje: Porocˇali so o hudih, življenjsko nevarnih ali smrtnih neželenih ucˇinkih srcˇnega popušcˇanja. Bolnike je treba spremljati glede pojavov znakov in simptomov srcˇnega popušcˇanja in ob pojavu simptomov zmanjšati odmerjanje ali prekiniti zdravljenje. Nevtropenija in levkopenija: V klinicˇnih študijah so porocˇali o nevtropeniji, levkopeniji in febrilni nevtropeniji; spremljati je treba popolno krvno sliko (pogostejše preiskave, cˇe se opazijo abnormalnosti stopnje 3 ali 4 ali cˇe se pojavi povišana telesna temperatura ali okužba). Perforacija v prebavilih: V klinicˇnih študijah so porocˇali o perforacijah v prebavilih, v obdobju trženja pa o smrtnih primerih perforacij v prebavilih. Krizotinib je treba pri bolnikih s tveganjem za nastanek perforacije v prebavilih uporabljati previdno; bolniki, pri katerih se razvije perforacija v prebavilih, se morajo prenehati zdraviti s krizotinibom; bolnike je treba poucˇiti o prvih znakih perforacije in jim svetovati, naj se nemudoma posvetujejo z zdravnikom. Vplivi na ledvice: V klinicˇnih študijah so opazili zvišanje ravni kreatinina v krvi in zmanjšanje ocˇistka kreatinina. V klinicˇnih študijah in v obdobju trženja so porocˇali tudi o odpovedi ledvic, akutni odpovedi ledvic, primerih s smrtnim izidom, primerih, ki so zahtevali hemodializo in hiperkaliemiji stopnje 4. Vplivi na vid: V klinicˇnih študijah so porocˇali o izpadu vidnega polja stopnje 4 z izgubo vida. Cˇe se na novo pojavi huda izguba vida, je treba zdravljenje prekiniti in opraviti oftalmološki pregled. cˇe so motnje vida trdovratne ali se poslabšajo, je priporocˇljiv oftalmološki pregled. Histološka preiskava, ki ne nakazuje adenokarcinoma: Na voljo so le omejeni podatki pri NSCLC, ki je ALK in ROS1 pozitiven in ima histološke znacˇilnosti, ki ne nakazujejo adenokarcinoma, vkljucˇno s plošcˇatocelicˇnim karcinomom (SCC). Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Zdravila, ki lahko povecˇajo koncentracije krizotiniba v plazmi (mocˇni zaviralci CYP3A4, npr. atazanavir, indinavir, nel. navir, ritonavir, sakvinavir, itrakonazol, ketokonazol, vorikonazol, klaritromicin, telitromicin, troleandomicin), tudi grenivke in grenivkin sok. Zdravila, ki lahko zmanjšajo koncentracije krizotiniba v plazmi (mocˇni induktorji CYP3A4, npr. karbamazepin, fenobarbital, fenitoin, rifampicin, šentjanževka). Zmerni induktorji CYP3A4, npr. efavirenz in rifabutin. Zdravila, katerih koncentracije v plazmi lahko krizotinib spremeni (midazolam, alfentanil, cisaprid, ciklosporin, derivati ergot alkaloidov, fentanil, pimozid, kinidin, sirolimus, takrolimus, bupropion, efavirenz, peroralni kontraceptivi, raltegravir, irinotekan, mor. n, nalokson, digoksin, dabigatran, kolhicin, pravastatin, metformin, prokainamid). Zdravila, ki podaljšujejo interval QT ali ki lahko KRIZOTINIB povzrocˇijo Torsades de pointes (antiaritmiki skupine IA (kinidin, disopiramid), antiaritmiki skupine III (amiodaron, sotalol, dofetilid, ibutilid), metadon, cisaprid, moksi. oksacin, antipsihotiki). Zdravila, ki povzrocˇajo bradikardijo (nedihidropiridinski zaviralci kalcijevih kanalcˇkov (verapamil, diltiazem), antagonisti adrenergicˇnih receptorjev beta, klonidin, gvanfacin, digoksin, me. okin, antiholinesteraze, pilokarpin). Plodnost, nosecˇnost in dojenje:Ženske v rodni dobi se morajo izogibati zanositvi. Med zdravljenjem in najmanj 90 dni po njem je treba uporabljati ustrezno kontracepcijo (velja tudi za moške). Zdravilo lahko škoduje plodu in se ga med nosecˇnostjo ne sme uporabljati, razen cˇe klinicˇno stanje matere ne zahteva takega zdravljenja. Matere naj se med jemanjem zdravila dojenju izogibajo. Zdravilo lahko zmanjša plodnost moških in žensk. Vpliv na sposobnost vožnje in upravljanja strojev: Lahko se pojavijo simptomatska bradikardija (npr. sinkopa, omotica, hipotenzija), motnje vida ali utrujenost; potrebna je previdnost. Neželeni ucˇinki: Najresnejši neželeni ucˇinki so bili hepatotoksicˇnost, ILD/pnevmonitis, nevtropenija in podaljšanje intervala QT. Najpogostejši neželeni ucˇinki (. 25 %) so bili motnje vida, navzea, diareja, bruhanje, edem, zaprtje, povecˇane vrednosti transaminaz, utrujenost, pomanjkanje apetita, omotica in nevropatija. Ostali zelo pogosti (. 1/10 bolnikov) neželeni ucˇinki so: nevtropenija, anemija, levkopenija, disgevzija, bradikardija, bolecˇina v trebuhu in izpušcˇaj. Nacˇin in režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet: P. zer Limited, Ramsgate Road, Sandwich, Kent CT13 9NJ, Velika Britanija. Datum zadnje revizije besedila: 11.11.2016 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih znacˇilnosti zdravila. Vir: 1. Povzetek glavnih znacˇilnosti zdravila Xalkori, 11.11.2016 XAR-03-17 Samo za strokovno javnost P. zer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J.F. Kennedy, L-1855, P. zer podružnica Ljubljana, Letališka cesta 3c, 1000 Ljubljana