adiology
AND
ncology
June 2009 Vol. 43 No. 2 Ljubljana
ISSN 1318-2099
ALIMTA
pemetreksed
Edina histološko usmerjena citostatična terapija
/
Pravi Izbor Pravi Bolnik Pravi Čas
ALIMTA/Cisplatin:
Zdravljenje prvega reda pri bolnikih z nedrobnoceličnim pljučnim karcinomom, ki nimajo pretežno luskaste histologije
Edina kombinirana terapija z signifikantno izboljšanim preživetjem: 12,6 meseca pri bolnikih z adenokarcinomom pljuč1
*vs. Gemcitabin/Cisplatin
1. Scagliotti GV et al. Phase III study comparing clsplatin plus gemcitabine with cisplatln plus pemetrexed In chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2008;26(21):3543-51,
SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA
Ime zdravila AÜMTA100 mg prašek za koncentrat za raztopino za infu ndiranje in ALIMTA 500 mg prašek za koncentrat za raztopino za infundiranje Kakovostna Inkollčlnaka «eatava ALIMTA 100 mg: vsaka viala vsebuje 100 mg pemetrekseda (v obliki dinatrijevega pemetrekseda). AUMTA 500 mg: vsaka viala vsebuje 500 mg pemetrekseda (voblid dinatrijevega pemetrekseda). Pomožne «novi: manitol, klorovodikova kislina, natrijev hidroksid. Terapevtske Indkadje ALIMTA je v kombinaciji S cispiatinom indtaVaruizazdravljenje bolnikov z nereseklabilnim
AUMTAje Indldrana kot monoteraplja za zdravljenje bolnikov z lokalno napredovalim ali metastatsklm nedrobnocellčnlm pljučnim karcinomom, Id nima pretežno luskaste histologije. Odmar] vija In način uporaba: ALIMTO smemo dajati le pod nadzorom zdravnica, usposobljenega za uporabo kemoterapije za zdravljenje raka. ALIMTA v kombinaciji s risplatlnom: Priporočeni odmerek ALIhTTE je 500 mg/m2 telesne površine (TP), dan kot Intravenska Infuzija v 10 minutah prvi dan vsakega 21 -dnevnega ciklusa, Priporočeni odmerek clsplatlna Je 75 mg/m2 TP, infundirán v dveh urah približno 30 minut po zaključku infuzije pemetrekseda prvi dan vsakega 21 dnevnega ciklusa. Prporočeni odmerek cisplatina Je 75 mg/m2 TP, infundirán v dveh urah približno 30 minut po zakf učku infuzije pemetrekseda prvi dan vsakega 21 dnevnega ciklusa. Bolniki morajo prejeti zadostno antiemetieno zdravljenje, pred in/ali po prejemanju cisplatina jih moramo tudi ustrezno hidrirat. ALIMTA kot monoterapija: Priporočeni odmerekALlMTE je 500 mg/mZTP, dan kot intravenska infuzija v 10 minutah prvi dan vsakega 21 dnevnega cilduaa-
tokslčnostl morajo bolniki dnevno Jemati tudi peroralno foho kislino al miiltMtamlnskl pripravek, ki Jo vsebuje (350 dcMOOO mlkrogramov). V sedmlhdneh pred prvim odmerkom pemetrekseda morajo vzeti vsajat odmerkov folne kisline! odmerjanje pa morajo nada|evatl ves čas zdravljenja in še 21 dra po zadnjem odmerku pemetrekseda. Bolniki morajo preJstituciintramuskulamoinjekcijovitaminaB12(1000 mikrogramov) v tednu pred prvim odmerkom pemetrekseda in enkrat vsake tri cikluse zatem. Kasnejše injekcije vitamina B12 lahko dajemo isti dan kot pemetrelaed. Kontraindikacije: Preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. Med zdravljenjem s pemetretoedom je treba dojenje prekiniti. Sočasno cepljenje proti rumeni mrzlici. Potobna opozorila In previdnostni ukrepi: Pemetreksed lahko zavre delovanje kostnega mozga, kar se kaže kot nevtropenija, trombodtopsnija in anemija (af pancitopenija). Pri bolnikih, ki pred zdravljenjem niso prejemali kortkosteroidov, so poročali o kožnih reakcijah uporabe pemetrekseda pri bolnikih z oSstkom kreatinina < 45 ml/min ne
ki jih lahko zdravimo s pemetreksedom, naj se Izogibajo jemanju NSAID-ov z dolgimi razpolovnimi časi Izločanja vsaj 5 dni pred dajanjem pemetrekseda, na dan dajanja In še vsaj 2 dni po dajanju pemetrekseda. Poročali so o rasnih ledvičnih primerih, vključno z akutno ledvično odpovedjo, s pemetreksedom samim ali v povezavi z drugimi kemoterapevtiki. Pri bolnikih s klinično pomembno tekočino tretjega prostora moramo razmisliti o drenaži izliva pred dajanjem pemetrekseda. Kot posledico toksičnosti pemetrekseda v kombinaciji s cispiatinom za prebavila
Sočasno dajanje nefrotoksičnih zdravil (denimo, aminoglikozidov, diuretikovza
»neriti nesteroidnih pretivnetnih zdravil (NSAlD-i, denimo, ibuprofen) in acetilsalicilna kislina v visokii odmedah zmanjšajo eliminacijo pemstrekseda in tako lahko povečajo pojavnost moramo Izogibati sočasnemu dajanju pemetrekseda z NSAID-1 (denimo, Ibuprofenom) ali acetllsallcllne kisline v visokih odmerkih 2 dni pred dajanjem pemetrekseda, na dan dajanja s pemetreksedom se moramo Izogibati vsaj 5 dni pred dajanjem pemetrekseda, na dan dajanja In še vsaj 2 dni po dajanju pemetrekseda.Vellka razKnost med posamezniki v
m po cepljenju. Odsvetovana sočasna uporaba: Živa oslabljena Cepiva (razen proti rumeni mrzlici): tveganje zj
la temperatura, povišanje SGPT (ALT), povišanje SGOT (AST), srbenje, alopeclja. Klinične študije nedrobnocellčnega pljučnega karclnoma - ALIMTA v kombinati s cispiatinom Zelo pogosti: znižan hemoglobin, znižani Ievtocltlt, it, bruhanje, anoreksija, zaprtje, stomatitis/fanngitis, diareja brez kolostomije, utrujenost, povišan kreatinin, alopecija, izpuščaj/luščenje. Pogosti: dispepsija^gaga, nevropatija-senzončna, motnje okusa. Občasno so v kliničnih ložilnih in možganskožjlnih dogodkov, vključno z miokardnim nfarktom, angino pektons, cerebrovaskulamim insultom in prehodnimi ishemičnimi atakami; primerih kolitsa ter o primerih interstitijake pljučnice z raspiratorno
Izozemska. Datum zadnje revizije besedila 06.01.2009
la lokalnem predstavništvu.	!
Eli Lilly farmacevtska družba, d.o.o. Dunajska 156,1000 Ljubljana, Slovenija Telefon:+386 [0)1 5800 010 Faks: +386 (0)1 5691 705

ADIOLOGY
AND
NCOLOGY

Editorial office Radiology and Oncology
Institute of Oncology Zaloska 2 SI-1000 Ljubljana Slovenia
Phone: +386 1 5879 369 Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si
June 2009 Vol. 43 No. 2 Pages 65-136 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM
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 Sersa Ljubljana, Slovenia
Executive Editor Viljem Kovac Ljubljana, Slovenia
Deputy Editors Andrej Cor Ljubljana, Slovenia
Igor Kocijančič Ljubljana, Slovenia
Geoffrey J Pilkington Portsmouth, UK Ervin B. Podgoršak Montreal, Canada Uroš Smrdel Ljubljana, Slovenia Primož Strojan Ljubljana, Slovenia Borut Štabuc Ljubljana, Slovenia Ranka Štern-Padovan Zagreb, Croatia Justin Teissie Tolouse, France Sandor Toth Oroshaza, Hungary Gillian M. Tozer Sheffield, UK Andrea Veronesi Aviano, Italy Branko Zakotnik Ljubljana, Slovenia
Advisory Committee
Marija Auersperg Ljubljana, Slovenia; Tomaž Benulič Ljubljana, Slovenia; Jure Fettich Ljubljana; Valentin Fidler Ljubljana, Slovenia; Berta Jereb Ljubljana, Slovenia; Vladimir Jevtič Ljubljana, Slovenia; Stojan Plesničar Ljubljana, Slovenia; Živa Zupančič Ljubljana, Slovenia
Editorial Board Karl H. Bohuslavizki Hamburg, Germany Maja Čemažar Ljubljana, Slovenia Christian Dittrich Vienna, Austria Metka Filipič Ljubljana, Slovenia Tullio Giraldi Trieste, Italy Maria Godény Budapest, Hungary Vassil Hadjidekov Sofia, Bulgaria Marko Hočevar Ljubljana, Slovenia Maksimilijan Kadivec Ljubljana, Slovenia
Miklos Kasler Budapest, Hungary Michael Kirschfink Heidelberg, Germany Janko Kos Ljubljana, Slovenia Tamara Lah Turnšek Ljubljana, Slovenia Damijan Miklavčič Ljubljana, Slovenia Luka Milas
Houston, USA Damir Miletič Rijeka, Croatia Maja Osmak Zagreb, Croatia Branko Palčič Vancouver, Canada Dušan Pavčnik
Portland, USA
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..
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ISSN 1581-3207
K ADIOLQGY
AND
(Jncology
m
Ljubljana, Slovenia June 2009 Vol. 43 No. 2
ISSN 1318-2099 UDC 616-006 CODEN: RONCEM
CONTENTS
REVIEW
Cancer cachexia-anorexia syndrome and skeletal muscle wasting	65
Mihaela Jurdana
RADIOLOGY
Xanthogranulomatous cholecystitis remains a challenge in medical practice:	76
experience in 24 cases
Mehmet Yildirim, Ozgur Oztekin, Fatih Akdamar, Savas Yakan, Hakan Postaci
Spinal subdural haematoma in von Willebrand disease	84
Artur Franko, Ronald Antulov, Sinisa Dunatov, Igor Antoncic, Damir Miletic
ONCOLOGY
Increased late urinary toxicity with whole pelvic radiotherapy after prostatectomy	88
Borut Kragelj
Usage of the standard and modified comet assay in assessment of DNA damage in human lymphocytes after exposure to ionizing radiation
Morana Miklos, Goran Gajski, Vera Garaj - Vrhovac
97
Cell size dynamics and viability of cells exposed to hypotonic treatment	108
and electroporation for electrofusion optimization
Marko Usaj, Katja Trontelj, Rosana Hudej, Masa Kanduser, Damijan Miklavcic
Cisplatin-induced non-convulsive posterior reversible encephalopathy syndrome	120
in a 41-year-old woman with metastatic malignant melanoma
Janja Ocvirk, Marko Boc, Martina Rebersek, Tanja Ros
Angiosarcoma of the liver after multimodality therapy for gallbladder carcinoma	126
Maikel Botros, J. Fernando Quevedo, Robert C. Miller
RADIOPHYSICS
How well are clinical gross tumor volume DVHs approximated	132
by an analytical function?
Pavel Stavrev, Colleen Schinkel, Nadia Stavreva and B. Gino Fallone
SLOVENIAN ABSTRACTS	I
NOTICES	X
Radiology and Oncology is covered in Science Citation Index Expanded (SciSearch®), Journal Citation Reports/Science Edition, Scopus, EMBASE/Excerpta Medica, Open J-gate, Chemical Abstracts, Biomedicina Slovenica
Radiol Oncol 2009; 43(2): 65-75.
doi:10.2478/v10019-009-0007-y
review
Cancer cachexia-anorexia syndrome and skeletal muscle wasting
Mihaela Jurdana
College of Health Care Izola, University of Primorska, Izola, Slovenia
Background. Cachexia-anorexia syndrome is a common and important indicator of cancer. It occurs in 30% to 80% of cancer patients. Cachexia means "bad condition" and may be present in the early stages of tumor growth, before any signs of malignancy. Cancer cachexia is a syndrome of progressive body wasting, characterized by loss of adipose tissue and skeletal muscle mass. In most cancer patients, cachexia is characterized by anorexia, which implies a failure of food intake, regulated through a complex system of hormones and neuropeptides. A decline in food intake relative to energy expenditure is a fundamental physiologic derangement leading to cancer associated weight loss. The weight loss in patients with cachexia-anorexia syndrome differs from that in caloric starvation or anorexia nervosa. The pathophysiology of cancer cachexia is not fully understood; however, studies have shown that cytokines are important in the alteration of the carbohydrate, lipid and protein metabolism. Cancer, prolonged bed rest, HIV infection and aging are conditions in which muscle wasting is a common feature. An intervention that may potentially attenuate the progression of muscle wasting in cancer patients is resistance exercise training, defined as multiple repetitions of static or dynamic muscular contractions that increase muscle mass.
Conclusions. The main components of the pathological state of cachexia are anorexia and metabolic abnormalities such as fat depletion and muscle protein catabolism. Future developments may concentrate on the molecular abnormalities of cachexia and on examination of the functional benefit of resistance exercise training for cancer related muscle wasting.
Key words: cancer cachexia; muscle wasting; cytokines; muscle
Introduction
Many patients with chronic or end-stage diseases, such as infection, cancer, acquired immunodeficiency syndrome (AIDS), congestive heart failure, cystic fibrosis, tuber-
Received 10 December 2008 Accepted 15 January 2009
Correspondence to: Mihaela Jurdana, PhD, University of Primorska, College of Health Care Izola, Polje 42, 6310 Izola, Slovenia. Phone: +386 5 662 64 69; Fax: +386 5 662 64 80; E-mail: mihaela.jurdana@vszi.upr.si
culosis, rheumatoid arthritis, and Crohn's disease, develop cachexia.1
The word "cachexia" is derived from Greek "kakos" meaning "bad" and "hexis" meaning "condition". Cachexia is characterized by weight loss involving depletion of host adipose tissue and skeletal muscle mass. Weight loss in cancer patients differs from that found in caloric starvation, where body fat is lost preferentially (Table 1). Cachexia is an important cause of mortality in cancer patients, between 10-22% of all cancer
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Jurdana M / Cancer cachexia-anorexia syndrome and skeletal muscle wasting
Table 1. Characteristics of starvation and cancer cachexia. Cancer associated weight loss arises from the loss of equal amounts of muscle and fat and is characterized by increased catabolism of skeletal muscle and decreased protein synthesis.
Starvation
Mobilization of fat, sparing of skeletal muscle Decreased basal metabolic rate Liver atrophy Normal lipoprotein lipase
Decreased protein breakdown
Reduced glucose turnover
deaths, as well as death from other causes such as infection.2 An effective therapy for cachexia should improve the quality of life of cancer patient and it may also be expected to extend the survival time. In general, patients with solid tumors have a higher frequency of cachexia.3 About 80% of patients with upper gastrointestinal cancer and 60% of patients with lung cancer have had substantial weight loss.3 Cachexia is more common in children and elderly patients and becomes more pronounced as the disease progresses.
Tumor growth is associated with profound metabolic and neurochemical alterations. About half of all cancer patients show a syndrome of cachexia, causing the clinical manifestations of anorexia, one of the most common symptoms in advanced cancer.4 Anorexia is defined as the loss of desire to eat, which frequently leads to reduced food intake. Based on various diagnostic tools, anorexia has been detected at the point of cancer diagnosis in 13-55% of patients.5 In cancer patients, the development of anorexia is associated with the presence of cachexia, resulting in cancer anorexia-cachexia syn-drome.6 Symptoms which are usually identified as part of cachexia-anorexia syndrome include weight loss, anorexia, early satiety, muscular weakness and anemia.7 Anorexia alone is unlikely to be responsible for the wasting seen in cancer patients, although it may be a contributing factor because the de-
Cancer cachexia
Equal mobilization of fat and skeletal muscle
Normal or increased basal metabolic rate
Increased liver size
Reduced lipoprotein lipase
Increased protein breakadown Increased synthesis of acute- phase protein
Normal or increased glucose turnover
gree of weight loss cannot be ascribed completely to reduced food intake. Anorexia and cachexia can co-exist in cancer patients. The muscle wasting observed in cancer patients occurs even in the presence of normal food intake, and increased muscle proteolysis is detectable even before weight loss occurs.8 The pathogenesis of anorexia is multifacto-rial and is related to disturbance of the central physiological mechanisms controlling food intake. The presence of anorexia can be characterized by identification of objective symptoms, including early satiety, taste and smell alterations, meat aversion and nausea/vomiting.9
The presence of anorexia is an extremely distressing syndrome, because appetite and the ability to eat have been reported to be the most important factors in the physical and psychological aspects of a patient's quality of life.10 In anorexic cancer patients, early satiety together with a reduced appetite has been postulated to be caused by the production of factors by the tumor that exert their effects by acting on the hypotha-lamic sensory cells.11
Loss of appetite can arise from decreased taste and smell of food, as well as from cytokine production.4 An alteration in the regulation of feeding, controlled by a complex of hormones and neuropeptides in the ventral hypothalamus, are also involved in cachexia-anorexia. For instance, neuropep-
Radiol Oncol 2009; 43(2): 65-75.
67	Jurdana M / Cancer cachexia-anorexia syndrome and skeletal muscle wasting

Tumor Products
Lipolysis
CANCER
Metabolic Abnormalities
Protein Loss __>
V'
CACHEXIA
Immune System
Cytokines
' J!
Anorexia
Figure 1. Metabolic abnormalities caused by cancer, resulting in losses of fat and skeletal muscle. Tumors produce factors inducing breakdown of adipose tissue into fatty acids, and protein degradation (amino acids) in skeletal muscle. Cytokines synthesised by tumor or host induced anorexia, the most common symptoms in advanced cancer. Progressive weight loss and skeletal muscle protein loss, profound anorexia characterized Cachexia-Anorexia Syndrome.
tide Y (NPY) is considered to be among the most potent of the feeding stimulatory peptides.12,13 Increased levels of leptin, a hormone secreted by adipocytes, can block NPY and induce satiety.14
Cytokines, in particular, appear to play a key role in satiety disturbances, and an emerging view is that anorexia-cachexia syndrome is caused predominantly by cy-tokines, either produced by cancer cells or released by the immune system of the host as a response to the presence of cancer.14
The wasting observed in cancer cachexia can arise from a decreased energy intake and increased energy expenditure (EE - the amount of calories required for a 24-hour period by the body during an active period) or a combination of both. Hypermetabolism and weight loss are significant predictors of decreased survival and they also indicate that weight loss cannot be explained solely by diminished dietary intake.15
Patients with cancer have a highly variable change in energy expenditure. Cancer associated weight loss arises from the loss
of equal amounts of muscle and fat and is characterized by increased catabolism of skeletal muscle and decreased protein synthesis.16 Weight loss can occur during any phase of the disease, and some tumors seem more capable than others of inducing the metabolic changes apparent in cancer cachexia syndrome. In comparison with control groups, patients with malignant disease have been reported to have reduced, normal or elevated energy expenditure.17-19 The metabolic abnormalities caused by cancer are shown in Figure 1.
Patients with lung and pancreatic cancer have an increased resting energy expenditure (REE - the amount of calories required for a 24-hour period by the body during a non active period) compared with healthy control subjects.20,21 However, patients with gastric and colorectal cancer were reported to have no elevation of REE.20 Cachexia can occur even with a normal EE, which suggests that tumor and host factors play an important role in depletion of body lipids (FAT) and protein during the process of cachexia.
The metabolic profile of cachexia is not the same as that of caloric starvation.7 During the first few days of starvation, glucose utilization by the brain and eryth-rocytes necessitates depletion of liver and muscle glycogen and increased glucose production by the liver, using gluconeo-genic amino acids derived from catabolism of muscle. This phase is replaced in long-term starvation by the use of fat as a fuel, in which free fatty acids released from adipose tissue are converted into ketone bodies, which are utilized for energy by peripheral tissues and, eventually, to some extent by the brain. This leads to prolonged conservation of muscle mass.6 In contrast, in cancer cachexia, there is equal loss of both fat and muscle, so that for a given degree of weight loss there is more loss of muscle in a patient with cancer cachexia than in caloric starvation.22 In starving persons, the body
Radiol Oncol 2009; 43(2): 65-75.
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Jurdana M / Cancer cachexia-anorexia syndrome and skeletal muscle wasting
quickly adapts to the lack of nutrients by decreasing its EE and by oxidizing store lipids for energy. This metabolic adaptation to a decreased food intake is critical for maintaining a functional muscle mass. Cancer-bearing hosts, however, fail to develop such metabolic adaptation and thus continue to deplete their skeletal muscle proteins.6 Metabolic abnormalities in cancer cachexia are described in Figure 1.
The role of cytokines
Cytokines are polypeptides synthesised and released primarily by activated monocytes and macrophages. They are also produced by some types of tumor cells.23
Skeletal muscle plays an important role in the immune system as one of the producers of cytokines. Cytokines are primarily responsible for cell-to-cell communication and stimulate the arrival of lymphocytes, neutrophils, monocytes and other healer cells to the injury site to repair the injured tissue.24 Three important cytokines, tumor necrosis factor-a (TNF-a), interleukin 1 (IL-1) and in-terleukin 6 (IL-6), are responsible for protein breakdown in skeletal muscle and increased production of prostaglandins. Cytokines are also involved in muscle hypertrophy and in the muscle regeneration process.25
Numerous cytokines, including (TNF-a), (IL-1), (IL-6), interferon y (IFN- y) and leukemia-inhibitory factor (LIF), play an important role in the etiology of cancer cachexia. Such cytokines may be produced by tumor or host tissue and are characterized by the induction of anorexia and a decrease in the clearing enzyme lipoprotein lipase.6
Cytokines are implicated in changes in sensory function in the chorda tympani (involved in the transduction of taste), leading to alterations in taste and in specific food preferences.4 Patients with end stage cancer have altered taste thresholds with
respect to the bitter modality and these changes are most apparent in patients with higher concentrations of C-reactive protein, ILP-1, IL-6 and TNF-a. The odor threshold was also lower in these patients than in healthy subjects.26 The described cytokines also sensitize the vagal afferent nerve fibers, resulting in increased activation of the mechanisms that mediate sensations of fullness and thus contribute to the process of satiety.26 Cytokines may inhibit feeding by causing nausea and vomiting and also by decreasing gastric motility and gastric emptying, intestinal motility or by modifying gastric acid secretion. Their effects may result from direct action on the gastrointestinal system or indirect effects mediated by cytokines (IL0-1, IL-2, IFN-y and TNF- a) on the central nervous system.12,27-33 Cytokines can also interact with prostaglandins (members of a group of lipid compounds that are derived enzymatically from fatty acids and have important functions in the body) and corticotrophin-releasing hormone (CRH), a polypeptide hormone and neurotransmitter involved in the stress response to inhibit gastric emptying.29,30
Cytokine interaction with neuropeptides may induce anorexia and can also modify serotonin and catecholamine pathways in the central and peripheral nervous system. Cytokines; TNF-a, IL-1, IL-6 and IFN-y have been implicated in the induction of cancer related muscle wasting.34
Interleukin-1 (IL-1)
IL-1 was observed to induce anorexia, weight loss and hypoalbuminemia in mice35 and satiety in rats as a result of activation of gluco-sensitive neurons in the ventromedial nucleus of the hypothalamus.36,37 Otterness et al.38 and Mrosovsky et al.39 observed cachexia-causing effects of IL-1 when it is administrated to animals. Transfection of a cachectic tumor cell line
Radiol Oncol 2009; 43(2): 65-75.
69	Jurdana M / Cancer cachexia-anorexia syndrome and skeletal muscle wasting

(coIon-26) with the gene for IL-1 receptor antagonist failed to abolish the capacity of the tumor to produce cachexia.40 These results confirmed a role of this cytokine in the induction of tissue wasting in cancer.
Tumor necrosis factor alpha (TNF- a)
Many of the metabolic disturbances associated with IL-1 are similar to those of TNF-a, a possible cachectic factor. Administration of TNF-a to laboratory animals induces a state of cachexia, with anorexia and depletion of adipose tissue and Iean body mass. Its effects are mediated centraIIy and in the gastrointestinal tract. TNF-a infusions di-rectIy reduce gastric emptying and peristaI-sis,41 they can induce lipolysis and inhibit lipid synthesis,42-46 and increase proteolysis in peripheral muscle.47 TNF-a has been shown in several studies to activate muscle protein degradation directly and induces IL-6 release.48 It has been demonstrated that IL-6 can impair TNF-a expression in cardiac muscle; one potential role of IL-6 expression in contracting skeletal muscle is therefore to downregulate TNF-a expression.49
Llovera et al.50 demonstrated that TNF-a administration to heaIthy, cancer free rats brought about an enhanced rate of degradation of skeIetaI muscIe protein, even though body weight Ioss was not apparent in the animaIs.
Hyperexpression of TNF-a has been identified as one of the key cytokine responses involved in cachexia.51 TNF-a appears to in-fIuence severaI other abnormaIities present during cancer cachexia: adipose and mus-cIe wasting, insuIin resistance, increased thermogenesis, and aIteration in Iipid and protein metabolism.52,53
Interleukine - 6 (IL-6)
The role of IL-6 in the development of cancer cachexia has mainly been provided from
animaI studies invoIving the use of murine coIon-26 adenocarcinoma model. Evidence of a causative roIe of IL-6 in the pathogen-esis of anorexia and cachexia comes from experiments reporting that treatment with anti-mouse IL-6 antibody was successfuI in reversing the key parameters of anorexia in mice bearing adenocarcinoma.54,55
EIevated serum concentration of IL-6 has been reported in cancer patients. IL-6 increased in Iung cancer patients and has been identified as a mediator of cachexia by the growth of a uterine cervical carcinoma (Yomoto) in nude mice.56 An eIevated IeveI of serum IL-6 has been reported in patients with coIon cancer and in acute-phase response,57 however, since aII patients had Iost weight, it is difficuIt to associate this eIevation with the induction of cachexia.
MuscIe atrophy has been observed in IL-6 transgenic mice, and another study reported that administration of IL-6 to rats acuteIy activated both totaI and myofibriIIar protein degradation in skeIetaI muscIe.58 It has been demonstrated that contracting human skeIetaI muscIe reIeased IL-6 but not TNF-a, because it negatively affects glucose uptake in skeIetaI muscIe.59
Glucocorticoids can prevent the stimulatory effects of proinfIammatory factors on IL-6 secretion, which in generaI stimuIate proIiferation at the earIiest, myobIast stage of muscle formation. Prelovsek et al.48 reported that a high dexametasone concentration prevents the stimuIatory effects of TNF-a and LPS on IL-6 secretion from the precursors of human muscIe regeneration. It resuIts in prevention of myobIast proIif-eration, Ieading to a reduced finaI mass of the regenerated muscIe.
The resuIts of human and animaI studies strongIy impIicate IL-6 in the cachectic process. IL-6 probabIy does not act aIone but may either induce or act in synergy with other cachectic factors.
Radiol Oncol 2009; 43(2): 65-75.
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Interferon gamma (IFN-y)
In mice bearing Lewis lung tumors, the development of tumors is associated with IFN-y production and with progressive weight loss. IFN-y antibodies counteract the wasting syndrome observed in cancer cachexia.60 In rats that had received transplants of MCG 101 sarcoma, anti-IFN-y antibody reduced weight loss and anorexia but the treatment was partial and short-lived, suggesting that IFN-y may not be the sole mediator.61 Such a result should not be interpreted to mean that IFN-y by itself can induce cachexia, since both IFN-y release and the presence of tumor cells were found to be required.
Leukemia- inhibitory factor (LIF)
LIF is proposed to be a mediator of cachexia through its ability to decrease lipoprotein lipase activity. LIF plays an important role in the development of cancer cachexia syndrome observed in melanoma-bearing nude mice. The expression of LIF mRNA was examined in four melanoma xenografts, SEKI, G361, A375 and MEWO, in nude mice. SEKI- and G361-bearing nude mice developed cancer cachexia syndrome, and their body weights decreased. A375- and MEWO-bearing nude mice, however, did not develop the syndrome. Northern blot analysis revealed that G361 as well as SEKI expressed a large amount of LIF mRNA, but A375 and MEWO did not, suggesting a close relationship between the expression of LIF mRNA and the development of the syndrome.62
Proteolysis-inducing factor (PIF)
Proteolysis-Inducing Factor, which induces muscle wasting, was purified from cachex-ia-inducing MAC-16, a murine adenocarci-noma. The exogenous administration of PIF to healthy mice resulted in a 50% decrease
in muscle protein synthesis and a 50% increase in muscle protein degradation.63
A PIF of identical characteristics and molecular weight was detected in the urine of persons losing weight due to pancreatic or gastrointestinal cancers but not in the urine of weight-stable patients with cancer or weight-losing non-cancer patients.64
PIF appears to induce muscle wasting via activation of the ubiquitin proteosome pathway.65 It seems unlikely that any of the cytokines alone are able to explain the complex mechanism of wasting seen in cancer cachexia, and other factors must be involved.
Effects of cachexia on skeletal muscle
Cancer cachexia is associated with perturbations in protein metabolism, leading to significant wasting of tissue proteins. Muscle wasting results from an imbalance between the rates of muscle protein synthesis and degradation. Body composition analysis shows that skeletal muscle is the major site of protein loss in patients with solid (non hematological) tumors.66 There are also changes in the concentration of plasma amino acids, and most studies report a decrease in gluconeogenic amino acids, in contrast with severe malnutrition, in which the concentration of branched-chain amino acids in plasma is normal or even increased. Protein degradation of amino acid results in the release of amino acids, particularly alanine and glutamine. The former is channeled to the liver for gluco-neogenesis and APP (acute phase protein) synthesis, whereas glutamine is taken up by the tumor to sustain energy and nitrogen demands.1
Intracellular protein breakdown can be mediated by three pathways; lysosomal, Ca2+-dependent and ATP-ubiquitin dependent proteolytic pathways. All three
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pathways may be involved in cachexia, although the ubiquitin-dependent system is considered to be the most important and has been most studied.67 Recent evidence suggests that PIF and TNF-a, but not other cytokines, can induce expression of the key regulatory components of this pathway.65,68
TNF-a induces muscle wasting via inhibition of pathways involved in muscle cell differentiation and regeneration.69 Exposure of myocites to TNF-a activates the transcription factor-nuclear factor kappa B (NF-kB), which in turn inhibits muscle cell differentiation by suppressing the synthesis of MyoD, a transcriptional factor that is essential for muscle cell differentiation and for the repair of damaged muscle tissue.69 The activation of NF-kB is also involved in upregulation of cytokine synthesis, which can contribute to paracrine effects of cy-tokines on skeletal muscle tissue, as previously described. Cytokine-induced skeletal muscle wasting is probably a multifactorial process, involving increased protein degradation and reduced myocite regeneration and repair.70
The consequence of skeletal muscle wasting is fatigue. Atrophy of skeletal muscle leads to asthenia and muscle weakness, which causes a reduction of physical activity level in affected persons. This leads to more muscle deconditioning and atrophy, which in turn aggravates the feeling of fatigue.
Physical exercise training may potentially attenuate muscle wasting and/or reduce fatigue in cancer patients. Data obtained from healthy humans and from experimental animals demonstrate that regular endurance exercise training of submaximal intensity (below maximal heartbeat) increases muscle endurance and resistance to fatigue.71 Resistance exercise training also increases the mass of healthy muscles and attenuates muscle wasting associated with some catabolic conditions. Resistance ex-
ercise training in cancer patients increases muscle mass by accelerating the rate of protein synthesis and by attenuating muscle protein breakdown and/or muscle wasting. The effect of resistance exercise training on increased muscle mass in cancer has not been adequately studied.
A mouse model of cancer cachexia (mice bearing the colon-26 adenocarcinoma) was used to test the hypothesis that resistance training, performed by electrical stimulation of the motor nerve would attenuate cancer-related wasting in the contracting muscles.72 The results of the study demonstrate that resistance training attenuates wasting of the extensor digitorum longus muscle (EDL) in tumor-bearing mice. This attenuation of wasting was paralleled by an increase in muscle weight, which was due to an increase in the actual mass of muscle and not merely due to edema. The findings of this study also suggested that the dose of training that attenuates wasting of the EDL muscle in tumor-bearing mice is not sufficient to induce hypertrophy in the EDL muscle in the control non-tumor-bearing mice. These data show that wasted muscles respond differently than non-wasted muscles to exercise training. Attenuation of muscle wasting may be easier to achieve than the induction of hypertrophy of healthy non-wasted muscles, which suggests that persons with wasted muscle may not need to exercise as vigorously in order to attenuate wasting of their muscles.
The loss of skeletal muscle mass that accompanies cancer cachexia is associated with changes in muscle functional properties, with a shift in muscle fiber type distribution or a shift in myosin isoform expression. Myosin heavy chain (MHC) and myosin light chain (MLC) expression are characterized by a decrease in the phenotypic expression of "slow" myosine isoforms (type I MHC and slow forms of MLC) and an increase in the phenotypic
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expression of "fast" myosine isoforms (type II MHC and fast MLC isoforms) in the so-leus muscle.68 These changes are likely to have a significant impact on the functional properties of muscles during cachexia. The exact mechanism of this effect on myosin isoform expression is unknown at present. Evidence suggests that the problem is a multifactorial process mediated by host-released and tumor-released factors. The activation of pro-inflammatory cytokines and various proteolytic pathways, particularly the ubiquitin proteasome pathway within skeletal muscle may be responsible for the problem.69 These factors apparently disturb the balance between the rates of skeletal muscle protein synthesis and muscle protein degradation, leading to muscle protein depletion and muscle wasting. Wasting of skeletal muscle contributes to muscle weakness, fatigue and to the morbidity and mortality of cancer.
ance exercises should be examined as a potential intervention for attenuating cancer-induced muscle wasting. Exercise-induced changes of skeletal muscle mass should be pursued in terms of changes in muscle protein synthesis and muscle protein degradation pathways in the exercised versus the unexercised muscles, both of which have been implicated in cancer induced muscle wasting.
Exercise that induces attenuation of muscle wasting is a result of an increase in muscle protein synthesis or a decrease in muscle protein breakdown. Its mechanisms need to be explored in future research. It would be important to examine the functional benefit of training-induced attenuation of cancer-related muscle wasting.
The intervention of preserving muscle mass has an important clinical implication, since it improves the prognosis and the quality of life of cachetic cancer patients.
Conclusions
There is a high incidence of cancer cachex-ia-anorexia syndrome in cancer patients. This syndrome is observed in 80% of patients in advanced stage cancer. Cachexia is characterized by wasting of skeletal muscle and depletion of host adipose tissue, while anorexia is associated with persistent loss of appetite and decrease in food intake. Both induce weight loss that differs from caloric starvation.
The cachectic state is particularly problematic in cancer. It typifies a poor prognosis and often lowers responses to chemotherapy and radiation treatment.74 Radiation damages mitotically active muscle satellite cells, prevents compensatory hypertrophy of skeletal muscle, and prevents small fiber formation.75
Skeletal muscle wasting during cancer cachexia is well documented, and resist-
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doi:10.2478/v10019-009-0007-y
research article
Xanthogranulomatous cholecystitis remains a challenge in medical practice: experience in 24 cases
Mehmet Yildirim1, Ozgur Oztekin2, Fatih Akdamar1, Savas Yakan1, Hakan Postaci3
1Department of Surgery, 2Department of Radiology, 3Department of Pathology, Izmir Bozyaka Teaching and Research Hospital, Izmir, Turkey
Background. Xanthogranulomatous cholecystitis (XGC) is a rare, benign, chronic inflammatory disease of the gallbladder. Its importance lies in the fact that imaging studies and intraoperative appearance that may be confused with tumour of the gallbladder. This study aimed to evaluate pre-and intraoperative findings of XGC and to remind it in difficult cholecystectomy patients.
Patients and methods. The clinical data of 24 patients with XGC over a period of 7 years were analyzed retrospectively (mean age, 53 years (32-68) M/F ratio 1:1.4).
Results. The clinical symptoms were abdominal pain, nausea and jaundice in 79%, 62% and 12% of the patients. Preoperative ultrasonography for 24 patients revealed gallstone (95.8%) and bile sludge (8%). Pericholecystic fluid, polyp and tumour of the gallbladder was present in 20%, 4% and 4% of the patients. The gallbladder was thickened (>3mm) in 10 patients. On computed tomography, all patients showed abnormal findings. The intraoperative findings were as follows: gallstones (100%), chronic cholecystitis (54%), hydropic gallbladder, emphysematous gallbladder, adhesions of the gallbladder to adjacent organs and tumoural mass of gallbladder.
Conclusions. XGC is difficult to diagnose pre-or intraoperatively and remains a challenge in medical practice. The definitive diagnosis depends on the histopathologic examination.
Key words: xanthogranulomatous; cholecystitis; gallbladder
Introduction
Xanthogranulomatous cholecystitis (XGC) is an uncommon infIammatory disease of the gallbladder characterized by the infil-
Received 23 January 2009 Accepted 10 April 2009
Correspondence to: Dr Mehmet Yildirim, Atakent Mah.Bergama 2 Apt.Giris:32 Daire: 1, Bostanli, Izmir, Turkey; Phone: +90 232 3625692; Fax: +90 232 2614444; E-mail: mehmetyildi@gmail.com
tration of pIasma ceIIs, Iipid-Iaden histio-cytes, and the proIiferation of fibrobIasts in the gallbladder wall.1
The term of xanthogranuIomatous choIe-cystitis was initiaIIy proposed by Goodman and Ishak in 1981.2 The pathogenesis of XGC is the rupture of Rokitansky-Aschoff sinuses and extravasation of bile into the muscuIar Iayer. The rupture of the serosa results in adhesion to the adjacent liver, duodenum, and transverse coIon. GaIIstones
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77
may have an important role in the patho-genesis, since they appear to be present in most patients.3
The clinical and laboratory findings of cases with XGC are similar to those of acute or chronic cholecystitis.4 The major intraoperative findings are frequently characterized by thickening of the gallbladder wall, tumour-like mass and adherence of the gallbladder to adjacent organs.5 Patients with XGC are frequently misdiagnosed with imaging studies and even during the operation as having carcinoma of the gallbladder.
The aim of this study is to evaluate pre-and intraoperative findings of XGC and to remind it in difficult cholecystectomy patients.
Patients and methods
Twenty four histologically confirmed cases of XGC were identified from the retrospective analysis of the patient records of 749 cholecystectomy operations over a period of 7 years (January 2000-April 2007). The study included 14 female and 10 male (male/female ratio 1:1.4) having a mean age of 53 years (range, 32- 68 years).
The clinical presentation, laboratory and radiological findings, surgical findings, his-topathological characteristics, morbidity and mortality were investigated from the surgeon's registry.
All patients underwent preoparative ultrasound examination with 3 MHz transabdominal probe (General Electric Logic5 Pro, Milwaukee, Wisconsin, USA). CT examination was performed for 6 patients with Toshiba spiral CT Asteion. A standard abdominal MRI protocol was employed to one patient. All images were acquired using on 1.5 Tesla MR units. MR studies were performed on a 1.5 T unit (Achieva; Philips Medical Systems, Eindhoven, The Netherlands).
Figure 1. 59-year-old woman with xanthogranulomatous cholecystitis. Contrast-enhanced CT scan shows hydropic gallbladder.
The US findings used for the diagnosis were presence of gallstone and bile sludge, pericholecystic fluid and thickness of the gallbladder wall. Thickening of the gallbladder wall was considered abnormal if it exceeded 3 mm to Kim et al.6 The CT feature used for the diagnosis were presence of gallstones; increased thickness of the gallbladder wall (>3mm), loss of interface between the gallbladder and the liver; peric-holecystic fluid; and choledocholithiasis. In one patient, a MRI study was made because of suspected gallbladder carcinoma in CT appearance.
The gallbladder was fixed with 10% formalin and specimens were stained with H and E. Descriptive statistics were used to describe the features of the data in our study.
Results
The clinical symptoms were right hypo-chondrial pain in 19 (79%) patients, nausea in 15(62%) icteric sclera in three (12%), and fever in two (8%). Main signs included positive Murphy's sign, palpable mass in the right upper quadrant and yellow skin were found in 17 (70%), 24 (100%) and three (12%) patients.
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Figure 2. Abdominal MR imaging demonstrating suspected gallbladder mass involving the liver in T1 weighted series(A), and a soft tissue mass in T2 weighted series (B).
Laboratory tests were within normal ranges except leucocytosis in five patients (>12.000/|l), elevated ALT-AST and bil-uribin levels were found in three patients in each group.
Preoperative US for 24 patients revealed gallstone in 23 (96%) patients, perichole-cystic fluid in five (20%), bile sludge in two (8%), polyp of gallbladder in one (4%), and tumour of gallbladder in one (4%) patient. The gallbladder was thickened (>3mm) in ten (41.6%) patients. Upper abdominal CT was performed in six (25%) patients. CT revealed hydrops of gallbladder in two (33.3%) patients (Figure 1), carcinoma of the pancreas also in two (33.3%) patients, pericholecystic abscess in one and one carcinoma of gallbladder. Thickening of the gallbladder wall was seen, more than 3 mm thick, in six patients. MRI showed suspected gallbladder cancer involving the liver in one patient (Figure 2A-B).
The intraoperative findings were as follows: chronic cholecystitis in 13 (54%), adhesions of the gallbladder to adjacent organs in four (adhesions to the transverse colon in two patients, the duodenohepatic ligament in one, and fundus of the gallbladder to abdominal wall in one), emphysema-tous gallbladder in three (perforation of the gallbladder in two patient and carcinoma
of the pancreas in one), hydrops of the gallbladder in two (carcinoma of the pancreas in one patient and enlarged gallbladder with a stone impacted in Hartmann's pouch in one), and tumoural mass of the gallbladder in two patients (Figure 3). The gallbladder stones were found in all patients. Furthermore, the frozen section was performed in one patient and malignancy was not found. The surgical treatment was elective open cholecystectomy in 23 (95.8%) patients and laparoscopic cholecystectomy (LC) in one (4.2%) patient. In one patient, who could not rule out the possibility of
Figure 3. Tumoular like deposits of xanthogranuloma-tous cholecystitis in surgical specimen.
Radiol Oncol 2009; 43(2): 76-83.
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79
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A B
B
Figure 4. Histiocytes, lymphocytes and giant cells present in a granulomatous focus of xanthogranulomatous inflammation. A; (HE X20) B; (HE X40)
carcinoma, cholecystectomy and wedge resection of adjacent liver was performed.
The postoperative complications were found in 7(29%) patients who included wound infection and pleural effusion in four and two patients, respectively. Acute renal failure developed in one patient with pancreas cancer. The average hospital stay time was 8 days (range, 3- 22 days). No mortality was seen.
Histopathologically there were a focal or diffuse inflammatory process with xanthogranulomatous changes, histiocytosis and giant cells of foreign body in all patients (Figure 4-5). The examination of specimen showed gallstones in 24 (100%) patients, presence of sludge in 13 (54.4%), thickening gallbladder wall (>5 mm) in 15 (62%), dysplasia in four (16%) and mucosal
ulcers in three (12%). In addition four (16%) patients had lymphadenopathy which showed reactive lymphadenitis.
Discussion
XGC was previously described as an uncommon form of chronic cholecystitis.7 Christensen AH and Ishak KG initially described it as a pseudotumour with destructive type of gallbladder inflammation, pericholecystic infiltration, hepatic involvement and lymphadenopathy. In 1981 the term xanthogranulomatous cholecystitis was proposed in a review of 40 cases collected over a 10-year period.2
Even though the number of published cases is not large, XGC not as rare as gen-
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Figure 5. Xanthogranulomatous inflammation of the gallbladder wall, characterized by histiocytes (HEx20).
erally believed.7 In the literature, the incidence of XGC is reported to be 0.7% to 13.2%.8 Higher incidence was reported from the Eastern countries.9-11 In our series, the incidence of XGC was 4% among 749 patients who were operated on gallstone. XGC mostly affects middle-aged women and old persons between 60-70 years. Similar to the previous reports the mean age was 63 years in our study.12,13 This suggests that age must be one of the significant factors in the development of the XGC. The male to female ratio range is from 2:1 to 1:2 in other series.14 A study from India12 reported a 1:9 male to female ratio while in our report male to female ratio was found 1:1.4. The different incidence of XGC may be due to misdiagnosis by clinicians.
Radiol Oncol 2009; 43(2): 76-83.
Clinically, XGC does not have a typical presentation. Our patients were presented with right upper quadrant pain, nausea, fever, icterus and palpable mass; they are similar to acute or chronic cholecystitis. These clinical features are not specific for XGC and there was no difference between the patients with cholecystitis and gallbladder carcinoma.11,15 We noted that all of the patients with these symptoms required an elective surgical procedure at first presentation. The association of XGC with a perforated gallbladder, abscess formation, enterobiliary fistula and Mirizzi syndrome were supported by increasing series.4,16 In our series, these lesions were found in 16% patients. In one case, the adhesion of fundus to abdominal wall was considered as the potential of XGC for fistula formation.
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The reported series support the existence of this comorbid factors seen in nearly 23% of patients.4
Preoperative biochemical tests or imaging studies are not suggestive of XGC.12 Neither the liver function abnormalities nor the tumour markers (CEA, CA-19.9) are suggestive of XGC, although Adachi et al}7 argued that serum level of CA-19.9 may be elevated in both carcinoma and XGC patients. It has been reported that thickening of the gallbladder, adhesion to neighbouring tissues or organs were specific findings to XGC, although there are other series that it is difficult to differentiate XGC from other lesions.18,19 All these findings can be seen in acute cholecystitis; however, the presence of intramural low-attenuation nodules, preservation of mu-cosal lining and degree of enhancement of the gallbladder wall are suggestive of the XGC.18 In our study none of the patients was diagnosed by radiologist as XGC with imaging findings. US misdiagnosed one case of XGC as carcinoma of the gallbladder (misdiagnose rate 4.3%), while CT mis-diagnosed two cases of XGC as carcinoma (misdiagnose rate 33.3%). In this study, high misdiagnosis rate may be related to our insufficient experience for imaging findings of XGC. Chun et al.18 concluded that a definitive diagnosis of gallbladder carcinoma is not possible with only imaging findings. In our study, thickening of the gallbladder wall was found in 24% and 46% patients with US and CT respectively. While thickening of the gallbladder wall was found in 62% of patients pathologically. This leads to the conclusion that thickening of the gallbladder wall in imaging findings itself is not a predictive factor in the diagnosis of XGC. Hatakenaka et al.20 have demonstrated that MR imaging may play an important role in differentiating XGC from carcinoma in patients with a thickened gallbladder wall. In our series,
MR imaging did not differentiate XGC from gallbladder carcinoma.
In our series as in others reported in the literature, all patients had gallstones (100%), frequently sludge of bile (54%) or biliary obstruction (12%).4 Its importance lies in the fact that gallstones can have an important role in the pathogenesis of XGC via extravasation of bile into the gallbladder wall. In contrary, in a series noted the presence of gallstone in only 85% of the XGC, which leads to the conclusion that the presence of gallstone is only an associated condition and not the cause of the inflammatory process.21 The small ulcerations in the mucosa reported to be a precipitating factor in other studies.7,8 The extravasation of bile causes that fibrous reaction and scarring healing within the gallbladder wall, response due to thickening of the gallbladder wall. The ulcerations in the mucosa were found pathologically in 12% of our patients.
Open cholecystectomy is the first choice for XGC, either complete or partial.14 In our series, single cholecystectomy was performed on 23 patients and cholecystectomy with partial hepatic wedge resection on one patient. LC may be contraindicated in XGC because of a high incidence of complications. According to the study carried by Guzman-Valdivia G, LC was not completed due to difficulty in dissecting the gallbladder and converted into the open procedure in 80% of the patients diagnosed as XGC.21 The necessity of radical surgery is not cleared with extra-gallbladder involve-ment.4 The intraoperative frozen section or fine needle aspiration has been suggested to confirm the diagnosis of XGC.9 The frozen section is valuable when there is no invasion of pericholecystic organs. In our series, the frozen section was used (negative result) in a case with an extensive invasion of liver. We believe it was not change the approach of surgeon. Nevertheless, in the patients with negative results, the radical
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surgery can be performed due to the coexistence of XGC and carcinoma of the gaII-bladder. On the other hand, studies report that the radicaI surgery may be associated with a high perioperative morbidity.22
Although XGC is a benign disease, patients usuaIIy have a Ionger hospitaI stay with a postoperative compIications.4 In our study, complications occurred in 7(29%) patients incIuding wound infection, pIeuraI effusion and renaI faiIure reIated to hepato-renaI faiIure. CompIications were reported incIuding Ieakage of biIe and biIe peritonitis which IargeIy reIated to the difficuIty in choIecystectomy.9 The compIications are thus reIated more to the technicaI difficuIty in stripping the gaIIbIadder, the mode of the operation and the cIinicaI condition of the patient than to the disease itseIf.
Conclusions
The pre- or intraoperative differentiaI diagnosis of XGC from other gaIIbIadder diseases remains a chaIIenge in medicaI practice. The presence of firm adhesions of the gaIIbIadder to neighbouring organs and tissues, thickened gaIIbIadder waII, together with gaIIstone in a patient with chronic disease, is highIy suggestive of XGC. The definitive diagnosis depends on the histopathoIogic examination. XGC can be treated successfuIIy with an accurate diagnosis and proper operation.
References
1.	Houston JP, Collins MC, Cameron I, Reed MWR, Parsons MA, Roberts KM. Xanthogranulomatous cholecystitis. Br J Surg 1994; 81: 1030-2.
2.	Goodman ZD, Ishak KG. Xanthogranulomatous cholecystitis. Am J Surg Pathol 1981; 5: 653-9.
3.	Benbow EW. XanthogranuIomatous choIecysti-
tis. Br J Surg 1990; 77: 255-6.
4.	Yang T, Zhang BH, Zhang J, Zhang YJ, Jiang XQ, Wu MC. SurgicaI treatment of xanthogranuIo-matous choIecystitis: experience in 33 cases. Hepatobiliary Pancreat Dis Int 2007; 6: 504-8.
5.	Spinelli A, Schumacher G, Pascher A, Lopez-Hanninen E, AI-Abadi H, Benckert C, et aI. Extended surgicaI resection for xanthogranuIo-matous choIecystitis mimicking advanced gaII-bIadder carcinoma: a case report and review of literature. World J Gastroenterol 2006; 12: 2293-6.
6.	Kim PN, Ha HK, Kim YH, Lee MG, Kim MH, Auh YH. US findings of xanthogranulomatous choIecystitis. Clin Radiol 1998; 53: 290 -2.
7.	Ros PR, Goodman ZD. XanthogranuIomatous choIecystitis versus gaIIbIadder carcinoma. Radiology 1997; 203: 10-2.
8.	Kwon AH, Matsui Y, Uemura Y. SurgicaI procedures and histopathoIogic findings for patients with XanthogranuIomatous choIecystitis. J Am Coll Surg 2004; 199: 204-10.
9.	KarabuIut Z, Besim H, Hamamci O, Bostano Iu S, Korkmaz A. XanthogranuIomatous choIecystitis. Retrospective anaIysis of 12 cases. Acta Chir Belg 2003; 103: 297-9.
10.	Dixit VK, Prakash A, Gupta A, Pandey M, Gautam A, Kumar M, et aI. XanthogranuIomatous cholecystitis. Dig Dis Sci 1998; 43: 940-2.
11.	Roberts KM, Parsons MA. Xanthogranulomatous choIecystitis: cIinicopathoIogicaI study of 13 cases. J Clin Pathol 1987; 40: 412-7.
12.	Belague C, Targarona EM, Sugra es G, Rey MJ, Arce Y, Viella P, et al. Xanthogranulomatous choIecystitis simuIating gaIIbIadder neopIasm: therapeutic implications. Gastroenterol Hepatol 1996; 19: 503-6.
13.	Eriguchi N, Aoyagi S, Tamae T, Kanazawa N, Nagashima J, Nishimura K et al. Xanthogranulomatous cholecystitis. Med J 2001; 48: 219-21.
14.	Guzman-Valdivia G. Xanthogranulomatous cholecystitis: 15 years' experience. World J Surg 2004; 28: 254-7.
15.	Wang D, Yamakawa T, Fukuda N, Maruno K, Ushigome S, Ish yama J. XanthogranuIomatous choIecystitis: report of a case and Iiterature review on cIinicaI differentiating factors from gallbladder carcinoma. Digest Endoscopy 2004; 16: 143-7.
16.	Benbow EW. XanthogranuIomatous choIecysti-tis. Br J Surg 1990; 77: 255-6.
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83
17.	Adachi Y, Iso Y, Moriyama M, Kasai T, Hashimato H. Increased serum CA 19-9 in patients with xanthogranulomatous cholecystitis. Hepatogastroenterol 1998; 45: 77-80.
18.	Chun KA, Ha HK, Yu ES, Shinn KS, Kim KW, Lee DH, et al. Xanthogranulomatous cholecystitis: CT features with emphasis on differentiation from gall-bladder carcinoma. Radiology 1997; 203: 93-7.
19.	Para A, Acinas O, Bueno J, Guezmes A, Fernandez MA, Farinas MC. Xanthogranulomatous cholecystitis clinical, sonographic, and CT findings in 26 patients. AJR 2000; 174: 979-83.
20.	Hatakenaka M, Adachi T, Matsuyama A, Mori M, Yoshikawa Y. Xanthogranulomatous cholecystitis: importance of chemical-shift gradient-echo MR imaging. Eur Radiol 2003; 13: 2233-5.
21.	Guzman-Valdivia G. Xanthogranulomatous cholecystitis in laparoscopic surgery. J Gastrointest Surg 2005; 9: 494-7.
22.	Srinivas GN, Sinha S, Ryley N, Houghton PW. Perfidious gallbladders - a diagnostic dilemma with xanthogranulomatous cholecystitis. Ann R Coll Surg Engl 2007; 89: 168-72.
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Radiol Oncol 2009; 43(2): 84-87.
doi:10.2478/v10019-009-0013-0
case report
Spinal subdural haematoma in von Willebrand disease
Artur Franko1, Ronald Antulov1, Sinisa Dunatov2, Igor Antoncic2, Damir Miletic1
1Department of Radiology, 2Department of Neurology, Clinical Hospital Centre Rijeka, Rijeka, Croatia
Background. Von Wilebrand disease (vWD) is the most common inherited disorder of hemostasis. Bleeding in patients with von Wilebrand disease is a frequently reported complaint. Patients with inherited bleeding disorders are also in a large number of cases infected with hepatitis C virus (HCV). Studies showed an increased risk factor for intracerebral hemorrhage (ICH) in patients infected with HCV receiving the combination treatment.
Case report. A 44-year old man reported to our Emergency Department with low back pain, headaches, neck pain, leg weakness and urinary retention. The neurological examination showed nuchal rigidity and spastic paraparesis. Thoracic spine MRI revealed a subacute subdural haematoma at T8-T11 level. Conclusions. To the best of our knowledge, this is the first report of a subdural haematoma of the thoracic spine in a patient with vWD and chronic HCV infection. The presented patient was receiving a combination treatment, a fact that has also to be taken in consideration as a possible risk factor for a bleeding episode.
Key words: von Willebrand disease; subdural haematoma; hepatitis C; complications of treatment; interferon; ribavirin
Introduction
Von Wilebrand disease (vWD) is an inherited bleeding disorder that results from quantitative and qualitative deficiencies of von Wilebrand factor (vWF), a glycopro-tein with an essential role in primary and secondary hemostasis.1 vWD is the most common inherited bleeding disorder, with
Received 23 December 2008 Accepted 23 March 2009
Correspondence to: Damir Miletic, MD, PhD, Department of Radiology, Clinical Hospital Centre Rijeka, Kresimirova 42, 51 000 Rijeka, Croatia; Phone: +385 (0)51 658 862; Fax. +385 (0)51 658 386; E-mail: damir.miletic@medri.hr
a prevalence from 0.6% to 1.2%.2 Patients with vWD are prone to complain of different bleeding episodes like mucocutaneus bleeding, menorrhagia, gastrointestinal bleeding with angiodysplasia or excessive postsurgical bleeding.1 The infection with hepatitis C virus (HCV) is a major co-morbidity in patients affected by bleeding disorders, with a prevalence of 39% in vWD patients.3 In patients with chronic HCV infection the combination therapy of pegylated interferon alpha-2b and ribavirin is becoming a standard treatment.4 Recent studies suggested that patients with chronic HCV infection receiving combination therapy were more prone to develop an in-tracerebral haemorrhage (ICH), indicating
Franko A et al. / Spinal subdural haematoma in von Willebrand disease
85
a possible association between HCV, HCV treatment and ICH.5, 6
Spinal subdural hematoma (SSDH) is a rare condition that can lead to spinal cord or cauda equina compression.7 A variety of causes, like bleeding disorders, anticoagulant therapy, arteriovenous malformations or underlying neoplasm have been described as possible pathogenic factors that could promote the formation of an acute SSDH.8 Therefore, SSDH is a neurological and neurosurgical emergency that has to be promptly diagnosed and treated in order to provide the best possible recovery.
We report on a 44-year old man with vWD and chronic HCV receiving the combination therapy that developed sudden leg weakness and low back pain as the initial presentation of spontaneous acute SSDH. The diagnosis was confirmed by MRI and the patient was treated with a conservative approach.
Here we discuss the clinical features, imaging findings, treatment decision and outcome.
Case report
A 44-year old man reported to our Emergency Department (ED) with acute low back pain, headaches, neck pain, leg weakness and urinary retention. During the last two days before he reported to our ED, the patient had high body temperature. Since childhood, he was diagnosed vWD. He was serologically diagnosed as infected with HCV in 1999, and had been treated with the combination therapy of pegylated interferon alpha-2b and ribavi-rin for a period of five months before he reported to our ED. A month and a half before this episode, he was admitted to our Gastroenterology Department because of a bleeding duodenal ulcer. There was no history of trauma.
The physical examination showed blood pressure 100/60 mm Hg, pulse 88/min and body temperature of 37.7 °C. The neurological examination showed nuchal rigidity and spastic paraparesis of the legs. The blood test revealed a platelet count of 89.000/mm3, a prothrombin time of 65% and a partial thromboplastin time of 54 s. Laboratory studies for vWD showed a factor VIII activity of 43% and a vWF activity (ristocetin factor) of 17%. A MRI of the thoracic spine revealed a high intensity signal in T1- and T2-weighted images (WI) extending from T8 to T11 that belonged to a dorsal subdural haematoma. There was no abnormal signal visible in the spinal cord (Figure 1). The treatment was conservative and cryoprecipitates were administered according to the level of factor VIII and vWF activity. The antiviral combination therapy was withdrawn. The patient responded well to our therapy and physiotherapy treatment followed after the initial mobilization.
After a month, on discharge from our centre, the patient was able to sit independently and his neurological improvement was satisfactory. On a follow-up MRI scan done 3 months after he reported to our ED there was a complete regression of the sub-dural hematoma and the neurological examination was normal.
Discussion
Acute SSDH is a rare condition that may result from iatrogenic factors, but also from coagulopathies, anticoagulant therapy, severe liver failure, underlying neoplasm, arteriovenous malformations or after poisoning with rodenticides of the coumarin group.8-10 It usually presents with acute back pain and signs of spinal cord and cauda equina compression. Bleeding episodes often accompany vWD, but they rarely lead to the formation of a hematoma.1 In two
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cases, patients with vWD reported with a subgaleal hematoma and an encapsulated hematoma of the thigh, both of them occurring after a trauma.11,12
Our patient with SSDH and vWD did not have any recent history of trauma, but he was on combination therapy for chronic HCV infection. Recent studies and case reports suggested that HCV infection alone or with combination therapy raised the possibility to develop an ICH.5,6,13 Taking this in consideration, we could hypothesize that chronic HCV infection and combination therapy could be a risk factor to develop SSDH. Therefore, we decided to withdraw the antiviral combination therapy from his treatment.
MRI is the imaging method of choice to determine the location, evolution, extent and shape of the hematoma, as well as the follow-up of the patient like at the patients with the others causes of spine cord com-presion.9,10,14,15 Our patient had a dorsal hematoma, with a crescent shape, extending from T8 to T11 and a high intensity signal in T1- and T2-weighted images indicating
a hematoma in the subacute phase. The epidural fat was well delineated, a fact that confirms the subdural location of the hematoma. Follow-up MRI scans were done in order to asses the evolution of the hematoma, that in our case in a 3 months period showed a complete regression, and to look for possible long term complications represented by arachnoidal fibrosis or spinal cord atrophy.10
The current literature suggests three treatment options in case of a subdural hematoma: surgical decompression, percutaneous clot drainage and conservative treat-ment.9,10,14 In a previous case of a subdural hematoma in a patient with idiopathic thrombocytopenic purpura a conservative approach was preferred, because of a significant risk of bleeding.16 Because our patient was diagnosed with vWD, a factor that could promote bleeding complications during a surgical procedure, we opted for a conservative treatment.
Reviewing the literature regarding spinal hematoma and vWD, Kakazu et al.17 reported of a spontaneous spinal epidural haem-
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87
orrhage associated with vWD. Therefore, to the best of our knowledge, this is the first report of a subdural hematoma of the thoracic spine in a patient with vWD and chronic HCV infection under the combination therapy. Our patient had an indicative clinical presentation, the diagnosis was confirmed by MRI and we decide to pursue a conservative treatment approach. We withdrew the antiviral combination therapy because of the possibility that it could be a risk factor for spontaneous bleeding. After three months, our patient showed a complete recovery.
References
1.	Ewenstein BM. Von Willebrand's disease. Annu Rev Med 1997; 48: 525-42.
2.	Rodeghiero F, Castaman G, Dini E. Epidemiological investigation of the prevalence of von Willebrand's disease. Blood 1987; 69: 454-9.
3.	Federici AB, Santagostino E, Rumi MG, Russo A, Mancuso ME, Soffredini R, et al. The natural history of hepatitis C virus infection in Italian patients with von Willebrand's disease: a cohort study. Haematologica 2006; 91: 503-8.
4.	Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001; 358(9286): 958-65.
5.	Ferencz S, Batey R. Intracerebral haemorrhage and hepatitis C treatment. J Viral Hepat 2003; 10: 401-3.
6.	Nishiofuku M, Tsujimoto T, Matsumura Y, Toyohara M, Yoshiji H, Yamao J, et al. Intracerebral hemorrhage in a patient receiving combination therapy of pegylated interferon alpha-2b and ribavirin for chronic hepatitis C. Intern Med 2006; 45: 483-4.
7.	Langmayr JJ, Ortler M, Dessl A, Twerdy K, Aichner F, Felber S. Management of spontaneous extramedullary spinal haematomas: results in eight patients after MRI diagnosis and surgical decompression. J Neurol Neurosurg Psychiatry 1995; 59: 442-7.
8.	Domenicucci M, Ramieri A, Ciappetta P, Delfini R. Nontraumatic acute spinal subdural hematoma: report of five cases and review of the literature. J Neurosurg 1999; 91(Suppl 1): 65-73.
9.	Boukobza M, Haddar D, Boissonet M, Merland JJ. Spinal subdural haematoma: a study of three cases. Clin Radiol 2001; 56: 475-80.
10.	Morandi X, Riffaud L, Chabert E, Brassier G. Acute nontraumatic spinal subdural hematomas in three patients. Spine 2001; 26: E547-51.
11.	Raffini L, Tsarouhas N. Subgaleal hematoma from hair braiding leads to the diagnosis of von Willebrand disease. Pediatr Emerg Care 2004; 20: 316-8.
12.	Wexler S, Edgar M, Thomas A, Learmonth I, Scott G. Pseudotumour in von Willebrand disease. Haemophilia 2001; 7: 592-4.
13.	Karibe H, Niizuma H, Ohyama H, Shirane R, Yoshimoto T. Hepatitis C virus (HCV) infection as a risk factor for spontaneous intracerebral hemorrhage: hospital based case-control study. J Clin Neurosci 2001; 8: 423-5.
14.	Kyriakides AE, Lalam RK, El Masry WS. Acute spontaneous spinal subdural hematoma presenting as paraplegia: a rare case. Spine 2007; 32: E619-622.
15.	Rajer M, Kovac V. Malignant spinal cord compression. Radiol Oncol 2008; 42: 23-31.
16.	Benito-Leon J, Leon PG, Ferreiro A, Martinez J. Intracranial hypertension syndrome as an unusual form of presentation of spinal subarachnoid haemorrhage and subdural haematoma. Acta Neurochir (Wien) 1997; 139: 261-2.
17.	Kakazu K, Ohira N, Ojima T, Oshida M, Akiyama M, Horaguchi M, et al. Extensive spinal epidural hemorrhage associated with von Willebrand's disease - a case report. Nippon Seikeigeka Gakkai Zasshi 1980; 54: 501-5.
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Radiol Oncol 2009; 43(2): 88-119.
doi:10.2478/v10019-009-0017-9
research article
Increased late urinary toxicity with whole pelvic radiotherapy after prostatectomy
Borut Kragelj
Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia
Background. Radiotherapy aimed at prostatic bed (PBRT) can prevent recurrence or reestablish remission in prostate cancer patients primarily treated with prostatectomy. In selected patients results may be improved with the additional irradiation of pelvic nodes (WPRT).
Patients and methods. The objective of the study was to evaluate late toxicity of postoperative radiotherapy in 43 patients - 21/43 treated with WPRT. Dysuria, haematuria, nocturia, continence and obstructive urination problems as well as urgency, continence, frequency, pain and bleeding of defecations were prospectively registered and converted to a modified Radiation Therapy Oncology Group (RTOG) - late effects normal tissue (LENT) scoring system. Median tumour dose (TD) for PBRT was 64.8 (59.4-70.0) Gy and for WPRT 50.4 (48.0-56.0) Gy.
Results. More important than the deterioration of intestinal function (worsening for 1 grade in 54% and ^2 grades in 5% of patients) was the deterioration of urinary function (worsening for 1 grade in 33% and ^2 grades in 26% of patients). This appeared to be more frequent in patients with WPRT than PBRT (67% vs. 50% of patients) especially in conjunction with WPRT TD >52 Gy (deterioration in 71% of patients). Conclusions. Although several factors may influence increased urinary toxicity after WPRT, it seems reasonable to lower the urinary bladder dose as it possible with novel radiation techniques.
Key words: prostate cancer; postoperative radiotherapy; whole pelvic radiotherapy; late toxicity
Introduction
Radiotherapy has a well established role in the treatment of patients after radical prostatectomy. Immediately after the operation it can prevent recurrence in patients with high risk features1-3 or reestablish the
Received 15 March 2009 Accepted 31 March 2009
Correspondence to: Borut Kragelj, MD. PhD, Department of Radiotherapy, Institute of Oncology Ljubljana, Zaloška c. 2, SI-1000 Ljubljana, Slovenia; Phone: +386 1 5879 489; Fax: +386 1 5879 400; E-mail: bkragelj@onko-i.si
remission when applied as a salvage treatment for biochemical or local recurrence.4 It can be used as a sole treatment or in combination with hormonal therapy.5
Regardless whether it is used immediately postoperatively, or as a salvage treatment, radiation fields are focused on a believable position of prostate and seminal vesicles as it was before the resection1-3,6,7 and/or areas of the most probable local recurrence.8 It is suggested that with broadening of the treatment fields with the inclusion of pelvic nodes in selected patients considered at high risk of lymph node involvement, the
Kragelj B / Toxicity of postoperative whole pelvic RT
89
improved biochemical relapse-free survival can be acquired.9
The objective of the study was to evaluate late toxicity of this treatment.
Patients and methods
Documents of 49 patients with prostate cancer that received the radiation treatment after prostatectomy between June 2001 and December 2005 at Institute of Oncology Ljubljana were analysed to evaluate the consequences of the radiation treatment. The treatment and the follow-up with the attentive evaluation of side effects were conducted by the author.
Radiation started 1.8-88.5 (median 6.1) months after prostatectomy. Forty-three % (21/49) of patients were treated a few months after prostatectomy because of a high risk of recurrence and with preradia-tion PSA below 0.2 ng/ml; the others (28/49 (57%) patients) were submitted to radiotherapy because of the biochemical or local recurrence.
Treatment fields were limited to pros-tatic bed (PBRT) in 24/49 (49%) of patients - in 25/49 (51%) patients pelvic nodes up to the lumbosacral (S1/L5) interspace were also included (WPRT). The median dose defined by 95% isodose, encompassing target volume applied to the prostatic bed, was 64.8 Gy (59.0-70.0 Gy) and 50.4 Gy (48.056.0 Gy) to the pelvic region in fractions of 1.8-2.2 Gy (median 2.0 Gy). Bioequivalent doses for a fraction of 2 Gy (BED2) were computed using a /p = 3. Median BED2 to the prostatic bed was 62.2 Gy (range 57.373.0 Gy) and to the whole pelvis 50 Gy (dose range 48.0-57.3 Gy). Treatments were delivered on the linear accelerator using four-field technique and standard fractiona-tion. A dose calculation with 2D planning was performed in 20/49 (41%) patients and 3D in 29/49 (59%) patients. The posterior
rectal wall was excluded on lateral fields otherwise no dose constraints were used to limit the dose to organs at risk. Regular portal images were used to limit set up errors.
Long term side effects were assessed in 43/49 (88%) patients still alive and with no sign of recurrence at the time of evaluation.
Side effects rising as a consequence of urinary damage were prospectively registered at each visit with regard to dysuria, haematuria, nocturia, incontinence and obstructive problems. Similarly, the consequences of intestinal toxicity were registered with regard to urgency, continence, frequency of defecations, as well as pain and bleeding problems.
Side effects were converted to late lower gastrointestinal and urinary toxicity scores according to Storey's modification of the Radiation Therapy Oncology Group (RTOG) - late effects normal tissue (LENT) late toxicity scoring system at the time of evaluation.10 Still some additional modification was performed regarding urinary frequency and obstruction primarily aimed to evaluate toxicity already present after prostatectomy. A minor modification was made also with regard to intestinal toxicity with the inclusion of urgency of defecations (Table 1). Erectile problems were not prospectively evaluated and therefore, they were not included and analysed.
Urination characteristics of the initial (before radiotherapy) and the final (at the last follow-up visit) evaluation were compared also in the sense of the possible improvement. On the other hand, worsening of urination characteristics for a grade was considered as a minor deterioration, while worsening of two or more grades was considered as a major deterioration. The same principle was applied also in the evaluation of late intestinal toxicity.
The Kaplan Meier survival method was used to assess the time of the appearance of toxicity. The first appearance of the most
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Kragelj B / Toxicity of postoperative whole pelvic RT
Table 1. Modifications of delayed radiation toxicity grading using Radiation Therapy Oncology Group (RTOG) and Late Effects Normal Tissue Task Force (LENT) criteria with regard to urinary and intestinal toxicity
Criteria
Grade1
Grade 2
Grade 3
Grade 4
Nocturnal
urinary
frequency
Obstructive
urinary
symptoms
Urinary incontinence
Urgency of defecations
2-3
occasional
occasional/drops
present/ without incontinence
3-4
regular
hourly or less
operative treatment / urethrotomy
regular us of up to regular use of 2 1 sanitary pad sanitary pads or more
intermittent use of regular use of sanitary pads	sanitary pads
dysfunction requiring
cystectomy/urinary
diversion/nephrostomy
dysfunction requiring
cystectomy/urinary
diversion/nephrostomy
dysfunction requiring
cystectomy/urinary
diversion/nephrostomy
dysfunction requiring surgery
pronounced change in urination and defecation characteristics in each patient was considered as the observed event. 1-cum-mulative survival curve was used to present the results graphically. Chi square test was used for the estimation of differences in late toxicity between patients with prostate only and additional pelvic radiation as well as between patients with different dose regi-mens.11 P-value <0.05 was considered significant in all statistical tests. The SPSS 15.0 for Windows was used as a tool for the analysis.
Results
Considerable urination difficulties already existed at the start of the radiation treatment. These were mostly related to the increased nocturnal urination frequency and difficulties with continence. Problems with increased nocturnal urinations experienced
21/43 (49%) patients - difficulties were considerable (grade 3) in 5/43 (12%). Even more pronounced were efforts to remain continent. More or less pronounced dripping of urine was evident in 24/43 (56%) patients. Urinary incontinence without the need for sanitary pads was evident in 14/43 (33%) patients, up to one sanitary pad per day was used by the next 6/43 (14%) patients while pronounced incontinence with 2 or more pads per day was evidenced in 4/43 (9%) patients.
Late toxicity was evaluated after a median follow up of 52 months. During follow up visits some of urination difficulties have actually resolved or became less pronounced (Table 2). The improvement in the sense of less frequent nocturnal urinations was noted in 9/21 (43%) patients with this problem after the surgery and the improvement in the sense of less pronounced incontinence was noted in 11/24 (46%) pa-
Table 2. Change in urination characteristics after postoperative radiotherapy comparing	the condition after prostatectomy and at the last follow-up
Change in urination Dysuria	Haematuria	Nocturia	Incontinence	Obstruction characteristics
Improvement 0	0	9	11	0
No change 40	37	18	20	40
Minor deterioration 2	3	12	7	1
Major deterioration 1	3	4	5	2
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tients who had continence difficulties after the surgery.
The comparison of all urination characteristics between the situation at the start of radiation and at the last follow up visit showed that some improvement, or no change, was evident in 18/43 (42%) patients, and minor deterioration was noticed in 14/43 (33%) patients. However, the major deterioration of urination characteristics was evident in 11/43 (26%) patients. These eleven patients included two patients with severe haemorrhagic cystis and 6 patients with G3 urinary toxicity. In both patients with haemorrhagic cystitis cystectomy was obligatory. However, in 2 of 6 patients with G3 toxicity symptoms have resolved after the endoscopic incision for the bladder neck obstruction.
As expected, in comparison to crude incidence rates, results of the survival analysis of toxicity events were even more unpromising showing the deterioration of urinary function in 73% of patients at 5 years (Figure 1).
The comparison of the incidence of deteriorations between patients that received PBRT and WPRT, as well as the comparison of BED2 for WPRT (lower or greater than 52 Gy) and of BED2 for prostate bed (lower or greater than 65 Gy) showed no statistical significance. However, differences were seen in favour of PBRT and also in favour of lower radiation doses to either prostate bed or pelvic nodes (Table 3).
Compared to urinary deterioration, the deterioration of the intestinal function was
1,0-
0,00	20,00	40,00	60,00	80,00	100,00
Time to genitourinary toxicity (months)
Figure 1. Survival without deterioration of urination characteristics,
markedly less pronounced. It appeared as more frequent defecations (in 23/43 (54%) patients), urgency to defecate usually as a consequence to some dietary offence (in 15/43 (35%), occasional bleeding (7/43 (16%) or occasional uncontrolled mucous discharge (in 4/43 (9%) patients). None of the patients experienced G3/G4 toxicity. Altogether defecation characteristics remained unchanged in 13/43 (30%) patients, the minor deterioration as G1 toxicity was evident in 28/43 (65%) and major as G2 toxicity in 2/43 (5%) patients.
Actuarial rates of intestinal radiation late effects were slightly lower than for the urinary tract (63% at 5 years) and with decreasing appearance of new cases after 40 months after the start of radiotherapy.
Table 3. Incidence of deterioration in urinary function regarding treatment fields and radiation doses Characteristics	Incidence of deteriorations	P
Prostate bed RT	11/22 (50%)	0.226
Whole pelvic RT	14/21 (67%)
Whole pelvic dose <52 Gy	8/13 (62%)	0.243
Whole pelvic dose ¿52 Gy	6/8 (75%)
Prostate bed dose <64 Gy 15/29 (52%) 0.259 Prostate bed dose ¿64 Gy_10/14 (71%)_
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No difference in the rate of side effects was found between peIvic and prostatic onIy radiation and aIso not between different dosages
Discussion
Postoperative radiotherapy is generaIIy reported as non toxic with a few severe Iate side effects. With treatment fieIds Iimited to prostatic bed and doses of 60-62 Gy, rates of serious side effects are Iower than 5%,2,12 and seems to remain within this range aIso with the dose escaIation to 70 Gy.13,14
However, low grade toxicity seems to be quite common. In the survey of 75 patients, Pearce reported the incidence of any toxicity registered at any point during the median foIIow up of 45 months, after the irradiation of 60-66 Gy to prostatic bed, to be 51% for intestinaI and 78% for urinary tract.15 Similarly in the report of EORTC triaI 22911 5-year cumuIative incidence rate of compIications (of any grade) based on the competive risk analysis in the radiotherapy arm is about 70%.2 Doses appIied in the trial were 60 Gy while treatment fields were Iimited more or Iess to prostatic areas. InitiaI 50 Gy were appIied to somewhat Iarger area incIuding the region of seminaI vesicIes and Iarger margins around pros-tatic bed. Compared to these reports our resuIts are disappointing. They do not differ so much in overaII toxicity as in the rate of severe compIications. A straightforward expIanation for these events is hard to give. EspeciaIIy if we consider that both patients that required cystectomy were treated with conventionaI doses of 60 and 64 Gy and fractionation of 2 Gy and with treatment fieIds Iimited to the initiaI position of prostate and seminaI vesicIes.
Long term intestinaI toxicity, aIthough common in our study, does not turn out to
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be an important probIem. In aII patients it was of a low grade. In the majority it was manifested with sIightIy increased number of defecations. They also needed some kind of diet, and in the case of an offence, urgency that may in some patients be aggravated to minor continence problems occurred. No difference was noted whether treatment fieIds were Iimited to a prostate region or they encompass also pelvic nodes.
PrevaIent probIems in our patients were reIated to postirradiation damage to urinary bladder. However, our results suggest that considerabIe probIems with urination were aIready present after the prostatectomy. Furthermore, results of already mentioned EORTC triaI suggest that probIems arise with time aIso when prostatectomy was the sole treatment. So, we can not look at the consequences of the postoperative radiation neglecting the impact of the surgical treatment. At least in a part, long term side effects of this combined treatment are due to the prostatectomy. The improvement of urination characteristics in 24% of patients after radiotherapy suggests a proIonged heaIing after the prostatectomy at Ieast in some patients. For these patients often used intervaI of 2-3 months to start adjuvant radiation after the surgery may be too short. According to this, Iower toxicity couId be expected in patients irradiated after an in-tervaI that wouId suggest a compIete heaI-ing after the prostatectomy. However, there was no difference in the rates of major deterioration of urination characteristics between patients irradiated with an intervaI to surgery shorter or longer than 2 years. It appeared in 5/15 (33%) and 6/28 (21%) patients respectively. Nevertheless, an un-compIeted heaIing after the surgery may be an additionaI factor contributing to Iong term toxicity. It seems reasonable to wait with irradiation untiI patients report no further improvement, perhaps jointIy with the introduction of the hormonal treatment.
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Further reasons for urination problems may ground in the properties of the postoperative irradiation. Limited data are available as a predictor of late urinary toxicity in the postoperative radiotherapy.12 Less obscured is a situation with the radical radiotherapy treatment of prostate cancer and some of these data may be valid also in the context of postoperative radiotherapy. Pinkawa stated in a study of 80 patients with the use of Expanded Prostate Cancer Index Composite questionnaire, that the patients' ability to fill the bladder has a major impact on the dose-volume histogram parameters and on both, acute and late urinary toxicity.16 Similar was the conclusion of Harsolia stating the importance of urinary bladder dose-volume parameters on 331 patients using National Cancer Institute Common Toxicity Criteria 2.0 for the evaluation of chronic urinary toxiciy.17 A further similarity of both studies is a finding of the importance of the exposure of urinary bladder to relatively low doses of radiation in the range of 30-40 Gy. In our study, due to the inclusion of pelvic nodes and the use of box technique, large parts of bladder were exposed to irradiation. Even when treatment fields were restricted to prostatic bed regularly about 80% of the bladder volume received dose equal or greater than 60 Gy (dose that is similar to target doses). The interdependence of late urinary toxicity and volume and dose of irradiation is also suggested by our results. The increase in urinary problems, although not statistically significant, was seen in patients with larger fields by the comparison of pelvic and prostate only irradiation. The increase in toxicity was also evident with larger doses - greater than 52 Gy to pelvic field and 64 Gy to prostatic bed. To reduce the radiation damage to urinary bladder it seems important not only to limit the target dose but also, and for most, to exclude as much as possible of the bladder out of the irradiated
volume and to limit the dose to the rest of the bladder wall. New radiation techniques such as intensity modulated radiotherapy (IMRT) can produce concave dose distribution and, as shown by planning studies, can reduce the volume of the bladder exposed to high doses for 20-50%.18,19 Less effective is IMRT in the low dose range. How effective will be this and other techniques that make it possible to reduce the dose to the urinary bladder, in the reduction of late urination toxicity with pelvic irradiation is still unclear.
Perhaps the easiest and most reliable way to solve the marked urinary toxicity is to reduce the target volume. EORTC giudelines8 for the target volume definition in postoperative radiotherapy for prostate cancer, with the limitation of target volume to the sites of most probable local recurrence, enable a considerable reduction in dose-volume parameters of the urinary bladder. However, certain assurance is needed to exclude pelvic nodes. Reliable information is offered by extended, and perhaps also, but with less trustworthiness, classic lymphadenec-tomy.20 In our study lymphadenctomy was performed in 77% of patients with a median 6 resected nodes. If, and how much, the omitment of pelvic radiotherapy in our patients would compromise the results of the treatment, could be anticipated from the initial results that suggest the significantly improved progression free survival (86% and 71% at 5 years) in patients treated with whole pelvic radiotherapy (WPRT) for the relapsed or progressive disease after the prostatectomy. They support the effectiveness and superiority of WPRT to prostatic bed radiation for patients with the risk of nodal invasion greater than 15% according to Roach equation21 and with no reliable information on the lymph node status through lymphadenectomy. More secure is to make some compromise in the target volume. One possibility is the less strict
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inclusion of the lymphatic region around a distal part of external iliacal vessels,22 and the other is to adapt target volume to the extensiveness of lymphadenectomy with the exclusions of regions that were already submitted to lymphadenectomy.
The hormonal therapy can be a component of postoperative treatment.23 In the majority of our patients, goserelin 10.8 mg every 3 months was used for one year. The treatment started at least one month before the commencement of radiotherapy, so castrate testosterone levels were achieved at the time of radiotherapy. Considering the study of Taussky in which a relationship between chronic toxicity and testosterone level at the time of radiotherapy was stated, hormonal therapy should also be considered as one of the possible factors that influence our results.24 What will be the risks of the exclusion of the immediate hormonal therapy will be eventually demonstrated by RTOG 96-01 trial results.
Nevertheless, some caution is needed with the interpretation of the results on late urinary toxicity. The fibrosis of the bladder wall and the loss of muscle function as a result of late radiation damage, were in the majority of our patients demonstrated with continence problems and increased urination frequency with the need to wake up at night. Common criteria used to evaluate and grade this problem, are toxic criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer.25 However, in RTOG scales these problems are almost completely ignored, as is the case with incontinence, or inexact (and subjective) for correct grading of the problem, such is the case with the increased urination frequency. Modifications of this scoring system as proposed by Storey10 also leave some uncertainties in the evaluation of mild, initial continence problems not necessitating the use of pads, and also severe
problems with the use of multiple pads per day. It also remains too broad to register changes in the urination frequency - especially in connection with the postoperative radiation of prostatic carcinoma with distinctive problems present already after the prostatectomy. Not so rare scenario of 2-3 wakening per night after the prostatectomy with the increase to 4-5 wakening can be scored as no toxicity (not twice baseline), G1 toxicity (twice baseline) and also as G2 or G3 (moderate or severe) toxicity depending on the impression of the inquirer. One can speculate that exaggerated urinary tox-icity in our study can be the consequence of more rigorous grading - even with the regard to, generally more exact, grading of serious toxicity.
As a conclusion we can say that pelvic radiotherapy in the postoperative treatment of prostatic carcinoma may result in the increased and perhaps unacceptable urinary morbidity at least in connection with a classic box technique of irradiation and concurrent testosterone deprivation. Like in other irradiated areas, toxicity is strongly correlated with the irradiated volume in conjunction with the dose.26 The quality of life became more and more important in the case of a good prognosis of patients' outcomes regarding a disease control and survival, therefore, we have to carefully look for late toxicity assessing restrictions in the combined modality treatment.27 However, there are several possibilities to make pelvic radiotherapy after the radical prostatectomy more acceptable. Nevertheless, the individual response to radiotherapy is important, even with regard to high grade morbidity.
Acknowledgement
The author thanks Dr. Lijana Zaletel-Kragelj from Department of Public Health, Ljubljana University Faculty of Medicine,
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Slovenia, for her help in preparation of this manuscript.
References
1.	Thompson IM, Tangen CM, Paradelo J, Lucia S, Miller G, Troyer D, et al. Adjuvant radiotherapy for pathologically advanced prostate cancer. JAMA 2006; 296: 2329-35.
2.	Bolla M, van Poppel H, Colette L, van Cangh P, Vekemans K, Da Pozzo L, et al. Postoperative radiotherapy after radical prostatectomy: a randomised controlled trial (EORTC trial 22911). Lancet 2005; 366: 52-8.
3.	Wiegel T, Bottke D, Willich H, Piechota H, Souchon R, Stoeckle M, et al. Phase III results of adjuvant radiotherapy (RT) versus »wait and see« (WS) in patients with pT3 prostate cancer following radical prostatectomy (RP)(ARO 96-02/AU0 AP 09/95). [Abstract]. J Clin Oncol 2005; 23(Part 1 Suppl): 381S.
4.	Stephenson AJ, Scardino PT, Kattan MW, Pisansky TM, Slawin KM, Klein EA, et al. Predicting the outcomes of salvage radiation therapy for recurrent prostate cancer after radical prostatectomy. J Clin Oncol 2007; 25: 2035-41.
5.	Jani AB, Sokoloff M, Shalhav A, Stadler W. Androgen ablation adjuvant to postprostatecto-my radiotherapy: complication-adjusted number needed to treat analysis. Urology 2004; 64: 97681.
6.	Zelefsky MJ, Aschkenasy E, Kelesen S, Leibel SA. Tolerance and early outcome results of post-prostatectomy three-dimensional conformal radiotherapy. Int J Radiat Biol Phys 1997; 39: 327-33.
7.	Cox JD, Gallagher MJ, Hammond EH, Kaplan RS, Schellhammer PF. Consensus statement on radiation therapy of prostate cancer: guidlines for prostate re-biopsy after radiation and for radiation therapy with rising prostate specific antigen levels after radical prostatectomy. American Society for Terapeutic Radiology and Oncology consensus panel. J Clin Oncol 1999; 17: 1155-63.
8.	Poortmans P, Bossi A, Vandeputte K, Bosset M, Miralbell R, Maingon P, et al. Guidlines for target volume definition in post-operative radiotherapy for prostate cancer, on behalf of the EORTC Radiation Oncology Group. Radiother Oncol 2007; 84: 121-7.
9.	Spiotto MT, Hancock SL, King CR. Radiotherapy after prostatectomy: improved biochemical re-laps-free survival with whole pelvic compared with prostate bed only for high-risk patients. Int J Radiat Biol Phys 2007; 69: 54-61.
10.	Storey MR, Pollack A, Zagars G, Smith L, Antolak J, Rosen I. Complications from radiotherapy dose escalation in prostate cancer: preliminary results of a randomized trial. Int J Radiat Biol Phys 2000; 48: 635-42.
11.	Parmar MKB, Machin D. Survival analysis. Chichester: John Wiley&Sons; 1995.
12.	Feng M, Hanlon AL, Pisansky TM, Kuban D, Catton C, Michalski J, et al. Predictive factors for late genitourinary and gastrointestinal toxic-ity in patients with prostate cancer treated with adjuvant or salvage radiotherapy. Int J Radiat Biol Phys 2007; 68: 1417-23.
13.	Valicenti RK, Gomella LG, Ismail M, Mulholland SG, Petersen RO, Corn BW. Effects of higher radiation dose on biochemical control after radical prostatectomy for pT3N0 prostate cancer. Int J Radiat Biol Phys 1998; 42: 501-6.
14.	Jereczek-Fossa BA, Zerini D, Vavassori A. Sooner or later? Outcome analysis of 431 prostate cancer patients treated with postoperative or salvage radiotherapy. Int J Radiat Oncol Biol Phys 2008 74: 115-25.
15.	Pearse M, Choo R, Danjoux C, Gardner S, Morton G, Szumacher E, et al. Prospective assessment of gastrointestinal and genitourinary toxicity of salvage radiotherapy for patients with prostate-specific antigen relaps or local recurrence after radical prostatectomy. Int J Radiat Biol Phys 2008; 72: 792-8.
16.	Pinkawa M, Fischedick K, Asadpour B, Gagel B, Piroth MD, Eble MJ. Low-grade toxicity after conformal radiation therapy for prostate cancer - impact of bladder volume. Int J Radiat Biol Phys 2006; 64: 835-41.
17.	Harsolia A, Vargas C, Yan D, Brabbins D, Lockman D, Liang J, et al. Predictors for chronic urinary toxicity after the treatment of prostate cancer with adaptive three-dimensional con-formal radiotherapy: dose-volume analysis of a phase II dose-escalation study. Int J Radiat Biol Phys 2007; 69: 1100-9.
18.	Portelance L, Chao KSC, Grigsby PW, Benet H, Low D. Intensity-modulated radiation therapy (IMRT) reduces small bowel, rectum and bladder dose in patients with cervical cancer receiving pelvic and para-aortic radiation. Int J Radiat Biol Phys 2001; 51: 261-6.
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19.	Roeske JC, Lujan A, Rotmensch J, Waggoner SE, Yamada D, Mundt AJ. Intensity-modulated whole pelvic radiation therapy in patients with gynecologic malignancies. Int J Radiat Biol Phys 2000; 48: 1613-21.
20.	Heidenreich A, Ohlmann CH, Polyakov S. Anatomical extent of pelvic lymphadenectomy in bladder and prostate cancer. Eur Urol Suppl 2005; 4: 15-24.
21.	Roach M 3rd, Marquez C, Yuo HS. Predicting the risk of lymph node involvement using the pre-treatment prostatic specific antigen and Gleason score in men with clinically localized prostate cancer. Int J Radiat Biol Phys 1994; 28: 33-7.
22.	Shih HA, Harisinghani M, Zietman AL, Wolfgang JA, Saksena M, Weissleder R. Mapping of nodal disease in locally advanced prostate cancer: rethinking the clinical target volume for pelvic nodal irradiation based on vascular rather than bony anathomy. Int J Radiat Biol Phys 2005; 63: 1262-9.
23.	Corn BW, Winter K, Pilepich MV. Does androgen suppression enhance the efficacy of postoperative irradiation? A secondary analysis of RTOG 85-13. Urology 1999; 54: 495-502.
24.	Taussky D, Bae K, Bahary JP. Does timing of androgen deprivation influence radiation-induced toxicity? A secondary analysis of radiation therapy oncology group protocol 9413. Urology 2008; 72: 1125-9.
25.	Cox JD, Stetz JA, Pajak TF. Toxicity criteria of the radiation therapy oncology group (RTOG) and the European organization for research and treatment of cancer (EORTC). Int J Radiat Biol Phys 1995; 31: 1341-6.
26.	Botros M, Quevedo JF, Miller RC. Angiosarcoma of the liver after multimodality therapy for gallbladder carcinoma. Radiol Oncol 2009; 43: 126-31.
27.	Velenik V, Oblak I, Anderluh F. Quality of life in patients after combined modality treatment of rectal cancer: Report of a prospective phase II study. Radiol Oncol 2008: 42: 207-14.
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Radiol Oncol 2009; 43(2): 97-119.
doi:10.2478/v10019-009-0017-9
research article
Usage of the standard and modified comet assay in assessment of DNA damage in human lymphocytes after exposure to ionizing radiation
Morana Miklos, Goran Gajski, Vera Garaj - Vrhovac
Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia
Background. Human organisms are extremely sensitive to ionizing radiation, which has the strong genoto-xic effect on the DNA molecule. The aim of the present study was to detect the type of DNA damage and cell death caused by ionizing radiation as well as the sensitivity of the standard and modified comet assay. Methods. The effect of gamma radiation (0.1 Gy and 4 Gy) on human lymphocytes was observed using the standard alkaline and Fpg-modified comet assay with ability to detect oxidized purines as well as with the DNA diffusion test.
Results. Parameters of the standard comet assay showed significantly higher values in samples exposed to 4 Gy than in samples exposed to radiation dose of 0.1 Gy and control sample. The Fpg-modified comet assay showed significantly higher values already at dose of 0.1 Gy as the result of oxidative DNA damage. The DNA diffusion test showed that gamma rays lead to apoptosis more often than to necrosis. Conclusions. This observation suggests that the standard alkaline and Fpg-modified comet assays as well as the DNA diffusion test are reliable techniques for estimation of DNA damage and form of the cell death caused by gamma radiation in vitro. In addition Fpg-modified comet assay prove to be more sensitive for detection of gamma irradiation induced DNA damage than the standard assay.
Key words: human peripheral blood lymphocytes; ROS; gamma radiation; alkaline comet assay; Fpg-modified comet assay; diffusion test
Introduction
Ionizing radiation is nowadays omnipresent in human lives because of the increasing development of technology, industry
Received 21 January 2009 Accepted 19 March 2009
Correspondence to: dr. Vera Garaj-Vrhovac, Institute for Medical Research and Occupational Health, Mutagenesis Unit, Ksaverska cesta 2, 10000 Zagreb, Croatia. Tel. +385 1 4673 188; Fax. +385 1 4673 303; E-mail: vgaraj@imi.hr
and medicine. People live and work near nuclear facilities and places of testing nuclear weapon and also use nuclear (gamma) radiation in medical purposes. In each case, human organism bears the strong genotoxic effect making the structure of the DNA molecule unstable and causing the genesis of many changes in it.1 The damage of the genetic material caused by ionizing radiation is one of the best precondition indicator for development of malign diseases such as breast, gall-bladder or thyroid can-
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cer.2,3 Gamma radiation affects the DNA structure directly, causing strand breaks, or indirectly causing cleavage of the water molecules and damaging the DNA molecule by reactive oxygen species (ROS).4 Strand breaks and oxidative damage in the DNA causes further cell death in the form of apoptosis or necrosis.5 These changes can be examined at human lymphocytes using different cytogenetic techniques. The most frequently used cytogenetic techniques are the comet assay, micronucleus test, chromatid exchange test, chromosomal aberrations test and fluorescence in situ hybridization (FISH) test.6-10
The comet assay (SCGE; single cell gel electrophoresis) is a rapid, simple, visual and sensitive technique for measuring and analyzing DNA damage at the single cell level.6,11-17 This technique can be performed at the level of individual cells and requires a small number of cells per sample. Single cells can be used in in vivo and in vitro as well as in biomonitoring of population exposed to radiation or chemical muta-gens.6,18,19 The comet assay detects single and double stranded breaks at the level of DNA molecule, sites of incomplete repair, alkali labile sites, DNA-DNA and DNAprotein cross-links. Besides, the comet assay can be used for detection of the level of the DNA fragmentation in apoptosis.17, 20-22 In addition; particular enzymes such as for-mamidopyrimidine glycosilase can be used for detection of oxidative damage at the level of the DNA molecule caused by ROS.23-25 The DNA diffusion assay, a simple, sensitive, and reliable cytogenetic method is often used for quantification of apoptosis, is based on the principle that nuclear DNA of apoptotic cells have abundant alkalilabile sites and under alkaline conditions small pieces of DNA thus generates diffuse in agarose, giving the appearance of a halo if stained with a sensitive fluorescent dye. Apoptotic cells show a circular gradient of
granular DNA with a dense central zone and a lighter and hazy outer zone, giving the overall appearance of a halo.26
Peripheral blood lymphocytes were exposed to gamma radiation doses of 0.1 Gy and 4 Gy in vitro. In that manner, the aim of this study was to detect the type of DNA damage caused by ionizing radiation. Considering that, two forms of the comet assay, standard alkaline and Fpg-modified were used. In addition, sensitivity of both techniques toward different doses of gamma radiation was measured. The DNA diffusion test was also used to detect the form of the cell death caused by gamma radiation.
Materials and methods
Blood sampling
The study was performed on peripheral blood samples obtained from a healthy female non-smoking donor (age 24 years). The donor was not exposed to ionizing radiation, vaccinated or used medicals for a year before blood sampling. Whole venous blood was collected under sterile conditions in heparinised vacutainer tubes (Becton Dickinson, NJ, USA) containing lithium heparin as anticoagulant. After collection, blood was divided into a large number of samples. All experiments were conducted on peripheral blood lymphocytes cultivated at 37°C in an atmosphere of 5% CO2 in air.
Exposure conditions
The whole blood samples were irradiated with gamma radiation on the ice. As a source of radiation Gammacell 220 (Institute "Ruder Boskovic", Zagreb, Croatia) was used. Vacutainers (volume 5 cm3) containing blood samples were exposed to radiation doses defined as doses in the water, but irradiation was performed
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in the air. Samples were irradiated with radiation doses of 0.1 Gy and 4 Gy that is equal to radiation periods of 32.3 s and 23 min and 9 s at temperature of 21 °C. Significance of the absorbed dose was 3%.27 To get homogenate samples, they were stirred after irradiation, cooled to 4°C, transported to the laboratory on ice and processed as quickly as possible.
Determination of cell viability
The indices of cell viability and necrosis were obtained from differential staining with acridine orange and ethidium bromide, using fluorescence microscopy.28 Lymphocytes were isolated using a modification of the Ficoll-Histopaque centrifuga-tion method.29 The slides were prepared using 200 |l of human peripheral blood lymphocytes and 2 |l of stain (acridine orange and ethidium bromide, both diluted in PBS). A total of 100 cells were analyzed to determine the percentage of viable cells using an Olympus AX-70 microscope with 60x magnification and 515-560 nm fluorescence filters. The cells were classified according the following description: live cells with a functional membrane, with uniform green staining of the nucleus and necrotic cells with uniform red staining of the nucleus.
The alkaline comet assay
To evaluate DNA damage after irradiation and to test sensitivity of the technique towards gamma radiation the comet assay was carried out under alkaline conditions, basically as described by Singh et al.14 Fully frosted slides were covered with agarose (Sigma) containing the whole blood sample. The slides were then immersed for 1 h in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% sodium sarcosinate (Sigma), pH 10) with 1% Triton X-100 (Sigma) and
10% dimethyl sulfoxide (Kemika, Zagreb, Croatia). The slides were then placed on a horizontal gel electrophoresis tank. The unit was filled with electrophoresis buffer (0.3 M NaOH, 1 mM Na2EDTA; pH 13) and the slides were placed in this alkaline buffer for 20 min. Denaturation and electrophoresis were performed at 4°C under dim light. Electrophoresis was carried out for 20 min at 25 V (300 mA). After electrophoresis the slides were rinsed with neutralization buffer (0.4 M Tris-HCl, pH 7.5). Each slide was stained with ethidium bromide (20 |g/ ml) and covered with a coverslip. The slides were then stored in sealed boxes at 4°C until analysis.
Fpg-modified comet assay
For evaluation of possible oxidative DNA-damaging effect of gamma radiation and to test the sensitivity of the technique, modified version of the comet assay was performed using an Fpg FLARE™ assay kit (Trevigen Inc, Gaithersburg, USA) with some modification.30 Within the kit the manufacturer provided all the reagents used. Fully-frosted microscopic slides were prepared. Each slide was covered with 1% normal melting point (NMP) agarose (Sigma). After solidification, the gel was scraped off the slide. The slides were then coated with 0.6% NMP agarose. A low melting point (LMP) agarose was melted and stabilized in a water bath at 37°C. For each sample and control, 5 |l of cell homogenate was mixed with 100 |l of LMP agarose and placed on the slides. After 10 min of solidification on ice, the slides were covered with 0.5% LMP agarose. The slides were then immersed in a pre-chilled lysis solution and kept in a refrigerator at 2°C for 60 min. Followed the immersion in the buffer, three times for 15 min. After lysis, the slides were treated with 100 |l of Fpg enzyme (1:500 in REC dilution buffer). The enzyme was di-
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luted right before use. Control slides were treated with 100 |l of REC dilution buffer only. The slides were placed horizontally in a humidity chamber at 37 °C for 30 min. All slides were then immersed in an alkali solution (0.3 M NaOH, 1 mM Na2EDTA; pH 12.1) for 40 min. Followed electro-phoresis in a pre-chilled alkali solution (0.3 M NaOH, 1 mM Na2EDTA; pH 12.1) at 1 V/cm for 20 min. After electrophoresis, the slides were rinsed gently three times with neutralization buffer (0.4 M Tris-HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 |g/ml) and covered with a coverslip. Slides were stored at 4°C in sealed boxes until analysis.
Comet capture and analysis
A total of one hundred randomly captured comets from each slide were examined at 250x magnification using an epifluores-cence microscope (Zeiss, Germany) connected through a black and white camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd., UK). The analysis did not include the edges and damaged parts of the gel as well as debris, superimposed comets, and comets without distinct head ("clouds", "hedgehogs", or "ghost cells").
Differences in the tail length, tail intensity and tail moment between samples obtained with standard alkaline comet assay (basic DNA damage) and Fpg-modified comet assay (total DNA damage) were considered as oxidative DNA damage in a single cell.
DNA diffusion test
For evaluation of the type of the cell death DNA diffusion assay was performed following the protocol described by Singh.12,31 The chemicals needed for the DNA dif-
fusion assay were provided by the Sigma Chemical Company. Agarose precoated slides were made by spreading 50 ml of 0.7% normal melting agarose on each slide and drying them at room temperature. Microgels were made on agarose-precoated slides by mixing 5 |l of whole blood culture with 50 |l of 0.7% high-resolution agarose and pipetting it onto the slide. The gel was immediately covered with a cover glass. The slides were coded and cooled on ice for 1 min. The cover glasses were removed, and 200 ml of 2% agarose solution was layered. After keeping the slides for 1 min on ice, the cover glasses were removed and the slides were immersed in a freshly made lysing solution (1.25 M NaCl, 1 mM tetra sodium EDTA, 5 mM Tris-HCl pH 10, 0.01% sodium lauroyl sarcosine, 0.2% DMSO freshly added, 0.3 M NaOH freshly added) for 10 min at room temperature. After lysis, the slides were twice immersed in a neutralising solution (50% ethanol, 1 mg/ml spermine, 20 mM Tris-HCl pH7.4) for 30 min at room temperature. The slides were air-dried and stored at room temperature. The slides were stained with ethidium bromide (20 |g/ml) and covered with a coverslip for 10 min. 1000 lymphocytes per slide were analysed. Lymphocytes undergoing apoptosis or necrosis were distinguished from normal cells in accordance with the figures and instructions given by Singh.31 Apoptotic cell nuclei have a hazy or undefined outline without any clear boundary due to nucleosomal-sized DNA diffusing into the agarose. Necrotic cell nuclei are bigger and are poorly defined. They have a clear, defined outer boundary of the DNA halo and a relatively homogeneous halo appearance.
Statistical analysis
Each experimental set contained duplicated slides. The various parameters measured in
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the exposed and control groups were evaluated using Statistica 5.0 package (StaSoft, Tulsa, USA). Each sample was characterized for the extent of DNA damage by considering the mean ± SE (standard error of the mean), median and range of the comet parameters. Multiple comparisons between groups were done by means of ANOVA on log-transformed data. Post-hoc analysis of differences was done by Scheffe test. As for the DNA diffusion test, values were analyzed using the chi-square test. The level of statistical significance was set at P<0.05.
Results
Cell viability test
The viability of the cells as determined by acridine orange and ethidium bromide, using fluorescence microscopy, was consistently above 89% in all the exposed samples and 100% in control samples. This is considered to be in acceptable range for conducting the comet assay.32,33
Comet assay and Fpg-modified comet assay
Results of the standard alkaline comet assay are presented in the Table 1. These results showed statistically significant increase of the mean values for all three parameters of the standard comet assay (P<0.05) in the sample irradiated with the dose of 4 Gy in contrast to control sample and sample irradiated with the dose of 0.1 Gy. Sample irradiated with the dose of 0.1 Gy showed slightly increased values for all parameters measured but there were no significant differences in compare to the corresponding control (Figure 1).
Results of the Fpg-modified comet assay are presented in the Table 2. These results showed statistically significant increase in
all three parameters of modified comet assay (P<0.05) for both irradiation doses in compare to the control sample (Figure 2).
Generally, the mean values of all three Fpg-modified comet assay parameters were significantly higher than of the standard comet assay. These findings suggest that the Fpg-modified comet assay is more sensitive to gamma irradiation than the standard comet assay.
DNA diffusion test
Percentage of apoptotic and necrotic cells gained for the diffusion test is presented in Table 3 whereas Figure 3 presents mi-crophotographs of viable, apoptotic, and necrotic cells from the un-exposed sample and samples irradiated with gamma radiation. These results showed statistically significant increase (P<0.05) of apoptotic cells in samples irradiated with the dose of 0.1 Gy and 4 Gy in comparison to the control sample. Significant increase in apoptotic cells was also found in sample irradiated with 4 Gy in comparison to the sample irradiated with 0.1 Gy. Number of necrotic cells significantly increased only in sample irradiated with 4 Gy in comparison to the control sample, while statistically significant increase in comparison to the sample irradiated with the dose of 0.1 Gy was not present.
Discussion
Our goal was to test the sensitivity of different protocols for comet assay, and to evaluate the type of cell death caused by different doses of gamma radiation. This type of radiation is a potent carcinogen mainly due to it potential as oxidative-induced damage agent. It produces variety of primary lesions in DNA such as single and double strand breaks, DNA-DNA
Radiol Oncol 2009; 43(2): 97-107.
Table 1. Results of the alkaline comet assay and DNA diffusion test in human peripheral blood lymphocytes after exposure to 0.1 Gy and 4 Gy gamma irradiation
						Alkaline	comet	assay				
		Tail				Tail				Tail		
Sample		length ()jm)				intensity				moment		
	Min	Max	Mean±SE	Median	Min	Max	Mean±SE	Median	Min	Max	Mean±SE	Median
4 Gy	25.00	92.31	55.7811.90*-'	54.49	5.74	49.21	22.5111.02*-'	20.53	1.09	11.99	5.0610.27*-'	4.52
0.1 Gy	13.46	25.00	17.09±0.31	16.03	0.00	19.21	3.14±0.38	1.91	0.00	2.46	0.43±0.05	0.25
Control	12.18	21.79	16.19±0.27	15.70	0.00	9.74	2.64±0.26	1.57	0.00	1.28	0.36±0.03	0.21
* statistically significant increase in compare to the corresponding control (P<0.05) t statistically significant increase in compare to the 0.1 Gy irradiated sample(P<0.05)
Table 2. Results of the Fpg-modified comet assay in human peripheral blood lymphocytes after exposure to 0.1 Gy and 4 Gy gamma irradiation
Fpg-comet assay
Sample		Tail length (l-im)				Tail intensity				Tail moment		
	Min	Max	Mean±SE	Median	Min	Max	Mean±SE	Median	Min	Max	Mean±SE	Median
4 Gy	31.41	90.38	60.87±1.47*-'-+	60.90	8.76	66.86	32.14±1.38*-'-+	29.92	1.57	22.29	8.15±0.48*-'-+	7.15
0.1 Gy	14.74	28.20	18.61±0.32*-+	17.95	0.00	17.98	4.81±0.40*-+	4.28	0.00	2.70	0.70±0.06*-+	0.57
Control	12.82	20.51	16.18±0.18	16.03	0.00	8.29	2.42±0.21	2.13	0.00	1.12	0.33±0.03	0.29
* statistically significant increase in compare to the corresponding control (P<0.05) t statistically significant increase in compare to the 0.1 Gy irradiated sample(P<0.05) + statistically significant increase in compare to the standard comet assay (P<0.05)
Miklos M. et al. / Comet assay in assessment of DNA damage
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Figure 1. Parameters of the alkaline comet assay (tail length, tail intensity and tail moment), for human peripheral blood lymphocytes exposed to 0.1 Gy and 4 Gy gamma irradiation.
Figure 2. Parameters of the Fpg-modified comet assay (tail length, tail intensity and tail moment), for human peripheral blood lymphocytes exposed to 0.1 Gy and 4 Gy gamma irradiation.


70



50
50

.ÏÏ 40
30



10
D E:
D E

Contro]
Control









D E
Control
Control





12

E
E
Control
0.1 Gy
4 Gy
Table 3. Results of the DNA diffusion test in human peripheral blood lymphocytes after exposure to 0.1 Gy and 4 Gy gamma irradiation.
DNA diffusion test
Sample	Apoptosis	Necrosis
0/ 0/ % %		
4 Gy	4.90*,#	4.10*
0.1 Gy	3.00*	2.50
Control	1.10	1.40
*	statistically significant increase in compare to the corresponding control (P<0.05)
#	statistically significant increase in compare to the 0.1 Gy irradiated sample(P<0.05)
and DNA-protein cross-links, alkali-labile sites and damage to purine and pyrimidine bases as well as oxidize bases and abasic sites.34'35'36'37
In that manner, standard alkaline and Fpg-modified version of the comet assay were used as well as the DNA diffusion test to access whether this type of radiation induces apoptotic or necrotic cell death. Techniques used in this study showed genotoxic effect of gamma rays on DNA molecule of peripheral blood human lym-
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Miklos M. et al. / Comet assay in assessment of DNA damage	104
M
Figure 3. DNA diffusion microphotographs of viable lymphocytes from the un-exposed sample (A), and apoptotic (B) and necrotic (C) cells from samples irradiated with gamma radiation.
phocytes in vitro. With the standard alkaline comet assay increase in DNA damage was noticed in both exposure doses but it was significant only at higher dose of 4 Gy whereas at lower dose there were no statistically significant increase in neither of the standard comet assay parameters.
Usage of the Fpg-modified protocol showed significant increase in all the parameters measured at both exposure doses indicating that the modified version of this assay is capable to detect wider scale of DNA damage induced by gamma irradiation. In addition, with modified protocol it is possible to detect ROS mediated DNA damage, thus significant increase in modified comet parameters in comparison to the standard one suggests that gamma radiation did induce oxidative damage in DNA molecule in vitro.
Some of the previous studies also showed that the Fpg-modified version of the comet assay is more sensitive for detection of DNA damage than the standard alkaline one.18,22, 24, 38-41 According to that fact, scientists revealed that using the standard alkaline comet assay it is possible to detect damages at radiation doses from 5 cGy to 10 cGy 42,43 and in some adapted experimental conditions (e.g. addition of the Fpg enzyme) it is possible to detect damages even at radiation doses of 0.6 cGy.44,45 The additional reason why the Fpg-modified comet assay is more sensitive than the standard alkaline one is that the Fpg enzyme helps to detect oxidative damage of the DNA molecule by cleavage of 8-oxodG, FaPyGua, FaPyAde and other ring-opened purines.46,47,48
In one of our previous research done on atorvastatin toxicity towards peripheral blood lymphocytes modified comet protocol also showed greater sensitivity in the manner of DNA damage. In that study all parameters obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes, but the Fpg-modified protocol showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay.18
Another comparison of the standard alkaline and Fpg-modified comet assay was made on leukocytes collected from Wistar
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rats after exposure to 915 MHz microwave radiation. Both the standard and the Fpg-modified comet assay detected increased DNA damage in blood leukocytes of the exposed rats. The significant increase in Fpg-detected DNA damage in the exposed rats suggests that oxidative stress is likely to be responsible and that Fpg-modified protocol is much more sensitive than the standard one. These results are in compliance with our results because of the higher values obtained with Fpg-modified comet assay than with standard alkaline indicating oxidative DNA damage.39Strand breaks and oxidative damage at the level of DNA molecule have further consequences for the cells. Depending on the kind and the level of the damage, these effects often lead to the cell death which appears in the form of apoptosis or necrosis.49 In our study this was evaluated by using the DNA diffusion test. Observing results, it was noticed that higher radiation dose increased the number of apoptotic cells in compare to the number of necrotic cells. That was because; gamma radiation damages the DNA molecule by producing reactive oxygen species more often than hitting the DNA molecule directly and causing strand breaks. These reactive oxygen species initiate cascade reactions in the cells witch leads to apoptosis, what makes apoptosis more frequent form of cell death than necrosis.50
Conclusion
In this study correlation between different protocols of the comet assay was made suggesting that Fpg-modified version is more sensitive to gamma radiation by virtue of measuring oxidative DNA damage in addition to basal DNA strand breaks. Moreover, evaluation of the type of cell death was made using DNA diffusion test indicating that gamma rays more often
leads to apoptosis. Results obtained lead to the same conclusion that gamma radiation affects the DNA molecule by ROS that are most frequent product of the gamma radiation effect. Human peripheral blood lymphocytes proved to be sensitive to ionizing radiation depending on the radiation dose and are suitable biomarkers for this type of research.
Acknowledgements
This investigation was supported by the Croatian Ministry of Science, Education and Sports (grant No. 0022-0222148-2125).
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Radiol Oncol 2009; 43(2): 108-119.
doi:10.2478/v10019-009-0017-9
research article
Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization
Marko Ušaj, Katja Trontelj, Rosana Hudej, Maša Kandušer, Damijan Miklavčič
University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia
Background. Various electrofusion parameters have to be adjusted to obtain the optimal electrofusion efficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treatment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and viability of four different cell lines in hypotonic buffer and to study the influence of the electroporation on the selected cell line in hypotonic buffer.
Materials and methods. Cell size dynamics of different cell lines exposed to hypotonic buffer and electroporation were analyzed by time-resolved cell size measurements. The viability of hypotonically treated or/and electroporated cells was determined 24 h after the experiment by a modified crystal violet (CV) viability assay.
Results. In our experimental conditions the hypotonic treatment at 100 mOsm was efficient for CHO, V79 and B16-F1 cell lines. The optimal duration of the treatment was between two and five minutes. On the other hand the same hypotonic treatment did not cause cell swelling of NS1 cells. Cell swelling was also observed after electroporation of B16-F1 in isotonic buffer and it was amplified when hypotonic buffer was used. In addition, the regulatory volume decrease was successfully inhibited with electroporation. Conclusions. Cell size dynamics in hypotonic conditions should be studied for each cell line since they differ in their sensitivity to the hypotonic treatment. The inhibition of cell regulatory volume decrease by electroporation may be beneficial in achieving higher electrofusion efficiency. The hypotonic treatment in itself did not significantly affect the cell viability; however, electric field parameters for electroporation should be carefully selected taking into account the hypotonically induced volume increase of cells.
Key words: hypotonic treatment; cell swelling; regulatory volume decrease; cell size measurements; viability; electrofusion; electroporation
Received 10 March 2009 Accepted 25 March 2009
Correspondence to: Prof. Dr. Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering, Tržaška 25, SI-1000 Ljubljana, Slovenia; Phone: +386 1 4768 456; Fax: +386 1 4264 658; E-mail: damijan.miklavcic@fe.uni-lj.si
Introduction
Electrofusion of two different types of cells generates a third polynuclear type, which displays hybrid characteristics of the two parental cells. Different methods
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are used to achieve cell fusion; however, electrofusion is gaining on its importance because it is easy to use, potentially highly efficient, reproducible and controllable.1-3 Electrofusion has great potential for clinical applications, with respect to viral and chemical methods, because it is a safe method that does not introduce any foreign substances into the body.
Cell fusion is a two-condition process: (I) cell membrane has to be brought into fu-sogenic state and (II) a close physical contact of two fusogenic membranes has to be established. The fusogenic state of cell membrane is achieved by electroporation.4-6 Electroporation is a method, widely used in medicine7-12, where a dramatic increase in membrane permeability is caused by cell exposure to short and intense electric pulses.13-15 With the appropriate selection of electrical parameters and media, taking into account biological characteristics of the treated cells, the reversible electroporation can be obtained. The reversible electropora-tion does not affect cell viability, because cell membranes reseal after the treatment.16-21
As mentioned before, the second condition required for cell fusion is a close physical contact between cells which has to be established during the fusogenic state of the membrane. A physical contact between cells can be achieved by the application of alternating electric filed which causes di-electrophoretic forces that result in cell migration and pearl chain formation.4,22
Even though electrofusion of biological cells is potentially a useful method, achieving the sufficient efficiency still requires extensive trial-and-error studies.23-26 One of the earliest approaches to improve electro-fusion efficiency was the use of hypotonic electrofusion buffer that resulted in the considerable fusion efficiency increase.27-34 To ensure the improvement of fusion efficiency in hypotonic buffer the duration and the os-molarity of the hypotonic treatment has to
be selected and used properly. Rapid cell swelling in the hypotonic environment due to influx of water namely triggers regulatory the volume decrease. If it is triggered before the induction of cell fusion, it can inhibit the positive effect of the hypotonic treatment on electrofusion by reducing cell size, restoring microvilli and excessive leaking of cytosolic electrolytes.33,35 The prolonged treatment thus leads to poor fusion efficiency and also decreases the cell viability.30,32-34
The aim of our study was first to determine cell size dynamics and the viability of four different cell lines in hypotonic buffer. The volume regulation of different cell lines exposed to strongly hypotonic buffers was analyzed by means of time-resolved cell size measurements. The second aim of our study was to determine the influence of the electroporation on the selected cell line in hypotonic buffer. B16-F1 cell line was selected because electroporation parameters and swelling induced by electroporation in isotonic buffer were previously well described for this cell line.21,36
Materials and methods
Chemicals, cell culture media
Eagle's minimal essential medium (EMEM), Ham's Nutrient Mixtures (F-12 HAM), Dulbecco's Modified Eagle's Medium (DMEM), trypsin, fetal bovine serum (FBS), L-glutamine, sucrose, phosphate (K2HPO4/ KH2PO4), MgCl2, crystal violet, trypsin and EDTA were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Germany). Antibiotics (crystacillin and gentamicin) were purchased from Lek (Ljubljana, Slovenia).
Cells
All cell lines were cultured in an incubator (Kambic, Slovenia) in the humidified
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Usaj M. et al. / Cell size dynamics after electroporation	110
Table 1. Chemical composition, conductivity and osmolarity of isotonic and hypotonic buffers used in our experiments.
Ingredients	Isotonic	Hypotonic
Phosphate buffer	10 mM	10 mM
MgCl2	1 mM	1 mM
Sucrose	250 mM	75 mM
pH	7,2	7,2
Conductivity	1.62 mS/cm	1.62 mS/cm
Osmolarity	260 mOsm	93 mOsm
atmosphere at 37 °C and 5% CO2 in the following culture media: murine melanoma (B16-F1) and Chinese hamster lung fibroblast (V79) in EMEM supplemented with 10% fetal bovine serum (FBS), antibiotics (gentamicin, crystacillin) and L-glutamine; Chinese hamster ovary cells (CHO) in F-12 HAM supplemented with 10% FBS, antibiotics and L-glutamine; mouse myeloma NS1 in DMEM supplemented with 13% FBS, antibiotics and L-glutamine. Cell lines were grown in 25 cm2 culture flask (TPP, Switzerland) until they reached 80-90% confluence. Adherent cells were exposed to 0.25% trypsin/EDTA solution for 1 minute. Trypsin solution was then removed and 5 ml of culture media was added. Cells were gently rinsed from the bottom with a plastic pipette and the homogenous cell suspension was prepared. For cell size dynamics and viability measurements in hypotonic buffer 5 x 105 cells were placed on Petri dish (see Cell size measurement and analysis section). For viability tests after the electroporation, cell suspension was centrifuged (290 x g, 5 min, 4°C) and then resuspended in the electroporation buffer (details explained in Cell viability section).
Isotonic and hypotonic buffers
Iso- and hypo-tonic buffers (phosphate buffer saline - PBS) of osmolarities 260 and 93 mOsm (mOsmol/kg) and conductivity 1.62 mS/cm were used (Table 1). The os-
molarity of solutions was determined with Knauer vapor pressure osmometer K-7000 (Knauer, Wissenschaftliche Gerätebau, Germany). Buffers pH was 7.2.
Cell size measurement and analysis
Cell volume changes were measured by protocol which allowed a rapid exchange of media. For that purpose we used 9.2 cm2 tissue culture Petri dishes (TPP, Switzerland). Before the microscopic measurement, cells in suspension were counted by haemocy-tometer and 5 x 105 cells were placed on Petri dish. Cells were incubated at 37 °C for 20-40 min (except for NS1, which were incubated overnight) in culture medium allowing cells to slightly adhere but still preserving a round shape. Culture medium was removed and cells were washed with 1 ml isotonic buffer leaving 300 |l to avoid drying of the sample during the acquisition of the first image (represents time t = 0 min). Cells were observed under the Axiovert 200 microscope (Zeiss, Germany) with 40 x objective in transmitted light. Phase contrast images were acquired with cooled CCD video camera VisiCam 1280 (Visitron, Germany) and PC software MetaMorph 7.0 (Molecular Devices, USA). After the first image was acquired 3.3 ml hypotonic buffer was added to the cells (in the control samples isotonic buffer was used). The resulting osmolarity of hypotonic buffer was calculated to 100 mOsm. Images of cells were then taken at various time intervals up to 30 min after the buffer exchange (every 15 s until 2 min and in minute steps after that). One to three sequences were recorded of each repetition of every experiment (cell line).
Cell diameters of 5-10 cells per image were determined at each time interval (37 images in every sequence). Cell size dynamic was defined as the relative change of cell diameter = d/d0, where d is an actual
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cell diameter and d0 is an initial one (at t = 0 min). The mean values (± STD) for a given experiment (cell line) were calculated from at least three independent repetitions of the experiments and plotted against time. The script was written in Matlab (Matlab 2008a, Math Works, USA) for all calculations to enhance calculation speed and graph performance. Observed differences between cell lines were statistically tested using ANOVA test or One-Sample T test (SPSS Statistics).
Electroporation
In the second part of our study, where we studied the effect of electroporation on B16-F1 cells swelling in isotonic and hypotonic buffers, the protocol was slightly altered from the one described previously. One minute after the buffer exchange from isotonic to hypotonic (or to isotonic again for control) electric pulses were delivered using two parallel electrodes (Pl/Ir = 90/10) and an electric pulse generator Cliniporator (IGEA, Italy). We used electrical parameters optimal for transient permeabilization of B16-F1 cells in the isotonic buffer (1000 V/cm, 8 x 100 |is, 1 Hz) described in our previous studies.21,36 Cell sizes were measured and analyzed as described previously.
Cell viability
The viability of hypotonically treated cells as well as electroporated cells was analyzed 24 h after experiments by means of a modified crystal violet (CV) viability assay.37 From prepared cell suspension 105 cells per well were plated to 24 - well microplates (TPP, Switzerland) and incubated at 37 °C for 20-40 min in culture medium to settle and to slightly adhere. After the incubation culture medium was carefully removed and cells were washed with 1 ml isotonic buffer. One ml of hypotonic buffer was added to
the cells in each well. In control 1 ml of culture medium was added. After a hypotonic treatment (30 min at room temperature) all media (buffer and culture medium in control) was carefully removed and 1 ml of fresh culture medium was added. Cells were then cultured in the incubator for 24 h. In described protocol washing, removing and adding medium were the same for treated cells as well as for the control ones.
After 24 h culture medium was removed and cells were washed with the isotonic buffer (1 ml per well) 0.1% CV solution prepared in the isotonic buffer was then added (200 |l per well). After 30 min of incubation at room temperature dye was carefully removed and cells were washed three times with the isotonic buffer per well (200 | l, 500 |l, 1000 |l). After the washing procedure cells were lysed by 10% acetic acid (1 ml per well). The same lysis procedure was also applied to wells without cells and their absorption values were used as the background. The absorption of lysate was measured with a microplate reader Infinite M200 (Tecan, Switzerland) at 595 nm wavelength controlled with PC software i-Control (Tecan, Switzerland) at maximum 1 h after the dying procedure. The viability of treated cells was defined as
Vc={Atc~Abs) x 100, i^cc ~ABg)
where
Vc ... viability [%]
Atc ... absorption value of treated cells
ACC . absorption value of control cells (100 % viability)
ABg ... absorption value of background
The mean Vc values (± STD) for a given cell line were calculated from three independent experiments.
In the second part of our study, where we studied the effect of electroporation on
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Figure 1. Swelling and regulatory volume decrease of B16-F1 cells in 100 mOsm buffer containing sucrose as the major osmolyte. The images show the same cells before (0 min) and during the hypotonic treatment at the indicated time intervals. After the rapid change of isotonic to hypotonic buffer at zero time, the cells swelled within 5 min and then shrank gradually within the observed time (5-30 min). Scale bar corresponds to 30 |m.
B16-F1 cells swelling, the viability protocol was slightly altered from the one described above. In order to expose cells to an electric field, electroporation cuvettes with 4 mm gap (Eppendorf, Germany) were used. The cell suspension prepared in cell culture media was counted and aliquots of 5 x 105 cells were prepared and centrifuged (290 x g, 5 min, 4°C). Supernatant was carefully removed and cells were resuspended in 1 ml of hypotonic buffer (or culture media for control). One minute after the hypotonic buffer was added, 800 |l of cell suspension was electroporated. In the control treatment no pulses were delivered. After the electropora-tion cells were kept at room temperature for 10 min and on 37°C for another 10 min to allow membrane resealing. From each cuvette 600 |l of cell suspension was plated in three microplate wells (24 - well microplates, TPP, Switzerland). Finally 1 ml of culture medium was added to each well and cells were incubated for 24 h. The cell viability was then determined with the crystal violet assay as
described above. Differences between elec-troporated and non-electroporated (control) cells were tested by the Paired samples T -test (SPSS Statistics).
Results
Cell size dynamics due to exposure to hypotonic buffer
We monitored cell size (diameter) changes in B16-F1, CHO, V79 and NS1 cells after the rapid change of isotonic to hypotonic buffer. From the microphotographs such as shown in Figure 1, the diameters of individual cells were evaluated and normalized to the original isotonic diameter (v = d/d0, Figure 2). The hypotonic treatment caused CHO, V79, B16-F1 cells to swell rapidly within first minutes from their initial isotonic diameters (d0) to their maximum diameters (dmax) due to a fast water uptake driven by the imposed osmotic gradient.
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1.5
0.9.................................
0	5	10	15	20	25	30
Time [min]
Figure 2. Cell dynamics (d/d0) in hypotonic buffer for three different cell lines. Cell dynamics was obtained by directly measuring the size of the cells. Each data point represents the mean ± STD of at least three repetitions. One-Sample T test showed that after 30 min in the hypotonic buffer cell size of V79 and CHO did not differ significantly from their initial isotonic size (P > 0,1) while B16-F1 cells remained significantly enlarged dend/d0=1.09 (P = 0,004).
We estimate that B16-F1 cells reached their maximal size 5 min after exposure to hypotonic buffer, while V79 and CHO cells reached their maximal size at approximately 2 min. The size increase was the smallest for B16-F1 cells (dmax/d0 = 1.18) and larger for V79 and CHO cells (dmax/ d0 = 1.26 and 1.29 respectively). However, described differences among cell lines were not statistically significant. Cells in the isotonic buffer did not change their size (data not shown).
After the initial swelling all cell lines underwent regulatory volume decrease and cells shrank gradually to diameters near original ones, despite the persisting hypotonic treatment, but with some differences between cell lines. During our observation, V79 and CHO cells shrank to their original size, while B16-F1 cells remained significantly enlarged dend/d0=1.09 (P = 0,004). Consequently, a regulatory volume decrease was faster with CHO and V79 cells and slower with B16-F1 cells.
A completely different behaviour was found in NS1 cell line under the same hypotonic treatment. The hypotonic treatment led NS1 cells to blebbing and expressing non-spherical shapes (Figure 3). Therefore, no cell swelling and consequent regulatory volume decrease was observed or measured for this cell line.
Cell size response to electroporation in isotonic and hypotonic buffer
We monitored cell size (diameter) changes in B16-F1 cells after the electroporation. Cells were exposed to the hypotonic buffer for 1 min before delivery of the pulses. In this study we used electrical pulse parameters that are optimal for the reversible elec-troporation of cells in the isotonic buffer as described in Materials and methods.
From the microphotographs the diameter of an individual cell was measured and normalized to the original diameter in the
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;f]!		k il
Hr		
Figure 3. Mouse myeloma cells NS1 in 100 mOsm buffer containing sucrose as the major osmolyte. The image shows the behaviour of the cells during the hypotonic treatment. These cells did not exhibit any regular size alterations. Instead, NS1 cells started to bleb and express non-spherical shapes. Scale bar corresponds to 10 |m.
isotonic buffer (v = d/d0, Figure 4). Within 1 min after the buffer change from isotonic to hypotonic, cells started to swell as in previous experiments without electroporation. We observed that the electroporation inhibited the effect of regulatory volume decrease completely during the 30 min of our observation. Cell swelling dynamics was also observed in B16-F1 cells electroporated in the isotonic buffer where a lower magnitude of swelling was measured (Figure 4).
Cell viability after hypotonic treatment
The cell viability of fusion partners needs to be preserved in order to produce viable hybrid cells. We, thus, analyzed the cell viability for all cell lines with a crystal violet (CV) viability assay. Results of the viability assay for hypotonically treated cells are shown in Figure 5. We observed no decrease in viability indicating that most of the cells survive the hypotonic treatment.
In the second part of our study, where we studied the effect of electroporation on
B16-F1 cells swelling in hypotonic buffer, cell the viability was also analyzed (Figure 6). The viability of electroporated cells significantly decreased in the hypotonic buffer to 63% (P = 0.002) while in the isotonic buffer the viability was not affected (P > 0.1).
Discussion
Electrofusion in the hypotonic buffer is a promising approach for improving the cell fusion efficiency. Improved electrofusion conditions can be achieved with the hypotonic treatment. However, the duration and the strength (the osmolarity) of the hypotonic treatment should be optimized for the specific cell type. In the first part of our study, we determined cell size dynamics and survival of different cell lines in the hypotonic buffer. In the second part, we determined the influence of the electroporation on B16-F1 cell size dynamics and the survival in hypotonic buffers.
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Figure 4. Time courses of relative cell diameter (d/d0) during the hypotonic treatment for B16-F1 cells. Squares (□) represent cells in the hypotonic buffer. Rhombs (0) represent cells in the hypotonic buffer, which were electroporated at t = 1 min. Circles (o) represent cells in the isotonic buffer, electroporated at the same time (t = 1 min). Cell diameter was obtained by directly measuring the size of the cells. Each data point represents the mean ± STD of at least three repetitions.
Electroporation was performed before a regulatory volume decrease took place.
In cell size dynamics we observe similar behaviour for cell lines B16-F1, V79 and CHO. In those cells as in many other mammalian cells the initial hypotonic swelling was accomplished within 2-5 min (Figure 2). 34,38-41 The maximal cell size increase was between 1.18 and 1.29 (Figure 2), the values that can be also found in the previously published studies.33-35,38,40-42 The only exception was cell line NS1 that will be described later on. Observed cell swelling is a desired phenomenon in electrofusion. The increase in cell size requires the unfolding of undulations and invaginations of cell membrane.42,43 This decrease in membrane undulations results in the decrease of undulation repulsive forces, yielding better cell-cell contacts.31,44-47
Differences among cell lines were found in regulatory volume decrease dynamics.
The expression of a regulatory volume decrease depends on the type of sugar that is used as osmolyte. Cells can express a regulatory volume decrease in buffers containing disaccharide as major osmolyte whereas when monosaccharide is used, the regulatory volume decrease is inhibited. The different effect of sugars was explained by volume-sensitive channels in the plasma membrane that are selectively transporting monomeric sugars but are poorly permeable for oligosaccharides.34,35,40 In our study such behaviour was observed for V79 and CHO cells that showed the fast regulatory volume decrease, mainly completed within 10-20 min (Figure 2).
In contrast to V79 and CHO, only a partial regulatory volume decrease was observed for B16-F1 cells (dend/d0=1.09, Figure 2). Similar phenomena was reported for the Jurkat leukemic cell line (dend/d0=1.11) and murine fibroblast (dend/d0=1.12).33,40 A
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100 -	T
B16-F1	V79	CHO	NS1
Figure 5. Viability of four different cell lines exposed to the hypotonic treatment (100 mOsm buffer containing sucrose as major osmolyte) for 30 minutes. Data represent mean ± STD of at least three independent experiments.
partial regulatory volume decrease in epithelial and cancerous cell lines was attributed to the fact that those lines may slowly uptake sucrose. This uptake of oligosaccha-rides48,49 can explain the partial inhibition of regulatory volume decrease observed in B16-F1.
According to published literature the same hypotonic treatment causes different response in different cell lines due to their different (hypotonic) sensitivity.33,40,41,43 In mouse myeloma cells NS1 no swelling and regulatory volume decrease were observed. Instead, NS1 cells started to bleb and express non-spherical shapes (Figure 3). A similar behaviour was reported earlier for different cell lines when too hypotonic treatment (30 to 60 mOsm) was used.41,43 Therefore, we conclude that NS1 cells are more sensitive to a hypotonic treatment than other cell lines used in our study. Higher buffer osmolarities may have to be used for NS1 cells in order to obtain spherical cell shape with smooth membrane favourable for the electrofusion.
Regardless to immediately observed effect of the hypotonic treatment on cell size and shape, the cell viability was not affected (Figure 5). Those results show that the duration of hypotonic treatment and buffer
120
260 mOsm	lOOmOsm
Figure 6. Effect of electroporation in both, hypotonic and isotonic buffer on viability of B16-F1 cells. Eight pulses of 100 |is duration at 1000 V/cm electric field intensity at 1 Hz pulse repetition frequency were applied to the cells in 100 mOsm and 260 mOsm buffer containing sucrose as major osmolyte. Data represent mean ± STD of at least three independent experiments. The Paired Sample T test showed that the viability of electroporated cells significantly decreased in the hypotonic buffer (P = 0.002) while in the isotonic buffer the viability was not affected (P > 0.1).
hypoosmolarity were properly selected and are in agreement with literature.40,50,51 Interestingly no significant decrease in cell viability was observed for NS1 cells, even though the hypotonic treatment caused cell blebbing.
In the second part of our study we determined the influence of the electroporation on B16-F1 cell size dynamics and the survival in the hypotonic buffer. Electroporation by itself causes swelling of electroporated cells in the isotonic buffer36, which was also observed in our experiment with B16-F1 cells (Figure 4). Electroporation induced cell swelling was significantly amplified if the hypotonic buffer was used and, therefore, regulatory volume decrease was inhibited (Figure 4).
However, electroporation in the hypotonic buffer was associated with a significant loss of cell viability (Figure 6) that was also observed in the study of Golzio et al.47 Electric field parameters for electroporation in the hypotonic buffer should be carefully selected in order to preserve cell viability since hypotonic treatment causes a higher
Electroporation buffer
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susceptibility due to the increase in cell
size 16,28,30-33,39,52
In conclusion, cell size dynamics should be carefully analyzed and observed for each cell line in order to obtain all potential benefits of using hypotonic buffer for electrofu-sion. Electroporation should be performed when cells are close to their maximal size i.e. before the regulatory volume decrease starts. In addition, electroporation parameters should be adjusted to hypotonically induced changes on cells in order to preserve the cell viability.
Acknowledgements
We thank Prof. Vladka Čurin Šerbec from Blood Transfusion Centre of Slovenia for providing us with NS1 cells. This research was supported by the Slovenian Research Agency (ARRS). The authors would also like to thank Dr. Tomaž Jarm for his help with statistics and to Duša Hodžic for manuscript reading and her useful comments.
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doi:10.2478/v10019-009-0017-9
case report
Cisplatin-induced non-convulsive posterior reversible encephalopathy syndrome in a 41-year-old woman with metastatic malignant melanoma
Janja Ocvirk1, Marko Boc1, Martina Rebersek1, Tanja Ros2
^■Department of Medical Oncology, Institute of Oncology Ljubljana, 2Unit of Neurology, Institute of Oncology Ljubljana, Ljubljana, Slovenia
Background. Cisplatin, a widely used antineoplastic agent usually induces peripheral neuropathy, but can rarely also complicate with encephalopathy, with or without seizures.
Case report. We report a case of a young patient with metastatic malignant melanoma with signs and symptoms of cisplatin-induced non-convulsive posterior reversible encephalopaty syndrome. Within the days shortly after the first cycle of cisplatin based chemotherapy the patient suffered from nausea, vomitus, headache, severe pain at the site of sub-cutaneous metastases and confusion. She later experienced somnolence, cortical blindness and aphasia, but without epileptic seizures.
Conclusions. Cisplatin is an effective chemotherapeutic drug but also very toxic one and physicians using it must also be aware of possible encephalopathy.
Key words: neurotoxicity; cisplatin; posterior reversible encephalopathy syndrome
Introduction
Cisplatin is an effective and also widely used chemotherapeutic drug; it is part of numerous chemotherapeutic schedules used in the treatment of many solid tumours. Most common side-effects that patients experience are nausea, vomiting, nephrotoxicity, ototoxicity and peripheral neurotoxicity.1,2 Central nervous system disorders such as seizures, cortical blind-
Received 27 November 2008 Accepted 6 December 2008
Correspondence to: Janja Ocvirk, MD, PhD, Institute of Oncology Ljubljana, Zaloska 2, Ljubljana. Phone: +386 1 5879 220; Fax: +386 1 5879 305; E-mail: jocvirk@onko-i.si
ness, aphasia, coma, also hemiparesis are rare, but have already been reported many times,3 cortical blindness and seizures being reported first time in 1980's.4
Case report
A 41-year-old female patient with meta-static malignant melanoma was admitted to our hospital because of deterioration of her mental and physical state after the first cycle of the second line chemotherapy (ChT) in combination with cisplatin.
Primary malignant melanoma on the left lumbal part of the back was surgically removed in September 2005 (Clark IV,
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Breslow 1.68, ulceration 2 mm, with signs of regression, without microsatelites, without vascular invasion, TNM stage T2bN1a(1/ 14)M0, stage IIIB). The sentinel node biopsy presented with a 2.5 mm wide metastasis; therefore the dissection of axilliary lymph nodes was performed.
From February 2006 to May 2007 she was receiving interferon in an adjuvant setting. During the adjuvant treatment there was clinically a locoregional relapse with a 12 cm bulky mass on the site of the primary tumour, further examination (CT, RTG, and UZ) has shown the dissemination in the lungs and pleura with multiple lung metastases.
She started her first line ChT with dakarabazine-DTIC (1000 mg/m2 i.v., day 1, 21 days) in May 2007. After two cycles the further progression of the disease was observed, locoregionally and systemically. The bulky mass on the left lumbal part of her back was now clinically 15 cm in diameter and was at least 3 cm above skin level.
The second line treatment was started in June 2007, she received the first cycle of ChT with cisplatin (80 mg/m2 i.v., day 2., 21 days), lomustin-CCNU (80 mg/m2 p.o., day 1., 21 days) and vinblastin (3 mg/m2 24 h infusion, day 1, 21 days) with premedication with dexamethason 20 mg i.v., granisetron 3 mg i.v., manitol 500 ml i.v. and MgSO4 5 ml i.v.. She was also hydrated with 3000 ml of 0.9% NaCI. She ended her first cycle without acute toxic event.
ShortIy after she got home, she started suffering from nausea, vomitus, headache, severe pain at the site of subcutaneous mass and confusion. On the day of the admission she deteriorated even more, foIIowed by somnolence, cortical blindness and aphasia, but without epiIeptic seizures. At home she was continuing with buprenor-fin transdermal patch (Transtec®) 35 |g, short acting oral morphine (Sevredol®) 10 mg for breaking through pain, dicIofenak tbl. (Olfen SR®) 100 mg/day, pregabalin
tbl. (Lyrica®) 75 mg/day, pantoprazol tbl. (Controloc®) 1 tbl/day, metoklopramid tbl. (Reglan®) 3x1 tbl/day in granisetron tbl. (Kytril®) 2 mg when needed.
WHO performance status on admission was 3-4, the patient was affected, she was in pain, aphasic, somnoIent, aImost comatose. BIood pressure was 110/70 mmHg, heart rate was 80/min, she was afebriIe. MeningeaI signs were negative, her breathing and heart signs were normaI. Abdomen was soft, without pathoIogic mass or Iiver paIpabIe, without tenderness. The buIky subcutaneous mass on the site of primary tumour was smaIIer, onIy about 10 cm in diameter and has fIattened.
The Iaboratory report has shown miId hypokaliemia (3.7) and hyponatriemia (132), without any signs of dehydration, with elevated WBC (14.9) and CRP (39). The Iaboratory report did not expIain the patient's state; electrolytes, magnesium, caIcium were within Iimits, her bIood sugar IeveI was normaI.
Differential diagnosis
At first we suspected that she developed metastasis in the CNS, as CNS is frequent site of dissemination in maIignant meIano-ma. She was presented with possibIe signs of increased intracranial pressure with severe headache and vomitus, confusion, aphasia, somnoIence and Iater stupor. So, she was started on antioedematic therapy with corticosteroids and manitoI soIution, but her state was improving sIower than expected in increased intracraniaI pressure.
To excIude the possibiIity of morfine intoxication, morphine therapy was discon-tinuated.
Urgent computer tomography (CT) of her brain did not show any proof of intracraniaI dissemination; bedsides, the neuroIogic exam showed that the patient was deepIy somnoIent, without speech contact, though
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Figure 1c. Oedema in the corpus callosum (T2, without contrast).
she did react on a call with movements and restlessness, but she did not open her eyes. There were neither focal signs of central nervous damage nor signs of pyramidal tract lesion; meningeal signs were negative. The lumbar punction was performed; cerebrospinal fluid was entirely normal, without malignant cells and with negative tests on bacterial and viral examination.
Figure 1d. Occipital cortex (T2, with contrast).
MRI presentation
At that time MRI of her brain was performed. She had typical neuroradiological changes in the form of oedema in supra-and infra-tentorial areas with hyperinten-sity on T2-weighted MRI imaging, seen in the occipital cortex, cerebellum, basal ganglia and corpus callosum (Figures 1a, 1b, 1c, 1d).
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Figure 3. Control MRI-occipital cortex (T1, with contrast), infarction on the site of previous oedema.
EEG presentation
Electroencephalogram showed diffuse slow-wave activity with frequent generalized paroxysms of sharp waves and slow
delta activity (1.5-2 Hz) (Figure 2), EEG findings were compatible with reversible non-convulsive encephalopathy.
Further course of disease
During hospitalization we continued with the symptomatic treatment, her condition slowly improved, on the third and fourth day she was more alert, but we disclosed blindness, she didn't complain about. The ophthalmologic examination at bedside was normal, except the possibility to test vision and visual fields. Her vision and other symptoms slowly improved and so did MRI (Figure 3) and EEG findings (Figure 4). Repeated MRI disclosed an ischemic lesion in the occipital cortex, otherwise rarely seen complication of PRES, with partly completed regression of other changes. After fifteen days she was discharged from the hospital, fully conscious, with much improved vision, and without dysphasia.
Figure 2. EEG two weeks after admission with diffuse slow wave activity and generalised paroxysms of sharp waves and slow delta activity.
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She received next two cycles of ChT with cisplatin after twelve days and the third one later on, both times in split doses. No central neurotoxicity reoccurred, but after the malignant disease progressed, ChT was stopped and the patient died three and a half months after starting cisplatin-based ChT.
Discussion
Cisplatin side effects are often predictable in terms of their onset and duration, being most common nausea, vomitus, kidney toxicity and peripheral neuropathy with numbness, paresthesias, and occasionally pain, which usually begins in the toes and fingers, spreading proximally to affect the legs and arms.5 In the presented patient, clinical signs and symptoms, MRI and EEG findings were compatible with rare central nervous toxicity of cisplatin-induced non-
convulsive posterior reversible encephalop-athy syndrome that occurred after the first dose of cisplatin-based ChT. The causal relationship to the agent was made after excluding other causes of the condition, such as progression of malignant disease, metabolic, iatrogenic, infectious and vascular causes. As in other cerebral diseases the MRI was essential in differential diag-nosis,6 but we found the main reason for the neurological disorder with contextual understanding of other clinical findings.
Of the other concurrent medications that are potentially central neurotoxic she also received lomustine, but according to data that we found, nitrosoureas are associated with little neurotoxicity at conventional doses, toxic effect is possible after cumulative and high iv doses,7,8 which is not the case in our patient.
According to published data the exact mechanism for the emergence of cispla-tin-induced encephalopathy is not known,
Figure 4. Control EEG after three months indicating improvement with disappearance of sharp and slow wave delta activity.
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125
one hypothesis is that it may be caused by vascular events, other is that mechanism may be toxic encephalopathy, e.g. heavy metal toxicity and demyelinization.9 Fever, thrombocytopenia, neutropenia, renal insufficiency, metabolic disturbances also contribute to CNS toxicity of cisplatin, but that was not the case in our patient. Neurotoxicity typically starts within two weeks after the treatment with cisplatin, with or without seizures, accompanied by acute or sub acute confusional state, central visual problems (even blindness) and often headache.10 Our patient suffered from nausea, vomitus, headache, severe pain at the site of sub-cutaneous metastases and confusion, she later experienced somnolence, cortical blindness and aphasia, but without epileptic seizures, a combination characteristic of a less common, non-convulsive encephalopathy. Her symptoms resolved, she received the treatment with another two cycles, both times with split doses of cisplatin, decided by intending medical oncologist according to good clinical practice for minimizing the side effects. Both cycles were administered according to the same schedule and adverse events did not repeat. On control MRi, an ischemic lesion, a rarely seen complication to be present also at autopsy of such patients, was found.
Conclusions
According to published cases, cisplatin-in-duced central nervous toxicity is a relatively rare complication in cancer patients, but with the widespread use of cisplatin this rare disorder should be considered, especially if neurologic symptoms as described in this article occur. When diagnosed at an early stage it is usually a reversible condition, which requires adequate treatment of seizures, symptomatic treatment and withdrawal or cautiousness with cisplatin
therapy.11 it is important do differentiate
neurotoxicity of cisplatin from signs of tu-12
mour progression.12
References
1.	New PZ. Neurological complications of chemo-therapeutic and biological agents. Continuum: lifelong learning in neurology. Neuro-Oncol 2005; 11(5):116-152.
2.	Kaplan RS, Wiernik PH. Neurotoxicity of antine-oplastic drugs. Sem Oncol 1982; 9: 103-30.
3.	Lyass O, Lossos A, Hubert A, Gips M, Peretz T. Cisplatin-induced non-convulzive encephalopa-thy. Anticancer Drugs 1998; 9: 100-4.
4.	Berman iJ, Mann MP. Seizures and transient cortical blindness associated with cis-platinum (ii) diamminedichloride (PDD) therapy in a thirty year old man. Cancer 1980; 45: 764-66.
5.	UpToDate online, version 16.3. Neurologic complications of platinum-based chemotherapy. Available: http://www/uptodateonline.com/. 30 October, 2008.
6.	Franko A, Holjar-Erlic I, Miletic D. Lateral ventricle epidermoid. Radiol Oncol 2008; 42: 66-8.
7.	Burger PC, Kamenar E, Schold SC, Fay JW, Phillips GL, Herzig GP. Encephalomyelopathy following high-dose BCNU therapy. Cancer 1981; 48: 131837.
8.	Posner JB. Neurologic complication of cancer. Philadelphia: FA Davis; 1995. p. 282-310.
9.	Connolly RM, Doherty CP, Beddy P, O'Byrne K. Chemotherapy induced reversible posterior leu-coencephalopathy syndrom. Lung Cancer 2007; 56: 459-63.
10.	Steeghs N, de Jongh E, Sillevis Smith PAE, van den Bent MJ. Cisplatin-induced encephalopathy and seizures. Anticancer Drugs 2003; 14: 443-6.
11.	Ros T, Rebersek M, Boc M, Ocvirk J, Jereb S, Dolenc-Groselj I, et al. Cisplatin induced encephalopathy. [Abstract]. EANO 2008: P152. Neuro-Oncol 2008; 10(6): 1119.
12.	Verschraegen C, Conrad CA, Hong WK. Subacute encephalopathic toxicity of cisplatin. Lung Cancer 1995; 13: 305-9.
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doi:10.2478/v10019-009-0017-9
case report
Angiosarcoma of the liver after multimodality therapy for gallbladder carcinoma
Maikel Botros, J. Fernando Quevedo, Robert C. Miller
Department of Radiation Oncology and the Department of Oncology, Mayo Clinic, Rochester, Minnesota, USA
Background. Treatment-induced angiosarcoma is a rare complication of cancer therapy and is best known for arising on the chest wall after a mastectomy. We report an unusual case of a treatment-induced angiosarcoma developing in the liver after adjuvant radiotherapy for gallbladder carcinoma. Case report. Radiation-induced angiosarcoma of the liver developed in a 46-year-old woman after radiotherapy for stage IIB (T3N1M0) adenocarcinoma of the gallbladder in 1991. The patient subsequently underwent resection, postoperative external beam radiotherapy, and multiagent chemotherapy. Several severe late adverse effects developed, including duodenal obstruction with fistula formation and chronic mesenteric ischemia secondary to occlusion of the superior mesenteric artery. Six years after her gallbladder resection and adjuvant treatment, a fatal grade 4 of 4 angiosarcoma of the liver developed within the radiation field. Conclusions. To our knowledge, this is the first case of radiation-induced angiosarcoma of the liver after radiotherapy for gallbladder carcinoma. Normal organ dose-volume limits should be considered carefully when delivering a course of external beam radiotherapy in the upper abdomen.
Key words: angiosarcoma; gallbladder carcinoma; liver; radiotherapy toxicity
Introduction
Angiosarcomas are rare malignant tumors arising from the vasculature.1 Angiosarcomas of the liver occur even less commonly; about 25 cases are report-
Received 26 January 2009 Accepted 14 February 2009
Correspondence to: Robert C. Miller, MD, MS, Department of Radiation Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: miller. robert@mayo.edu
Conflict of interest: The authors have no conflicts of interest to disclose.
ed in the United States every year. Most angiosarcomas arising from the liver are secondary to long-term exposure to thorium dioxide, arsenic, or vinyl chloride.2 To our knowledge, radiotherapy-induced liver angiosarcomas have not been previously reported. The purpose of this paper is to describe the first case of a radiotherapy-induced liver angiosarcoma.
Case report
A 46-year-old woman presented with acute pain in the right upper quadrant of her
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127
abdomen in 1991. Ultrasonography at that time revealed a thickened gallbladder wall. A computed tomographic (CT) scan of the abdomen demonstrated a mass arising in the gallbladder and extending beyond the gallbladder wall into the porta hepatis and the left hepatic lobe. Her staging work-up, which included a barium enema study, sig-moidoscopy, and pelvic ultrasonography, revealed no sites of metastatic disease. Levels of a-fetoprotein and cancer antigen 125 were within normal limits. Carcinoembryonic antigen (CEA) and alkaline phosphatase values were slightly elevated at 4.4 ng/mL and 126 units/L, respectively. The patient had a 20-pack-year history of smoking. There was no familial history of cancer or other genetic conditions.
The patient underwent a cholecystecto-my and partial hepatectomy at another institution. Pathologic review of the resected specimen revealed a well- to moderately differentiated mucinous adenocarcinoma of the gallbladder, extending beyond the gallbladder wall to involve the liver capsule, stage IIB (T3N1M0). Three of 3 evaluated lymph nodes were involved with tumor. The patient's postoperative recovery was unremarkable and adjuvant therapy was recommended, given the primary tumor's adherence to the liver and the involved lymph nodes. The patient elected to receive adjuvant therapy. A course of external beam radiotherapy to a total dose of 54 Gy in 1.8-Gy daily fractions was administered using a 4-field technique. The patient received adjuvant chemotherapy consisting of fluorouracil, leucovorin, and mitomycin C concurrently with her radiotherapy. Her radiotherapy course was marked by grade 3 gastrointestinal tract toxicity, and she required hospital admission and support with intravenous hydration secondary to nausea, vomiting, and dehydration.
Over the next year, the patient had progressive upper abdominal pain with con-
comitant weight loss. Approximately 1 year after radiotherapy, an esophagogastroduo-denoscopy revealed friable gastritis involving the distal antrum along with duodenitis involving the duodenal bulb, secondary to the radiation effect. In the next year, the patient continued to experience symptoms of persistent gastritis and duodenitis, and the following year she underwent a vagotomy and gastrojejunostomy to bypass a stricture of the duodenum, with an associated fistula. Four years after radiotherapy, in 1995, a diagnosis of chronic mesenteric ischemia was made. Mesenteric angiography revealed a 3-cm proximal occlusion of the superior mesenteric artery. She underwent aortic-to-superior mesenteric artery bypass at that time, and her symptoms resolved.
Six years after radiotherapy, in 1997, right upper quadrant pain and persistent nocturnal fevers up to 38.9°C developed. On clinical examination, she had right upper quadrant tenderness with abdominal distension and a palpable mass in the right upper quadrant. A CT scan subsequently revealed a 12-cm mass involving the anterior segment of the right hepatic lobe and medial left segment of the left hepatic lobe, with additional satellite lesions within the left lateral hepatic segment. The tumor extended into the porta hepatis, with occlusion and thrombus of the anterior segmental division of the right portal vein, and involved the prior gallbladder fossa. There was no evidence of neoplastic spread outside the liver. A biopsy revealed a spindle cell neoplasm consistent with angiosar-coma, grade 4 of 4. Immunohistochemical stains for CD31 and vimentin were positive and negative for keratin, CEA, and S-100.
The patient was referred to Mayo Clinic in Rochester, Minnesota, for evaluation and treatment at this point. She was not considered a surgical candidate because of the multifocal presence of tumor within the liver. A symptomatic thrombus of her supe-
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rior mesenteric artery bypass graft developed approximately 14 days after the diagnosis of angiosarcoma. A course of salvage chemotherapy, consisting of ifosfamide, mitomycin C, doxorubicin, and cisplatin, was recommended, but the patient succumbed to fatal mesenteric ischemia before initiation of any systemic therapy.
Discussion
Gallbladder carcinoma is a rare malignancy. The prognosis is often poor because of the advanced stage at the time of diagnosis. Surgery remains the treatment of choice for gallbladder carcinoma.3 For gallbladder carcinoma that is limited to the gallbladder mucosa, cholecystectomy is often sufficient to eradicate all malignant disease. However, for locally and regionally advanced disease, a radical cholecystectomy is recommended. For patients in whom an incidental carcinoma is found at the time of routine cholecystectomy, re-resection with abdominal exploration and lymph node dissection is often considered.
Given the high risk for systemic as well as regional recurrence in locally advanced tumors, we consider adjuvant therapy after resection of tumors that are locally extensive, invade adjacent organs, or involve regional lymph nodes. No randomized trials have been performed to quantify the benefit of such adjuvant therapy, however.4 Retrospective studies have suggested that postoperative chemoradiotherapy has a survival benefit in patients with high-risk tumors, ie, tumors that show extensive local infiltration outside the wall of the gallbladder or those with regional lymph node involvement, in the absence of metastatic disease.4-6 A study from Mayo Clinic, published in 2002, described 12 patients who underwent adjuvant chemoradiotherapy for gallbladder cancer after resection with
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negative margins and had 5-year survival of 64%.5 Historical surgical controls from Mayo Clinic and other institutions had 5-year survival of 33% for patients treated with surgery alone.5 A more recent evaluation at Mayo Clinic showed survival rates were equivalent for patients undergoing surgery and for those undergoing surgery and adjuvant radiotherapy.4 However, the majority of patients who underwent resection alone did so for stage I gallbladder carcinoma (88%), whereas patients receiving adjuvant therapy had more advanced stages. Despite having more advanced stages, patients receiving adjuvant therapy, primarily concurrent fluorouracil and external beam radiotherapy, had a survival rate comparable to those undergoing surgery alone for stage I disease.4 A recent large Surveillance, Epidemiology, and End Results analysis showed a benefit in overall survival rates for selected patients undergoing postoperative adjuvant chemotherapy.7 Given the relative rarity of the disease in the Western world, however, it is unlikely that a definitive randomized trial will address this question. Retrospective studies from Mayo Clinic and elsewhere suggest the benefit of this treatment, but these studies are limited by known biases regarding patient selection with respect to performance status and other factors.
The difficulties encountered in case studies highlight the potential pitfalls in delivering external beam radiotherapy in the right upper quadrant. Modern image-guided radiotherapy techniques, coupled with 4-di-mensional CT-based treatment planning, now provide radiation oncologists with tools to avoid excess doses in critical organs. The chronic adverse effects in the patient described here—duodenal strictures, chronic gastritis, and arterial ischemia—are known complications resulting from highdose and large-volume irradiation of the organs in the gastrointestinal tract. Also,
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the risk of chronic, radiation-induced liver disease must be considered in treatment planning.8
The long-term adverse effects of radiation on the stomach may include gastritis with stenosis, dyspepsia, and ulceration. Gastritis may appear from 1 to 12 months after radiation treatment and is usually accompanied by antral stenosis. Endoscopy may also reveal atrophy of the mucosal wall. The onset of dyspepsia varies and may occur up to 4 years after treatment. Radiation-induced gastric ulcerations may heal spontaneously but are usually accompanied by fibrosis. Management of chronic gastritis has not been extensively studied. Some physicians elect to prescribe antiulcer medications such as H2-receptor block-ers.9
The volume of small bowel receiving high doses of radiotherapy is typically the limiting factor in radiotherapy planning for the upper abdomen. Radiation effects include fibrosis and chronic ischemia after vascular injury. Multifocal adhesions and areas of stenosis can occur in a dose-dependent fashion.9 Radiotherapy can decrease the number of mucosal stem cells in the small intestine, causing inflammation and shortening of the villi and leading to a decrease in intestinal absorption. Additionally, radiation can induce, independent of injury to the villi, an inflammatory cytokine cascade in which proinflammatory cytokines such as tumor necrosis factor a and interleukins 1 and 6 contribute to a cycle of initiation of injury, upregulation with generation of messenger agents, and ulceration with further inflammation.10 Vascular damage caused by radiation may involve hyalinized thickening and foam cell deposition in small-sized (<100 |im) and medium-sized (100-500 |im) arteries. Lymphocytic infiltration can occur in the tunica media and tunica adventitia. These changes are typical of small-vessel injury from radiotherapy and
may, in rare cases, occur in larger vessels.11 Symptoms of enteritis may take years to develop after irradiation of the abdomen, but the reported median is 8 to 12 months.9 Toxicity is strongly correlated with the volume of small bowel irradiated in conjunction with the dose.9,12,13
At Mayo Clinic, our current approach to postoperative treatment of locally advanced gallbladder carcinomas involves careful reevaluation of the patient's nutritional status and cancer staging status approximately 1 month after surgery, followed by consideration of concurrent radiotherapy and chemotherapy. For patients who have adequately recovered from their resection and who have no evidence of metastatic disease, either at surgery or at the time of restaging after surgery, consideration is given to systemic chemotherapy with gem-citabine as initial treatment. This is followed by concurrent continuous infusional fluorouracil chemotherapy with 50.4 Gy in 1.8-Gy daily fractions of conformal external beam radiotherapy to the gallbladder fossa and regional lymphatics. Then gemcitabine-based chemotherapy is resumed 1 month after completion of radiotherapy. This regimen is similar to the adjuvant therapy delivered postoperatively with gemcitabine for pancreatic cancer during the Radiation Therapy Oncology Group 97-04 trial14, with the exception of a change in radiotherapy target volume. A total radiation dose of 50.4 Gy is delivered to the tumor bed and structures involved by the tumor at the time of resection, with a lower dose of 45.0 Gy delivered to uninvolved lymph node regions at risk in the porta hepatis, pancreatoduo-denal lymph nodes, and celiac axis. The radiotherapy must be carefully planned to provide adequate planning target volume (PTV) coverage (95% of the PTV receiving >50.4 Gy) while respecting liver tolerance (mean liver dose in patients without preexisting liver disease <34 Gy; volume of liver
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receiving >30 Gy is <50%); kidney tolerance (volume of kidney receiving >18 Gy is <33% for at least 1 kidney and preferably both); and limiting maximum small bowel dose to no more than 52 Gy. Radiotherapy beam arrangements typically consist of opposed laterals, 2 anterior oblique angles 45° off vertical, a noncoplanar anterior beam angled 30° superior, and a noncoplanar posterior beam angled 20° toward the feet, with the angle of the posterior beam limited by the linear accelerator and couch design. A 4-dimensional CT scan for radiotherapy planning is performed at the time of simulation to track the target volume at risk with the breathing cycle.
Ionizing radiation can produce sarcomas in irradiated tissues. Cahan et al.15 have published a well-recognized set of criteria to describe radiotherapy-induced sarcomas. First, the sarcoma should arise in an area that was previously irradiated. Second, a period of latency must exist between the radiotherapy and onset of the sarcoma. Finally, histologic confirmation of the sarcoma should be obtained.16 Applying the criteria of Cahan et al.15, the angiosarcoma reported here would meet the criteria for a radiation-induced malignancy. Previously, angiosarcomas have been reported in organs such as the heart, liver, spleen, and adrenal glands and have had a poor prognosis. Angiosarcomas have also been reported after mastectomy in patients with breast cancer due to long-standing lymphedema (Stewart-Treves syndrome).1
In summary, the role of adjuvant therapy in the treatment of locally advanced, resected gallbladder carcinoma remains to be optimally defined. On the basis of limited retrospective evidence, chemotherapy, with or without concurrent chemoradiotherapy, may reduce the risk of recurrence for patients with risk factors for local and regional recurrence. The chronic adverse effects of treatment emphasize the need for careful
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consideration of normal-organ dose-volume limits when delivering a course of external
beam radiotherapy in the upper abdomen.
References
1.	Koch M, Nielsen GP, Yoon SS. Malignant tumors of blood vessels: angiosarcomas, hemangioendotheliomas, and hemangioperictyomas. J Surg Oncol 2008; 97: 321-9.
2.	Centers for Disease Control and Prevention (CDC). Epidemiologic notes and reports: angio-sarcoma of the liver among polyvinyl chloride workers-Kentucky. 1974. MMWR Morb Mortal Wkly Rep. 1997; 46: 97-101.
3.	Reid KM, Ramos-De la Medina A, Donohue JH. Diagnosis and surgical management of gallbladder cancer: a review. J Gastrointest Surg 2007; 11: 671-81.
4.	Gold DG, Miller RC, Haddock MG, Gunderson LL, Quevedo JF, Donohue JH, et al. Adjuvant therapy for gallbladder carcinoma: the Mayo Clinic experience. Int J Radiat Oncol Biol Phys 2009 Mar 16. [Epub ahead of print]
5.	Kresl JJ, Schild SE, Henning GT, Gunderson LL, Donohue J, Pitot H, et al. Adjuvant external beam radiation therapy with concurrent chemotherapy in the management of gallbladder carcinoma. Int J Radiat Oncol Biol Phys 2002; 52: 167-75.
6.	Czito BG, Hurwitz HI, Clough RW, Tyler DS, Morse MA, Clary BM, et al. Adjuvant external-beam radiotherapy with concurrent chemotherapy after resection of primary gallbladder carcinoma: a 23-year experience. Int J Radiat Oncol Biol Phys 2005; 62: 1030-4.
7.	Wang SJ, Fuller CD, Kim JS, Sittig DF, Thomas CR Jr, Ravdin PM. Prediction model for estimating the survival benefit of adjuvant radiotherapy for gallbladder cancer. J Clin Oncol 2008; 26: 2112-7.
8.	Gunderson LL, Martenson JA, Smalley SR, Garton GR. Upper gastrointestinal cancers: rationale, results, and techniques of treatment. Front Radiat Ther Oncol 1994; 28: 121-39.
9.	Coia LR, Myerson RJ, Tepper JE. Late effects of radiation therapy on the gastrointestinal tract. Int J Radiat Oncol Biol Phys 1995; 31: 1213-36.
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10.	von Bultzingslowen I, Brennan MT, Spijkervet FK, Logan R, Stringer A, Raber-Durlacher JE, et al. Growth factors and cytokines in the prevention and treatment of oral and gastrointestinal mucositis. Support Care Cancer 2006; 14: 519-27.
11.	Fajardo LF. Morphology of radiation effects on normal tissues. In: Perez CA, Brady LW, editors. Principles and practice of radiation oncology. 3rd ed. Philadelphia: Lippincott-Raven; c1998. p. 143-54.
12.	Hauer-Jensen M, Wang J, Denham JW. Bowel injury: current and evolving management strategies. Semin Radiat Oncol 2003; 13: 357-71.
13.	Letschert JG, Lebesque JV, Aleman BM, Bosset JF, Horiot JC, Bartelink H, et al. The volume effect in radiation-related late small bowel complications: results of a clinical study of the EORTC Radiotherapy Cooperative Group in patients treated for rectal carcinoma. Radiother Oncol 1994; 32: 116-23.
14.	Regine WF, Winter KA, Abrams RA, Safran H, Hoffman JP, Konski A, et al. Fluorouracil vs gemcitabine chemotherapy before and after flu-orouracil-based chemoradiation following resection of pancreatic adenocarcinoma: a randomized controlled trial. JAMA 2008; 299(9):1019-26. Erratum in: JAMA 2008; 299(16): 1902.
15.	Cahan WG, Woodard HQ, Higinbotham NL, Stewart FW, Coley BL. Sarcoma arising in irradiated bone: report of 11 cases. Cancer 1948; 1: 3-29.
16.	Morgan MA, Moutos DM, Pippitt CH Jr, Suda RR, Smith JJ, Thurnau GR. Vaginal and bladder angiosarcoma after therapeutic irradiation. South Med J 1989; 82: 1434-6.
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doi:10.2478/v10019-009-0017-9
technical note
How well are clinical gross tumor volume DVHs approximated by an analytical function?
Pavel Stavrev2, Colleen Schinkel1,2, Nadia Stavreva2 and B. Gino Fallone1-3
^-Department of Physics, University of Alberta, 2Department of Medical Physics, Cross Cancer Institute, 3Department of Oncology, University of Alberta, Edmonton, Alberta, Canada
The dose heterogeneity in the tumor is often described as being normally distributed. Besides the normal distribution we propose the Fermi function describing Fermi statistics as a possible dose heterogeneity descriptor. In order to demonstrate the adequacy of the proposed functions as dose distribution descriptors 30 clinical gross tumor volume (GTV) dose-volume histograms (DVHs) are gathered and fit with the examined functions.
Key words: dose-volume histograms; gross tumor volume; Gaussian and Fermi statistics
In order to theoretically investigate a given tumor control probability (TCP) model for the case of heterogeneous irradiation, it is often necessary to simulate tumor dosevolume histograms (DVHs) that closely resemble clinical ones. Some authors1-3 have assumed that tumor dose inhomogeneities are normally distributed around the target dose. In this case the integral DVH, iDVH, is represented by the erfc function:
iDVH \ v(d \fi,6) = 0.5erfc
e4ïn
(d-n)
[1]
where v is the relative tumor volume irradiated to a maximum dose d, | is a parameter
Received 14 January 2009 Accepted 15 February 2009
Correspondence to: Pavel Stavrev, Department of Medical Physics, Cross Cancer Institute, 11560 University Ave, Edmonton, Alberta, Canada. Phone: + 1 780 989 4334; Fax: +1 780 432-8615; E-mail: pavel.stavrev@gmail.com
corresponding to the mean (target) dose delivered to the tumor, and 9 is a parameter related to the slope of the erfc function. However, no investigations of how well this function describes the clinical tumor DVHs are reported in the literature.
We propose the parallel use of the Fermi statistics function for the description of clinical tumor DVHs:
iDVH: v(d\ju,0) = -
1
1 + exp
d-fi 0
[2]
This function describes the filling up of free energy levels in a Fermi system. The parameters d, ¡J, and 0 have the same meaning as in eq. [1].
To investigate this problem, we gathered 30 clinical gross tumor volume (GTV) DVHs for different treatment sites - lung, head & neck, prostate, etc., that were either
Stavrev P et al. / Analytical tumor DVH representation
133
obtained in the treatment planning process at the Cross Cancer Institute (CCI) or reported in the literature.4-14 They were fit with the erfc [Eq. 1] and Fermi [Eq. 2] functions.
The fit was performed using the %2 criterion for goodness of fit, presuming a log-log-normal distribution for the integral DVHs. Correspondingly, the function to be minimized is:
x2=X
The log-log form of the %2 criterion was used to account for the fact that an integral DVH is defined in the interval [0,1], while the standard %2 criterion deals with normally distributed random variables defined in (—oo,+oo).15 The experimental error is
^experiments which unfortunately is not reported in the literature. Therefore, we substituted ^experimental with a percentage band
- In (- In (Vtheoretical )) + ln(-ln (v;xperimental))
log experimental
-cr.
experimental
log experimental
experimental
^ (^experimental)
Figure 1. Fits to four clinical (head & neck) DVHs from CCI with the erfc function - a) and with the Fermi function - b) for the case of a 2% error band (^^^„j = 2%). On each subplot the p-value of the fit are shown, along with the statistics (number of data points, -/Vp0ints) and the corresponding best fit values of the model parameters (^ mid 6).
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10 -
8 -
<D w co o
? 6-a;
■Q £ =j
■z.
4 -
2 -
0I-----------—
-20 0 20 „ 40 60 80 100 2 2 x erfc " x Fermi
Figure 2. Distribution of the difference of x2 values for the fits obtained with the erfc and the Fermi functions, (xlfC ~xlermi), built on the bases of fits to 30 clinical DVHs.
in which a statistically acceptable fit could be obtained. We initially presumed a 2% error band and found that in 24 out of 30 cases Eq. [2] produced acceptable fits, while Eq. [1] produced acceptable fits in 19 out of 30 cases. We also investigated a 4% error band and found out that Eq. [2] produced statistically acceptable fits in all considered cases, while Eq. [1] produced poor fits in 2 of all cases. As an illustration, fits of four head & neck DVHs obtained at CCI with the erfc function [Eq. 1] and with the Fermi function [Eq. (2] are shown in Figure 1a and Figure 1b, respectively. The fits correspond to the case of ^experimental =2%. As can be seen from the obtained x2 and p-values (shown in each subplot of Figure 1), the Fermi function produces excellent fits in all four cases, while the erfc function produces a poor fit in one case. It can be therefore concluded that the Fermi function describes clinical integral DVHs better than the erfc function. To fur-
ther illustrate this conclusion a distribution of the difference of the x2 values of the fits obtained with the two proposed functions is shown in Figure 2. This distribution is constructed on the bases of the 30 clinical DVHs used in this study. As can be seen from Figure 2, the average of the distribute of [zifc -xlermi) is greater than zer^
showing that in most cases the Fermi function indeed produces better fits to clinical DVHs than the erfc function.
References
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2.	Stavrev P, Schinkel C, Stavreva N, Warkentin B, Fallone B. Population TCP estimators in case of heterogeneous irradiation. Radiother Oncol 2007; 84: S279-S80.
3.	Nahum AE, Sanchez-Nieto B. Tumour control probability modelling: basic principles and applications in treatment planning. Physica Medica 2001; XVII(Suppl 2): 13-23.
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6.	De Gersem WR, Derycke S, De Wagter C, De Neve WC. Optimization of beam weights in conformal radiotherapy planning of stage III non-small cell lung cancer: effects on therapeutic ratio. Int J Radiat Oncol Biol Phys 2000; 47: 255-60.
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8.	Teh BS, Mai WY, Uhl BM, Augspurger ME, Grant WH 3rd, Lu HH, et al. Intensity-modulated radiation therapy (IMRT) for prostate cancer with the use of a rectal balloon for prostate immobilization: acute toxicity and dose-volume analysis. Int J Radiat Oncol Biol Phys 2001; 49: 705-12.'
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10.	Stanescu T, Hans-Sonke J, Stavrev P, Fallone B. 3T MR-based treatment planning for radiotherapy of brain lesions. Radiol Oncol 2006; 40: 125-32.
11.	Bos LJ, Damen EMF, DeBoer RW, Mijnheer BJ, McShan DL, Fraass BA, et al. Reduction of rectal dose by integration of the boost in the large-field treatment plan for prostate irradiation. Int J Radiat Oncol Biol Phys 2002; 52: 254-65.
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14.	14. Olafsson A, Jeraj R, Wright SJ. Optimization of intensity-modulated radiation therapy with biological objectives. Phys Med Biol 2005; 50: 5357-79.
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Radiol Oncol 2009; 43(2): 132-135.
Slovenian abstracts
I
Radiol Oncol 2009; 43(2): 120-125.
Sindrom rakave kaheksije-anoreksije in izguba skeletno mišične mase
Jurdana M
Izhodišča. Sindrom rakave anoreksije-kaheksije je skupen in pomemben zaplet povezan z rakom. Pojavi se pri 30% do 80% vseh rakavih bolnikov. Beseda kaheksija pomeni „slabo stanje" in je lahko prisotna v zgodnji fazi rakavega razvoja, preden se pojavijo znamenja malignosti. Sindrom rakave kaheksije je postopno hujšanje telesa, za katerga je značilna izguba maščobnega tkiva in skletne mišične mase. Pri mnogih rakavih bolnikih je kaheksija povezana z anoreksijo, ki je posledica pomankljivega vnosa hrane. Tovrstno odklanjanje hrane, ki je pri raku povezano z energijsko porabo je ena temeljnih fizioloških motenj, ki vodi v izgubo telesne teže. Izguba teže pri pacientih s sindromom rakave anoreksije-kahe-ksije se razlikuje od izgube, ki je posledica stradanja in anoreksije nervoze. Patofiziologija rakave kaheksije še ni popolnoma pojasnjena, čeprav so študije pokazale pomembno vlogo citokinov v presnovi ogljikovih hidratov, maščob in beljakovin. Rak, dolgotrajno ležanje, HIV infekcija in staranje so označena kot stanja, katerih skupna lastnost je izguba mišične mase. Ukrep, s katerim bi lahko postopno zmanjšali izgubo mišične mase, je vadba proti uporu, definirana kot ponovitev statičnih in dinamičnih mišičnih kontrakcij. Na ta način želimo povečati mišično maso.
Zaključek. Ključni, sestavni deli patološkega stanja kaheksije so anoreksija in metabolne anomalije, kot sta izguba maščobe in katabolizem mišičnih beljakovin. Prihodnje raziskave bodo osredotočene na molekularne anomalije kaheksije ter na preiskovanje funkcijske koristi treninga z vajami proti uporu pri preprečevanju izgube mišične mase.
Radiol Oncol 2009; 43(2): I-IX.
II
Slovenian abstracts
Radiol Oncol 2009; 43(2): 126-131.
Ksantogranulomatozni holecistitis ostaja izziv v medicinski praksi
Izkušnje s 24 bolniki
Yildirim M, Oztekin O, Akdamar F, Yakan S, Postaci H
Izhodišča. Ksantogranulomatozni holecistitis je redka, benigna, kronična vnetna bolezen žolčnika. S slikovnimi preiskavami pa tudi intraoperativno lahko vidimo tumorozno raščo. Raziskava je želela pri bolnikih, kjer smo načrtovali holecistektomijo, oceniti vrednost pre-operativnih priskav in jih primerjati z intraoperativnim stanjem.
Bolniki in metode. Retrospektivno smo analizirali klinične podatke 24 bolnikov s ksantogra-nulomatoznim holecistitisom, ki so se zdravili v obdobju 7 let (srednja starost 53 let (32-68), razmerje med moškimi in ženskami 1:1,4).
Rezultati. Pogosti klinični simptomi so bile bolečine v trebuhu (79%), slabost (62%) in zlatenica (12%). Preoperativna ultrazvočna preiskava je pokazala žolčne kamne (95,8%) in žolčni drobir (8%) ter periholecistično tekočino (20%), polip (4%) in tumor žolčnika (4%). Stena žolčnika je bila zadebeljena (>3mm) pri 10 bolnikih. Računalniška tomografija je pri vseh bolnikih pokazala patološke spremembe. Intraoperativno pa smo našli: žolčne kamne pri vseh bolnikih (100%), kronični holecistitis pri več kot polovici bolnikov (54%), redkeje pa hidropični žolčnik, emfizematozni žolčnik, priraščanje žolčnika na sosednje organe in tumorske mase.
Zaključki. Ksantogranulomatozni holecistitis je težko ugotoviti pre- ali intraoperativno in ostaja izziv v klinični praksi. Končno diagnozo lahko postavimo le s histopatološko priska-vo.
Radiol Oncol 2009; 43(2): I-IX.
Slovenian abstracts
III
Radiol Oncol 2009; 43(2): 120-125.
Subduralni hematom hrbtenice pri von Willebrandovi bolezni
Franko A, Antulov R, Dunatov S, Antončic I, Miletic D
Izhodišča. Von WiIIebrandova bolezen je najpogostejša prirojena motnja strjevanja krvi, ki se najpogosteje manifestira s spontanimi krvavitvami. Bolniki s prirojenimi motnjami strjevanja krvi so neredko okuženi tudi z virusom hepatitisa C. Raziskave so pokazale, da je pri bolnikih, ki so okuženi z virusom hepatitisa C in prejemajo kombinirana zdravila, večja nevarnost nastanka znotrajmožganske krvavitve.
Prikaz primera. 44 letni moški je bil pripeljan v urgentni center z bolečinami v spodnjem predelu hrbta, glavobolom, bolečinami v vratu in z znaki spastične parapareze. Magnetnoresonančna preiskava prsnega dela hrbtenice je pokazala subduralni hematom v višini T8 do T 11.
Zaključki. Kolikor nam je znano, je to prvi opisani prikaz subduralnega hematoma v prsnem predeIu hrbtenice pri boIniku z von WiIIebradovo boIeznijo in s kronično okužbo z virusom hepatitisa C. Bolnik je prejemal kombinirano zdravljenje, ki je lahko dodatni dejavnik tveganja za nastanek krvavitve.
Radiol Oncol 2009; 43(2): I-IX.
IV
Slovenian abstracts
Radiol Oncol 2009; 43(2): 126-131.
Porast kronične poškodbe sečnika pri pooperativnem obsevanju karcinomov prostate z vključitvijo področnih bezgavk
Kragelj B
Izhodišča. Z obsevanjem ležišča prostate (PBRT) lahko pri bolnikih s karcinomom prostate, ki so zdravljeni s prostatektomijo, preprečimo ponovitev bolezni ali pa ob ponovitvi ponovno vzpostavimo remisijo bolezni. Uspešnost obsevanja lahko izboljšamo z dodatnim obsevanjem področnih bezgavk (WPRT).
Metode. Namen raziskave je bilo oceniti pozne posledice pooperativnega obsevanja pri 43 bolnikih (21 z dodatnim WPRT). Opazovali smo težave pri odvajanju urina in blata. Stopnjo zapleta smo ocenjevali s prilagojeno RTOG/LENT lestvico. Ležišče prostate je bilo obsevano z mediano dozo 64,8 (59,4-70,0) Gy in področne bezgavke z mediano dozo 50,4 (48,0-56,0) Gy. Rezultati. Pozne posledice zdravljenja so bile predvsem okvare sečnika (poslabšanje delovanja za 1 stopnjo pri 33% in >2 stopnji pri 26% bolnikov) in manj črevesne okvare (poslabšanje delovanja za 1 stopnjo pri 54% in >2 stopnji pri 5% bolnikov). Okvare sečnika so bile pogostejše pri WPRT kot pri PBRT (67% in 50% bolnikov), zlasti, če je bila doza za WPRT >52 Gy (71% bolnikov).
Zaključki. Kljub temu, da so okvare sečnika po prostatektomiji in pooperativnem obsevanju verjetno posledica več dejavnikov, lahko z zmanjšanjem obsevanosti sečnika, kot ga omogočajo sodobne tehnike, pričakujemo zmanjšanje poznih posledic zdravljenja.
Radiol Oncol 2009; 43(2): I-IX.
Slovenian abstracts
V
Radiol Oncol 2009; 43(2): 120-125.
Primerjava standardnega in modificiranega testa komet pri ugotavljanju poškodb DNA na humanih limfocitih po izpostavitvi ionizirajočemu sevanju
Mikloš M, Gajski G, Garaj-Vrhovac V
Izhodišča. Človeški organizem je zelo občutljiv na ionizirajoče sevanje, ki ima močno geno-toksično delovanje na molekulo DNA. Namen raziskave je bil ugotoviti vrsto DNA poškodb in način umiranja celic po obsevanju z ionizirajočimi žarki ter primerjati senzitivnost standardiziranega in modificiranega testa komet.
Metode. Učinki sevanja gama (0,1 Gy in 4 Gy) so bili proučevani na humanih limfocitih s standardnim in alkalnim Fpg- modificiranim testom komet, kot tudi s testom DNA difuzije. Rezultati. Parametri standardnega testa komet so pokazali značilno višje vrednosti pri vzorcih, ki so bili obsevani s 4 Gy, kot pri vzorcih, ki so bili obsevani z 0,1 Gy in kontrolnih vzorcih. Modificirani Fpg test komet je pokazal značilno višje vrednosti že pri nižji dozi obsevanja 0,1 Gy, zaradi oksidativne poškodbe DNA pri obsevanju in večje sensitivnosti testa. Test DNA difuzije je pokazal, da sevanje gama vodi celice v apoptozo bolj kot v nekrozo. Zaključki. Raziskava nakazuje, da sta oba testa komet, standardni in modificirani, kot tudi test DNA difuzije, zanesljivi za oceno poškodb DNA in vrsto celične smrti povzročene z ionizirajočim sevanjem in vitro. Poleg tega je modificirani Fpg test komet bolj občutljiv za zaznavo poškodb DNA kot standardni test komet.
Radiol Oncol 2009; 43(2): I-IX.
VI
Slovenian abstracts
Radiol Oncol 2009; 43(2): 126-131.
Vpliv hipotoničnega pufra in elektroporacije na dinamiko nabrekanja in viabilnost celic za optimizacijo elektrofuzije
Ušaj M, Trontelj K, Hudej R, Kandušer M, Miklavčič D
Uvod. Na elektrofuzijo vplivajo različni parametri. Do sedaj objavljeni rezultati so pokazali, da hipotonični fuzijski pufer pripomore k večji učinkovitosti elektrofuzije. Takšna optimizacija elektrofuzije vključuje določitev primerne osmolarnosti pufra in inkubacijskega časa, pri katerem nabrekla celica še ne vzpostavi mehanizmov regulacije volumna. Osmolarnost pufra in inkubacijski čas morata biti določena za vsako celično linijo posebej, ne da bi vplivala na njihovo preživetje. V naši raziskavi smo določili dinamiko nabrekanja celic in njihovo viabilnosti v hipotoničnem pufru za štiri celične linije. Za izbrano celično linijo smo proučili tudi vpliv elektroporacije v hipotoničnem pufru.
Materiali in metode. Dinamiko nabrekanja celičnih linij CHO, V79, B16-F1 in NS1 smo določili z meritvami velikosti celic v 30 minutnem časovnem intervalu po začetku inkubacije v hipotoničnem pufru. Viabilnost celic po inkubaciji v hipotoničnem pufru smo določili 24 ur po poskusu z modificirano kristal vijolično metodo za določanje viabilnosti. Rezultati. Pri naših eksperimentalnih pogojih je bila inkubacija v 100 mOsm pufru učinkovita za nabrekanje celičnih linij CHO, V79 in B16-F1. Najustreznejši inkubacijski čas celic v hipotoničnem pufru je bil od 2 do 5 minut. Enaki eksperimentalni pogoji pa niso povzročili želenega nabrekanja celic NS1. Za razliko od celic elektroporiranih v izotoničnem pufru, smo pri celicah elektroporiranih v hipotoničnem pufru opazili povečano nabrekanje, pri čemer pa po elektroporaciji celic v hipotoničnem pufru nismo opazili zmanjšanja volumna i.e. regulacije volumna.
Zaključek. Ker se različne celične linije razlikujejo po svoji občutljivosti na inkubacijo v hi-potoničnih pogojih, moramo dinamiko nabrekanja v hipotoničnem pufru določiti za vsako celično linijo posebej. Povečan volumen celic v hipotoničnem pufru, ki je zaradi elektroporacije dolgotrajnejši, lahko prispeva k izboljšanju učinkovitosti elektrofuzije. Viabilnost celic se po inkubaciji v hipotoničnem pufru ni zmanjšala. Pri izbiri parametrov elektroporacije v hipotoničnem pufru moramo upoštevati tudi povečanje celic.
Radiol Oncol 2009; 43(2): I-IX.
Slovenian abstracts
VII
Radiol Oncol 2009; 43(2): 120-125.
Nekonvulzivna posteriorna reverzibilna encefalopatija povzročena s cisplatinom pri 41-letni bolnici z metastatskim
malignim melanomom
Ocvirk J, Boc M, Reberšek M, Roš T
Izhodišča. Cisplatin, široko uporabljen cistostatik, v veliki večini povzroča periferno nevro-patijo, redko pa lahko povzroči zaplete v obliki encefalopatije ali brez krčev. Prikaz primera. Predstavljamo primer mlade bolnice z metastatskim malignim melanomom, z znaki in simptomi nekonvulzivne posteriorne reverzibilne encefalopatije povzročene s cisplatinom. Pri bolnici je v nekaj dneh po prvem krogu kemoterapije na osnovi cisplatina prišlo do slabosti, bruhanja, glavobola, hudih bolečin na mestu podkožnih metastaz in zmedenosti. Simptomi in znaki so se kasneje stopnjevali do somnolence, kortikalne slepote in afazije, brez epileptičnih krčev.
Zaključki. Cisplatin je učinkovit citostatik, vendar tudi zelo toksičen. Zdravniki, ki ga uporabljajo, morajo biti pozorni tudi na možnost encefalopatije.
Radiol Oncol 2009; 43(2): I-IX.
VIII
Slovenian abstracts
Radiol Oncol 2009; 43(2): 126-131.
Angiosarkom jeter po kombiniranem zdravljenju raka žolčnika
Botros M, Quevedo JF, Miller RC
Izhodišča. Angiosarkom, ki je nastal zaradi zdravljenja raka, je redek zaplet obravnave bolnika. Najbolj pogosto je opisan v prsni steni po mastektomiji. Poročamo o redkem primeru z zdravljenjem povzročenem angiosarkomu, ki je nastal v jetrih po pooperativni kemoradi-oterapiji raka žolčnika.
Prikaz primera. Angiosarkom jeter se je razvil pri 46-Ietni bolnici, ki je bila leta 1991 operirana zaradi žleznega raka žolčnika, stadij IIB (T3N1M0). Dodatno smo jo zdravili z obsevanjem in nato s kombinirano kemoterapijo. Po operaciji so se razviIi pozni zapIeti z zaporo dvanajsternika in fistulo ter zaradi zapore zgornje mezenterične arterije še kronična mezenterična ishemija. Šest let po operaciji in dodatnem zdravljenju smo odkrili lokalno napredovali angiosarkom jeter, gradus 4, ki se je razvil v področju obsevalnega polja. Zaključki. Po našem vedenju je to prvi opisani primer angiosarkoma jeter, ki je bil povzročen s pooperativnim obsevanjem zaradi raka žoIčnika. Pri obsevanju zgornjega deIa trebuha moramo tako zeIo skrbno načrtovati dozno-voIumske omejitve zdravih organov.
Radiol Oncol 2009; 43(2): I-IX.
Slovenian abstracts
IX
Radiol Oncol 2009; 43(2): 120-125.
Kako dobro lahko DVH makroskopskega tumorskega volumna opišemo z analitično funkcijo?
Stavrev P, Schinkel C, Stavreva N, Fallone BG
Dozno heterogenost v tumorju pogosto opišemo z normalno porazdelitvijo. Kot drugo možnost za opis dozne heterogenosti predlagamo uporabo Fermijeve funkcije, ki opisuje Fermijevo statistiko. Da bi prikazali primernost predlagane funkcije, obe funkciji prilagodimo 30 dozno volumskim histogramom (DVH) makroskopskih tumorskih volumnov (GTV) in ju med seboj primerjamo.
Radiol Oncol 2009; 43(2): I-IX.
X
Notices
Notices submitted for publication should contain a mailing address, phone and/or fax number and/or e-mail of a Contact person or department.
Oncology
July 2-5, 2009
The Educational Cancer Convention (ECCLU) will be held in collaboration with European Society for Medical Oncology in Lugano, Switzerland, E-mail www.cmelcher@eso.net; or see www.eso.net
Lung cancer
July 31 - August 4, 2009
The "13th World Conference on Lung Cancer" will be offered in San Francisco, USA.
Contact Conference Secretariat International Conference Services Ltd., Suite 2101 - 1177 West Hastings Street, Vancouver, BC Canada V6E 2K3; or call +1 604 681 2153; or e-mail wclc2009@meet-ics. com; or see http://www.2009worldlungcancer.org/
Radiotherapy
August 30 - September 3, 2009
The 19th Biennial ESTRO Conference" will be held in Maastricht, the Netherlands,
Phone +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http://www/estro-events.org
Oncology
September 4-8, 2009
The "34th ESMO Congress" will take place in Vienna, Austria.
Contact ESMO Head Office, Congress Department, Via La Santa 7, CH-6962 Viganello-Lugano, Switzerland; or +41 (0)91 973 19 19; or fax +41 (0)91 973 19 18; or e-mail congress@esmo.org; or see http:// www.esmo.org
Medical physics
September 7-12, 2009
The "World Congress 2009 - Medical Physics and Biomedical Engineering" will take place in Munich, Germany.
See http://www. wc2009.org/world-congress-2009
Brachytherapy
September 10-12, 2009
The ESTRO teaching course ''3D Image-Based Brachytherapy for Gynaecological Malignancies'' will be offered in Amsterdam, The Netherlands. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org
Radiation oncology
September 13-16, 2009
The "8th International Conference on Dose, Time and Fractionation in Radiation Oncology" will be held in Madison, Wisconsin, USA,
Contact ASTRO, 8280 Willow Oaks Corporate Dr., Suite 500, Fairfax, VA 22031; or call +1 703 502-1550; see http://www/astro.org
Oncology
September 20-24, 2009
The "15th ECCO and 34th ESMO Multidisciplinary Congress" will be offered in Berlin, Germany. See http://www.ecco-org.eu
Radiol Oncol 2009; 43(2): X-XII.
Notices
XI
Nuclear medicine
October 10-14, 2009
The "EANM'09 Annual Congress of the European Association of Nuclear Medicine" will take place in Barcelona, Spain.
Contact EANM Executive Secretariat and call +43 1 212 80 30; or fax +43 1 212 80 309; or e-mail office@ eanm.org; or see http://www. eanm.org
Radiation oncology
October 11-16, 2009
The ESTRO teaching course Evidence Based Radiation Oncology: Methodological Basis and Clinical Application '' will be offered in Vienna, Austria. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org
Lung Cancer
October 15-17, 2009
The ESTRO multidisciplinary teaching course on lung cancer will be held in Prague, Czech Republic. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org or http://www.estro-education. org/courses/Pages/Prague2009.aspx
Radiobiology
October 18-23, 2009
The ESTRO teaching course on basic clinical radiobio-logy will be offered in Toledo, Spain. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org
Therapeutic radiology and oncology
November 1-5, 2009
The "American Society for Therapeutic Radiology and Oncology Annual Meeting ASTRO" will take place in Chicago, USA.
Contact ASTRO, 8280 Willow Oaks Corporate Dr., Suite 500, Fairfax, VA 22031; or call +1 703 502-1550; or see http://www.astro.org
Radiotherapy
November 15-19, 2009
The ESTRO teaching course on IMRT and other conformal techniques in practice will take place in Gliwice, Poland.
Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org
PET in radiation oncology
November 21-22, 2009
The ESTRO / EANM educational seminar on PET in radiation oncology will take place in Vienna, Austria. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org
PET in radiation oncology
December 13-17, 2009
The ESTRO teaching course on image-guided radiotherapy in clinical practice will take place in Brussels, Belgium.
Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2775 93 40; or fax 32 2779 5494; or e-mail events@estro.org; or see http:// www/estro-events.org
Head and neck cancer
February 25-27, 2010
The multidisciplinary symposium on head and neck cancer will be offered in Chandler, Arizona, USA. Contact ASTRO, 8280 Willow Oaks Corporate Dr., Suite 500, Fairfax, VA 22031; or call +1 703 502-1550; see http://www/astro.org
Clinical oncology
June 4-8, 2010
The American Society of Clinical Oncology Conference (ASCO 2010) will be offered in Chicago, USA. E mail enews@asco.org; or see http://www/asco.org
Radiol Oncol 2009; 43(2): X-XII.
XII
Notices
Oncology
October 8-12, 2010
The "35th ESMO Congress" will take place in Milan, Italy.
Contact ESMO Head Office, Congress Department, Via La Santa 7, CH-6962 Viganello-Lugano, Switzerland; or call +41 (0)91 973 19 19; or fax +41 (0)91 973 19 18; or e-mail congress@esmo.org; or see http://www.esmo.org
Nuclear medicine
October 15-19, 2011
The "EANM'11 Annual Congress of the European Association of Nuclear Medicine" will take place in Birmingham, United Kingdom.
Contact EANM Executive Secretariat and call +43 1 212 80 30; or fax +43 1 212 80 309; or e-mail office@ eanm.org; or see http://www. eanm.org
Nuclear medicine
October 9-13, 2010
The "EANM'10 Annual Congress of the European Association of Nuclear Medicine" will take place in Vienna, Austria.
Contact EANM Executive Secretariat and call +43 1 212 80 30; or fax +43 1 212 80 309; or e-mail office@ eanm.org; or see http://www. eanm.org
Oncology
September 27 - October 1, 2013
The "17th ECCO and 38th ESMO Multidisciplinary Congress" will be offered in Amsterdam, The Netherlands,
See http://www.ecco-org.eu
Lung cancer
Therapeutic radiology and oncology
October 31 - November 4, 2010
The "American Society for Therapeutic Radiology and Oncology Annual Meeting ASTRO" will take place in San Diego, California, USA.
Contact ASTRO, 8280 Willow Oaks Corporate Dr., Suite 500, Fairfax, VA 22031; or call +1 703 502-1550; or see http://www.astro.org
2013
The "15th World Conference on Lung Cancer" will be offered in Sydney, Australia. See http://www.iaslc.org
Clinical oncology
June 3-7, 2011
The American Society of Clinical Oncology Conference (ASCO 2010) will be offered in Chicago, USA. E mail enews@asco.org; or see http://www/asco.org
Lung cancer
July 3-7, 2011
The "14th World Conference on Lung Cancer" will be offered in Amsterdam, The Netherlands. See http://www.iaslc.org
Oncology
September 23-27, 2011
The "16th ECCO and 36th ESMO Multidisciplinary Congress" will be offered in Stockholm, Sweden. See http://www.ecco-org.eu
As a service to our readers, notices of meetings or courses will be inserted free of charge.
Please send information to the Editorial office, Radiology and Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia.
Radiol Oncol 2009; 43(2): X-XII.
Fundacija dr. J. cholewa
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
ŽR: 02033-0017879431
Fundacija dr. J. cholewa
Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education - a report for the second quarter of 2009
Dr. J. Cholewa Foundation for Cancer Research and Education is of opinion that excellent and unorthodox ideas in cancer research and education must not be prevented to succeed for the simple lack of funding. The Foundation thus supports cancer research and education activities in Slovenia and continues to assess the requests for research grants and scholarships submitted by Slovenian experts in oncology and other associated scientific activities. As much as possible, the Foundation helps putting resulting advances in cancer therapy in practice. One of the goals of the Foundation is to transmit the latest diagnostic and therapy methods and knowledge to everyday research and clinical environment in Slovenia. This activity is directly beneficial for the increasing number of patients with various types of cancer in Slovenia, since the incidence rates of many types of cancer, like breast and prostate carcinoma, have kept rising in this country in recent decades. The "Dr. L. Cholewa Foundation for Cancer Research and Education« continues to support the regular publication of "Radiology and Oncology" international medical scientific journal, that is edited, published and printed in Ljubljana, Slovenia. This support emphasizes the need for the spread of information about advances in experimental and clinical oncology to professionals and interested individuals in public in Slovenia and elsewhere. "Radiology and Oncology" is an open access journal, available free of charge on its own website, thus allowing its users and readers to access it free of expense. In this way the Foundation promotes cancer education in general and among scientists with a particular interest in cancer research. The Foundation also supports the publication of results of cancer research in Slovenia and from Slovenian authors elsewhere in international scientific journals and other means of communication worldwide. It is important to know that results of cancer research, supported by the Foundation, have in many cases found its way to the practical application in hospital wards across Slovenia in a significantly easier manner in recent years than before. Careful assessment of requests and proposals for research grants and scholarships submitted by experts in oncology and other scientists is an essential part of the Foundation activities in spreading advanced knowledge of therapy and education in cancer.
Andrej Plesničar, MD
Tomaž Benulič, MD
Borut Štabuc, MD, PhD
SIEMENS
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Bežigrajski dvor ftirlčeva 29, Ljubljana Tel.: (0)1 436 49 01 Fax: (0)1 436 49 05
info<S)labormed.si
www.labormed.sl
Erbitux 5 mg/ml raztopina za infundiranje (skrajšana navodila za uporabo)
Cetuksimab je monoklonsko IgG1 protitelo, usmerjeno proti receptorju za epidermalni rastni faktor (EGFR). Terapevtske indikacije: Zdravilo Erbitux je indicirano za zdravljenje bolnikov z metastatskim kolorektalnim rakom in nemutiranim tipom KRAS; v kombinaciji s kemoterapijo in kot samostojno zdravilo pri bolnikih, pri katerih zdravljenje z oksaliplatinom in irinotekanom ni bilo uspešno. Zdravilo Erbitux je v kombinaciji z radioterapijo indicirano za zdravljenje bolnikov z lokalno napredovalim rakom skvamoznih celic glave in vratu. Odmerjanje in način uporabe: Zdravilo Erbitux pri vseh indikacijah infundirajte enkrat na teden. Začetni odmerek je 400 mg cetuksimaba na m2 telesne površine. Vsi naslednji tedenski odmerki so vsak po 250 mg/m2. Kontraindikacije: Zdravilo Erbitux je kontraindicirano pri bolnikih z znano hudo preobčutljivostno reakcijo (3. ali 4. stopnje) na cetuksimab. Posebna opozorila in previdnostni ukrepi: Če pri bolniku nastopi blaga ali zmerna reakcija, povezana z infundiranjem, lahko zmanjšate hitrost infundiranja. Priporočljivo je, da ostane hitrost infundiranja na nižji vrednosti tudi pri vseh naslednjih infuzijah. Če se pri bolniku pojavi huda kožna reakcija (> 3. stopnje po kriterijih US National Cancer Institute, Common Toxicity Criteria; NCI-CTC), morate prekiniti terapijo s cetuksimabom. Z zdravljenjem smete nadaljevati le, če se je reakcija pomirila do 2. stopnje. Priporoča se določanje koncentracije elektrolitov v serumu pred zdravljenjem in periodično med zdravljenjem s cetuksimabom. Po potrebi se priporoča nadomeščanje elektrolitov. Posebna previdnost je potrebna pri oslabljenih bolnikih in pri tistih z obstoječo srčno-pljučno boleznijo. Neželeni učinki: Zelo pogosti (> 1/10): dispneja, blago do zmerno povečanje jetrnih encimov, kožne reakcije, blage ali zmerne reakcije povezane z infundiranjem, blag do zmeren mukozitis. Pogosti (> 1/100, < 1/10): konjunktivitis, hude reakcije povezane z infundiranjem. Pogostost ni znana: Opazili so progresivno zniževanje nivoja magnezija v serumu, ki pri nekaterih bolnikih povzroča hudo hipomagneziemijo. Glede na resnost so opazili tudi druge elektrolitske motnje, večinoma hipokalciemijo ali hipokaliemijo. Posebna navodila za shranjevanje: Shranjujte v hladilniku (2 °C - 8 °C). Ne zamrzujte. Vrsta ovojnine in vsebina: 1 viala po 20 ml ali 100 ml. Imetnik dovoljenja za promet: Merck KGaA, 64271 Darmstadt, Nemčija. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila (EMEA) http://www.emea.europa.eu.
Dodatne informacije so vam na voljo pri: Merck d.o.o., Dunajska cesta 119, 1000 Ljubljana, tel.: 01 560 3810, faks: 01 560 3831, el. pošta: ¡nfo@merck.si www.oncology.merck.de
.MERCK
SERONO
Skrajšan povzetek glavnih značilnosti zdravila Arimidex® 1 mg filmsko obložene tablete
Sestava zdravila: Ena tableta vsebuje 1 mganastrozola.
Indikacije: Adjuvantno zdravljenje žensk po menopavzi, ki imajo zgodnji invazivni rak dojke S pozitivnimi estrogenskimi receptorji. Adjuvantno zdravljenje zgodnjega raka dojke s pozitivnimi estrogenskimi receptorji pri ženskah po menopavzi, ki so se dve do tri leta adjuvantno zdravile s tamoksifenom. Zdravljenje napredovalega raka dojke pri ženskah po menopavzi. Učinkovitost pri bolnicah z negativnimi estrogenskimi receptorji ni bila dokazana razen pri tistih, ki so imele predhodno pozitiven klinični odgovor na tamoksifen.
j merjenje In način uporabe: Odrasle (tudi starejše) bolnice: l tableta po 1 mg peroraino, enkrat na dan. Odmerka zdravila ni treba prilagajati pri bolnicah z blago ali zmerno ledvično odpovedjo ali blagi m jetrnim odpovedovanjem. Pri zgodnjem raku je priporočljivotrajanjezdravljenja5let. Glavni neželeni učlnld: Zelo pogosti (> 10 %): navali vročine, običajno blagi do zmerni. Pogosti (ä 1 % In < 10 %): astenlja, bolečine/okorelost v sklepi h, suhost vagine, razredčenje las, Izpuščaji, slabost, d ia reja, glavobol (vsi običajno blagi d o zmerni)
Posebna opozorila In previdnostni ukrepi: Uporabe Arimidexa ne priporočamo pri otrocih, ker njegova varnost in učinkovitost pri njih še nista raziskani. Menopavzo je potrebno biokemično določiti pri vseh bolnicah, kjer obstaja dvom o hormonskem statusu. Ni podatkov o varni uporabi Arimidexa pri bolnicah z zmerno ali hudo jetrno okvaro ali hujšo ledvično odpovedjo (očistek kreatinina manj kakor 20 ml/min (oziroma 0,33 ml/s)). Pri ženskah z osteoporozoali pri ženskah s povečanim tveganjem za razvoj osteoporoze je treba določiti njihovo mineralno gostoto kosti z denzitometrijo, na pri mers slikanjem DEXA na začetku zdravljenja, pozneje pa v rednih intervalih. Po potrebi Je treba začeti z zdravljenjem ali preprečevanjem osteoporoze In to skrbno nadzorovati. NI podatkov o uporabi anastrozola z analogi LH RH. Arimidex znižuje nivo estrogena v obtoku, zato lahko povzroči zmanjšanje mineralne kostne gostote. Trenutno ni na voljo ustreznih podatkov o učinku bifosfonatov na izgubo mineralne kostne gostote, povzročene z anastrozolom, ali njihovi koristi, če se uporabijo preventivno. Zdravilo vsebuje laktozo.
Kontraindikacije: Arimidex je kontraindiciran pri: ženskah pred menopavzo, nosečnicah in doječih materah, bolnicah s hujšo ledvično odpovedjo (očistek kreatinina manj kot20 ml/min (oziroma 0,33 ml/s)), bolnicah z zmernim do hud i m jetrnim obolenjem, bolnicah, ki imajo znano preobčutljivost za anastrozol ali za katerokoli pomožno snov. Zdravila, ki vsebujejo estrogen, ne smete dajati sočasno z Arimidexom, ker bi se njegovo farmakološko delovanje izničilo. Sočasno zdravljenje s tamoksifenom.
Medsebojno delovanje z drugimi zdravili In druge oblike Interakcij: Klinične raziskave o interakcijah z antipirinom in cimetidinom kažejo, da pri sočasni uporabi Arimidexa in drugih zdravil klinično pomembne interakcije, posredovane s citokromom P450, niso verjetne. Pregled baze podatkov o varnosti v ki i nični h preskušanjih pri bolnicah, ki so se zdravile z Arimidexom in sočasno jemale druga pogosto predpisana zdravila, ni pokazal klinično pomembnih interakcij. Imetnik dovo||enJa za promet: AstraZeneca UK Limited, 15 Stanhope Gate, London, WIK 1LN, Velika Britanija
Režim predpisovanja zdravila: Rp/Spec Datum priprave Informacije: april 2007
Pred predpisovanjem, prosimo, preberite celoten povzetek glavni h značilnosti zdravila.
Dodatne informacije in literatura so na voljo pri AstraZeneca U K Limited Podružnica v Sloveniji Verovškova ulica 55 1000 Ljubljana
o- in na spletnih straneh: 6 www.arimidex.net
g www.bco.org
tč www.breastcancersource.com
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Oiij'DLO^iJA
adjuvant [ae 'cCžuv *nt]
1. adjective pomagCjiv, pristen; ~ treatttient ZUitfl
adjuvantno zdravljenje žensf^po menopavzi, ki imajo zgodnji invazivni rak^dojke s pozitivnimi estrogenskimi receptoiji.
advanced[*cCm:nst]
1. adjective napreden; zvišan (cene); to 6e ~ napredovati; ~ inyears visoke starosti; treatment of ~ frreast cancer Zlri-th Arimi(k7(: Zdravljenje napredovalega rakg. dojke pri ženskah po menopavzi. Ilčinkgvitost pri Bolnicah z negativnimi estrogenskimi receptoiji ni 6iCa dokazana razen pri tistih, ki so imele predhodno pozitiven klinični odgovor na tamoksifen.
suntch [sziHc]
1. transitive verB udariti, Bičati s šiSo (z repom); šibati z, hitro mahati z; naglo pograbiti; raihvay ranžirati, zapeljati (usmeriti) (vlak) na drug tir; electricaC vključiti, vklopiti; spremeniti (pogovor), oSrniti drugam (tokjnisU); to ~ 6ack_ to figurutivehj (v mislih) vrniti se na; ~ tO ftritl Adjuvantno zdravljenje zgodnjega rakg. dojki s pozitivnimi estrogenskimi receptoiji pri ženskah po menopavzi, ki so se dve do tri leta adjuvantno zdravite s tamoksifenom.
SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Gemcitabin Lek 200 mg in 1 g prašek za raztopino za infundiranje
Sestava: Vsaka viala vsebuje 200 mg ali 1000 mg gemcitabina (v obliki gemcitabinijevega klorida). Vsak ml zdravila vsebuje 40 mg gemcitabina po redčenju na 5 ml (Gemcitabine Lek 200 mg) ali 40 mg gemcitabina po redčenju na 25 ml (Gemcitabiln Lek 1 g). Indikacije: Zdravilo prve izbire za zdravljenje bolnikov z lokalno napredovalim ali metastazirajočim nedrobnoceličnim pljučnim rakom. Za zdravljenje lokalno napredovalega ali metastazirajočegaadenokarcinoma trebušne slinavke. Za zdravljenje lokalno napredovalega ali metastazirajočega raka na mehurju v kombinaciji z drugimi citostatičnimi zdravili. V kombinaciji s paklitakselom za zdravljenje bolnikovz neoperabilnim, lokalno ponavljajočim se ali metastazirajočim rakom na dojki, pri katerih seje bolezen ponovno pojavila po adjuvantni/neoadjuvantni kemoterapiji, kije moralavključevati antraciklin. razen če ni bil klinično kontraindiciran. Odmerjanje in način uporabe: Zdravljenje mora začeti zdravnik, ki ima precej izkušenj z zdravljenjem s citotoksičnimi zdravili. Nedrobnocelični pljučni rak pri odraslih: Kombinirana uporaba: Pri tritedenskem načrtu gemcitabin v odmerku 1250 mg/m2 v 30-minutni intravenski infuziji pivi in osmi dan vsakega 21-dnevnega ciklusa. Pri štiritedenskem načrtu gemcitabin v odmerku 1000 mg/m2 v 30-minutni intravenski infuziji prvi, osmi in petnajsti dan vsakega 28-dnevnega ciklusa. Cisplatin v odmerkih med 75 in 100 mg/m2, enkrat na vsake tri ali štiri tedne. Uporaba enega samega zdravila: Priporočeni odmerek gemcitabina znaša 1000 mg/m2 v 30-minutni intravenski infuziji, ponavljajoče enkrat tedensko v obdobju treh tednov, čemur sledi en teden premora. Štiritedenski ciklus se nato ponovi. Rak trebušne slinavke: Priporočeni odmerek znaša 1000 mg/ta2 v 30-minutni intravenski infuziji, ponavljajoče enkrat tedensko v obdobju do sedem tednov, čemur sledi en teden premora. Naslednji ciklusi morajo biti sestavljeni iz injiciranj enkrat tedensko v obdobju treh zaporednih tednov izmed vsakih štirih tednov. Rak na mehurju: Priporočeni odmerek znaša 1000 mg/m2 v 30-minutni infuziji. Odmerek je treba dati prvi, osmi in petnajsti dan vsakega 28-dnevnega ciklusa v kombinaciji s cisplatinom. Cisplatin se daje v priporočenem odmerku 70 mg/m2 prvi dan po dajanju gemcitabina oziroma drugi dan vsakega 28-dnevnega ciklusa. Ta štiritedenski ciklus se zatem ponovi. Rak na dojki: Priporočljiva je uporaba gemcitabina v kombinaciji s paklitakselom, pil čemer se paklitaksel (v odmerku 175 mg/ta2) uporabi pivi dan v tri ure trajajoči intravenski infuziji, čemur sledi gemcitabin (v odmerku 1250 mg/m2) v 30 do 60 minut trajajoči intravenski infuziji prvi in osmi dan vsakega 21-dnevnega ciklusa. Pred začetkom dajanja kombinacije gemcitabin + paklitaksel mora pri bolnikih absolutno število granulocitovznašati najmanj 1,5 (* 109A). Preverjanja: Pri bolnikih, ki prejemajo gemcitabin, je treba pred dajanjem vsakega odmefta preverjati število trombocitov, levkocitov in granulocitov. Če je potrebno, se odmerek gemcitabina ob prisotnosti hematološke toksičnosti lahko zmanjša ali se ga preneha uporabljati. Treba je izvajati redne klinične preglede in preverjati delovanje jeter in ledvic, da bi lahko zaznali nehematološke škodljive vplive.
Kontraindikacije: Preobčutljivost za gemcitabin ali katerokoli pomožno snov. Uporaba med dojenjem pri ženskah, ki otroke dojijo. Sočasna uporaba s cepivom proti mmeni mrzlici. Kombinacija gemcitabina s cisplatinom pil bolnikih s hudo ledvično okvaro.
Posebna opozorila in previdnostni ukrepi: Gemcitabin lahko kratkotrajno zavre delovanje kostnega mozga, kar se kaže v levkopeniji. trombocitopeniji in anemiji.
Gemcitabin je treba uporabljati previdno pri bolnikih z okvarjeno ledvično funkcijo. Z uporabo gemcitabina je treba prenehati ob pivem pojavu kakršnihkoli znakov mikroangiopatske hemolitične anemije, kot je na primer hitro padajoča raven hemoglobina s spremljajočo trombocitopenijo, povečanje koncentracije bilirubina in kreatinina v serumu, povečanje ravni sečninskega dušika v krvi ali LDL, kar lahko nakazuje razvoj hemolitičnega uremičnega sindroma. Odpoved ledvic je lahko tudi po prenehanju zdravljenja ireverzibilna in lahko je potrebna dializa. Ne glede na to, ali zdravilo uporablja moški ali ženska, je treba med zdravljenjem upoštevati ukrepe za preprečevanje nosečnosti. Medsebojno delovanje z zdravili in druge oblike interakcij: Sočasno zdravljenje z obsevanjem (ki se izvaja obenem ali s časovnim presledkom < 7 dni). Zaporedno zdravljenje z obsevanjem (ki se izvaja s časovnim presledkom > 7 dni). Gemcitabin deluje radiosenzitizirajoče. Zaradi povečanega tveganja za trombozo pri bolnikih z rakom je uporaba antikoagulacijskega zdravljenja pogosta. Velika razlika v koagulacijskem statusu med posamezniki v času bolezni in možnost medsebojnega delovanja oralnih antikoagulantov in kemoterapije zahteva bolj pogosto spremljanje INR-ja v primeru uporabe antikoagulantov. Konttaindicirana sočasna uporaba: cepivo proti rumeni mrzlici. Nepriporočljiva sočasna uporaba: živa, oslabljena cepiva (razen rumene mizlice). Sočasna uporaba, ki zahteva premislek: ciklosporin, takrolimus.
Vpliv na sposobnost vožnje in upravljanja s stroji: Gemcitabin lahko povzroči blago do zmerno zaspanost. Bolnike je zato treba posvariti pred vožnjo ali upravljanjem s stroji, dokler se ne izkaže, da zdravilo nanje nima omenjenega vpliva.
Neželeni učinki: Na pogostnost in hudost neželenih učinkov vplivajo odmerek, hitrost infundiranja in časovni presledki med odmerki. Zelo pogosti (> 1/10): anemija, levkopenija, trombocitopenija. nevtro-penija. dispneja. navzea, bruhanje, povečane vrednosti jetrnih transaminaz (AST in ALT) in alkalne fosfataze, alergijski kožni izpuščaj, ki ga pogosto spremlja srbenje; plešavost - običajno blaga, he-maturija. proteinurija. edemi/periferni edemi, gripi podobni simptomi (povečana telesna temperatura, glavobol, bolečine v hrbtu, drgetanje, bolečine v mišicah, astenija, pomanjkanje teka, kašelj, rinitis, občutek slabosti, znojenje, motnje spanja). Pogosti (> 1/100 do < 1/10): febrilna nevtropenija. zaspanost, Stomatitis in razjede v ustih, driska, zaprtje, povečana koncentracija bilirubina, povečana telesna temperatura, astenija. Neželeni učinki, zaradi katerih je treba odmerek omejiti, so zmanjšanja števila trombocitov, levkocitov in granulocitov. Oprema: Škatla z eno vialo s praškom. Način izdaje zdravila: H
Imetnik dovoljenja za promet: Lekfaimacevtska družba d.d.,Verovškova 57,1526 Ljubljana, Slovenija, nformacija pripravljena: julij 2008
lek
član skupine Sandoz
Lek farmacevtska družba d. d., Verovškova 57,1526 Ljubljana, Slovenija • www.lek.si
Temodal 20 mg, 100 mg, 140mg, 180 mg, 250 mg.
Sestava zdravila: Vsaka kapsula zdravila Temodal vsebuje 20 mg, 100 mg, 140 mg, 180 mg aH 250 mg temozolomida. Terapevtske indikacije Temodal kapsule so indicirane za zdravljenje bolnikov z:
-	za zdravljenje novo diagnosticiranega glioblastoma multiforme, sočasno z radioterapijo in kasneje kot monoterapija
-	malignim gliomom, na primer multiformnim glioblastomom ali anaplastičnim astrocitomom, ki se po standardnem zdravljenju ponovi ali napreduje. Odmerjanje in način uporabe Temodal smejo predpisati le zdravniki, ki imajo izkušnje z zdravljenjem možganskih tumorjev. Odrasli bolniki z novo diagnosticiranim glioblastomom multiforme Temodal se uporablja v kombinaciji z žariščno radioterapijo (faza sočasne terapije), temu pa sledi do 6 ciklov monoterapije z temozolomidom. Faza sočasne terapije Zdravilo Temodal naj bolnik jemlje peroralno v odmerku 75 mg/m2 na dan 42 dni, sočasno z žariščno radioterapijo (60 Gy, danih v 30 delnih odmerkih). Odmerka ne boste zmanjševali, vendar se boste vsak teden odločili o morebitni odložitvi jemanja temozolomida ali njegovi ukinitvi na podlagi kriterijev hematološke in nehematološke toksičnosti. Zdravilo Temodal lahko bolnik jemlje ves čas 42-dnevnega obdobja sočasne terapije do 49 dni, če so izpolnjeni vsi od naslednjih pogojev: absolutno število nevtrofilcev > 1,5 x 109/l, število trombocitov > 100 x 109/l, skupni kriteriji toksičnosti (SKT) za nehematološko toksičnost < 1. stopnje (z izjemo alopecije, slabosti in bruhanja). Med zdravljenjem morate pri bolniku enkrat na teden pregledati celotno krvno sliko. Faza monoterapije Štiri tedne po zaključku faze sočasnega zdravljenja z zdravilom Temodal in radioterapijo naj bolnik jemlje zdravilo Temodal do 6 ciklov monoterapije. V 1. ciklu (monoterapija) je odmerek zdravila 150 mg/m2 enkrat na dan 5 dni, temu pa naj sledi 23 dni brez terapije. Na začetku 2. cikla odmerek povečajte na 200 mg/m2, če je SKT za nehematološko toksičnost za 1. cikel stopnje < 2 (z izjemo alopecije, slabosti in bruhanja), absolutno število nevtrofilcev (AŠN) > 1,5 x 109/l in število trombocitov > 100 x 109/l. Če odmerka niste povečali v 2. ciklusu, ga v naslednjih ciklusih ne smete povečevati. Ko pa odmerek enkrat povečate, naj ostane na ravni 200 mg/m2 na dan v prvih 5 dneh vsakega naslednjega ciklusa, razen če nastopi toksičnost. Med zdravljenjem morate pregledati celotno krvno sliko na 22. dan (21 dni po prvem odmerku zdravila Temodal). Ponavljajoči se ali napredujoči maligni gliom Odrasli bolniki Posamezen ciklus zdravljenja traja 28 dni. Bolniki, ki še niso bili zdravljeni s kemoterapijo, naj jemljejo Temodal peroralno v odmerku 200 mg/m2 enkrat na dan prvih 5 dni, temu pa naj sledi 23-dnevni premor (skupaj 28 dni). Pri bolnikih, ki so že bili zdravljeni s kemoterapijo, je začetni odmerek 150 mg/m2 enkrat na dan, v drugem ciklusu pa se poveča na 200 mg/m2 enkrat na dan 5 dni, če ni bilo hematoloških toksičnih učinkov (glejte poglavje 4.4). Pediatrični bolniki Pri bolnikih starih 3 leta ali starejših, posamezen ciklus zdravljenja traja 28 dni. Temodal naj jemljejo peroralno v odmerku 200 mg/m2 enkrat na dan prvih 5 dni, potem pa naj sledi 23-dnevni premor (skupaj 28 dni). Otroci, ki so že bili zdravljeni s kemoterapijo, naj prejmejo začetni odmerek 150 mg/m2 enkrat na dan 5 dni, s povečanjem na 200 mg/m2 enkrat na dan 5 dni v naslednjem ciklusu, če ni bilo hematoloških toksičnih učinkov (glejte poglavje 4.4). Bolniki z motnjami v delovanju jeter ali ledvic Pri bolnikih z blagimi ali zmernimi motnjami v delovanju jeter je farmakokinetika temozolomida podobna kot pri tistih z normalnim delovanjem jeter. Podatki o uporabi zdravila Temodal pri bolnikih s hudimi motnjami v delovanju jeter (razred III po Child-u) ali motnjami v delovanju ledvic niso na voljo. Na podlagi farmakokinetičnih lastnosti temozolomida obstaja majhna verjetnost, da bo pri bolnikih s hudimi motnjami v delovanju jeter ali ledvic potrebno zmanjšanje odmerka zdravila. Kljub temu je potrebna previdnost pri uporabi zdravila Temodal pri teh bolnikih. Starejši bolniki: Analiza farmakokinetike je pokazala, da starost ne vpliva na očistek temozolomida. Kljub temu je potrebna posebna previdnost pri uporabi zdravila Temodal pri starejših bolnikih. Način uporabe Temodal mora bolnik jemati na tešče. Temodal kapsule mora bolnik pogoltniti cele s kozarcem vode in jih ne sme odpirati ali žvečiti. Predpisani odmerek mora vzeti v obliki najmanjšega možnega števila kapsul. Pred jemanjem zdravila Temodal ali po njem lahko bolnik vzame antiemetik Če po zaužitju odmerka bruha, ne sme še isti dan vzeti drugega odmerka. Kontraindikacije Temodal je kontraindiciran pri bolnikih, ki imajo v anamnezi preobčutljivostne reakcije na sestavine zdravila ali na dakarbazin (DTIC). Temodal je kontraindiciran tudi pri bolnikih s hudo mielosupresijo. Temodal je kontraindiciran pri ženskah, ki so noseče ali dojijo. Posebna opozorila in previdnostni ukrepi Pilotno preskušanje podaljšane 42-dnevne sheme zdravljenja je pokazalo, da imajo bolniki, ki so sočasno prejemali zdravilo Temodal in radioterapijo, še posebej veliko tveganje za nastanek pljučnice zaradi okužbe s Pneumocystis carinii (PCP). Profilaksa proti tovrstni pljučnici je torej potrebna pri vseh bolnikih, ki sočasno prejemajo zdravilo Temodal in radioterapijo v okviru 42-dnevne sheme zdravljenja (do največ 49 dni), ne glede na število limfocitov. Če nastopi limfopenija, mora bolnik nadaljevati s profilakso, dokler se limfopenija ne povrne na stopnjo < 1. Antiemetična terapija: Z jemanjem zdravila Temodal sta zelo pogosto povezana slabost in bruhanje. Laboratorijske vrednosti: Pred jemanjem zdravila morata biti izpolnjena naslednja pogoja za laboratorijske izvide: ANC mora biti > 1,5 x 109/l in število trombocitov > 100 x 109/l. Na 22. dan (21 dni po prvem odmerku) ali v roku 48 ur od navedenega dne, morate pregledati celotno krvno sliko in jo nato spremljati vsak teden, dokler ni ANC nad 1,5 x 109/l in število trombocitov nad 100 x 109/l. Če med katerimkoli ciklusom ANC pade na < 1,0 x 109/l ali število trombocitov na < 50 x 109/l, morate odmerek zdravila v naslednjem ciklusu zmanjšati za eno odmerno stopnjo. Odmerne stopnje so 100 mg/m2, 150 mg/m2 in 200 mg/m2. Najmanjši priporočeni odmerek je 100 mg/m2. Moški bolniki Temozolomid lahko deluje genotoksično, zato morate moškim, ki se zdravijo z temozolomidom svetovati, da naj ne zaplodijo otroka še šest mesecev po zdravljenju. Interakcije Sočasna uporaba zdravila Temodal in ranitidina ni povzročila spremembe obsega absorpcije temozolomida ali monometiltriazenoimidazol karbok-samida (mTiC). Jemanje zdravila Temodal s hrano je povzročilo 33 % zmanjšanje Cmax in 9 % zmanjšanje površino pod krivuljo (AUC). Ker ne moremo izključiti možnosti, da bi bila sprememba Cmax lahko klinično pomembna, naj bolniki jemljejo zdravilo Temodal brez hrane. Analiza populacijske farmakokinetike v preskušanjih druge faze je pokazala, da sočasna uporaba deksametazona, proklorperazina, fenitoina, karbamazepina, ondansetrona, antagonistov receptorjev H2 ali fenobarbitala ne spremeni očistka temozolomida. Sočasno jemanje z valprojsko kislino je bilo povezano z majhnim, a statistično značilnim zmanjšanjem očistka temozolomida. Uporaba zdravila Temodal v kombinaciji z drugimi mielosupresivnimi učinkovinami la hko poveča verjetnost mielosupresije. Nosečnost Študij na nosečih ženskah ni bilo. Predklinične študije na podganah in kuncih z odmerkom 150 mg/m2 so pokazale teratogenost in/ali toksičnost za plod. Zato naj noseče ženske načeloma ne bi jemale zdravila Temodal. Če pa je uporaba v času nosečnosti nujna, morate bolnico opozoriti na možne nevarnosti zdravila za plod. Ženskam v rodni dobi svetujte, naj med zdravljenjem z zdravilom Temodal preprečijo zanositev. Dojenje Ni znano, ali se temozolomid izloča v materino mleko, zato ženske, ki dojijo ne smejo jemati zdravila Temodal. Neželeni učinki V kliničnih preskušanjih so bili najpogostnejši neželeni učinki, povezani z zdravljenjem, prebavne motnje, natančneje slabost (43 %) in bruhanje (36 %). Oba učinka sta bila ponavadi 1. ali 2. stopnje (od 0 do 5 epizod bruhanja v 24 urah) in sta prenehala sama, ali pa ju je bilo mogoče hitro obvladati s standardnim antiemetičnim zdravljenjem. Incidenca hude slabosti in bruhanja je bila 4 %. Laboratorijski izvidi: Trombocitopenija in. nevtro-penija 3. in. 4. stopnje sta se pojavili pri 19 % in. 17 % bolnikov, zdravljenih zaradi malignega glioma. Zaradi njiju je bila potrebna hospitalizacija in/ ali prekinitev zdravljenja z zdravilom Temodal pri 8 % in. 4 % bolnikov. Mielosupresija je bila predvidljiva (ponavadi se je pojavila v prvih nekaj ciklusih in je bila najizrazitejša med 21. in 28. dnem), okrevanje pa je bilo hitro, ponavadi v 1 do 2 tednih. Opazili niso nobenih dokazov kumulativne mielosupresije. Trombocitopenija lahko poveča tveganje za pojav krvavitev, nevtropenija ali levkopenija pa tveganje za okužbe. Imetnik dovoljenja za promet SP Europe 73, rue de Stalle B-1180 Bruxelles Belgija. Način in režim izdaje Zdravilo se izdaja samo na recept, uporablja pa se pod posebnim nadzorom zdravnika specialista ali od njega pooblaščenega zdravnika. Datum priprave informacije marec 2009.
Dunajska 22, 1000 Ljubljana tel: 01 300 10 70 fax: 01 300 10 80
Temodal
®
€05 Schering-Plough
temozDlomid
Resnični napredek
Pomembno izboljšanje preživetja potrjeno tudi ob daljšem spremljanju bolnikov
Izboljšanje celokupnega preživetja pri bolnikih z novo odkritim glioblastomom multiforme
Celokupno preživetje
n=11 n=39
2	leti: 27,3 % (RT/TMZ) vs 11,2 % (RT)
3	leta: 16,7 % (RT/TMZ) vs 4,3 % (RT)
4	leta: 12,9 % (RT/TMZ) vs 3,8 % (RT)
Literature: 1 Stupp R, Mason WP, van den Bent MJ, s sod. RADIOTHERAPY PLUS CONCOMITANT AND ADJUVANT TEMOZOLOMIDE FOR GLIOBLASTOMA N Engl J Med.2005;352:987-996. 2 Mirmanoff RO et al; IS LONG-TERM SURVIVAL IN GLIOBLASTOMA POSSIBLE?; 49th annual meeting of the ASRO, Los Angeles, okt. 2007.
30
25
RT
RT + TMZ
20
S 15
10
5
0
2. leto
5 jakosti v 5 barvah za lažje in natančnejše dnevno odmerjanje
Dunajska 22, 1000 Ljubljana
Schering-Plough
te 01 300 10 70
fax: 01 300 10 80
Novartis Oncology prinaša širok spekter inovativnih zdravil, s katerimi poskuša spremeniti življenje bolnikov z rakavimi in hematološkimi obolenji.
Ta vključuje zdravila kot so Glivec® (imatinib), Tasigna® (nilotinib), Exjade® (deferasiroks), Zometa® (zoledronska kislina), Sandostatin® LAR® (oktreotid/i.m. injekcije) in Femara® (letrozol).
Novartis Oncology ima tudi obširen razvojni program, ki izkorišča najnovejša spoznanja molekularne genomike, razumskega načrtovanja in tehnologij za odkrivanje novih učinkovin.
glivec
imatinib
^Tasi
asigna
(nilotinib)
O EXJADE
deferasiroks
zoledronska kislina
I Sandostatin lak
' oktreolid/l.m. Inletclic
if&rutoas
V	(letrozol)
11
NOVARTIS
ONCOLOGY
Novartis Pharma Services Inc, Podružnica v Sloveniji • Tivolska 30, 1000 Ljubljana Samo za strokovno javnost.
NVS-JA-01/09-SI
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Ime zdravila: Tarceva 25 mg/100 mg/150 mg filmsko obložene tablete
Kakovostna in količinska sestava: Ena filmsko obložena tableta vsebuje 25 mg, 100 mg ali 150 mg erlotiniba (v obliki erlotinibijevega klorida). Terapevtske indikacije: Nedrobnocelični rak pljuč: Zdravilo Tarceva je indicirano za zdravljenje bolnikov z lokalno napredovalim ali metastatskim nedrobnoceličnim rakom pljuč po neuspehu vsaj ene predhodne kemoterapije. Pri predpisovanju zdravila Tarceva je treba upoštevati dejavnike, povezane s podaljšanim preživetjem. Koristnega vpliva na podaljšanje preživetja ali drugih klinično pomembnih učinkov zdravljenja niso dokazali pri bolnikih z EGFR-negativnimi tumorji. Rak trebušne slinavke: Zdravilo Tarceva je v kombinaciji z gemcitabinom indicirano za zdravljenje bolnikov z metastatskim rakom trebušne slinavke. Pri predpisovanju zdravila Tarceva je treba upoštevati dejavnike, povezane s podaljšanim preživetjem. Koristnega vpliva na podaljšanje preživetja niso dokazali za bolnike z lokalno napredovalo boleznijo.
Odmerjanje in način uporabe: Zdravljenje z zdravilom Tarceva mora nadzorovati zdravnik z izkušnjami pri zdravljenju raka. Zdravilo Tarceva vzamemo najmanj eno uro pred zaužitjem hrane ali dve uri po tem. Kadar je potrebno odmerek prilagoditi, ga zmanjšujemo v korakih po 50 mg. Pri sočasnem jemanju substratov in modulatorjev CYP3A4 bo morda potrebna prilagoditev odmerka. Pri dajanju zdravila Tarceva bolnikom z jetrno okvaro je potrebna previdnost. Če se pojavijo hudi neželeni učinki pride v poštev zmanjšanje odmerka ali prekinitev zdravljenja z zdravilom Tarceva. Uporaba zdravila Tarceva pri bolnikih s hudo jetrno ali ledvično okvaro ter pri otrocih ni priporočljiva. Bolnikom kadilcem je treba svetovati, naj prenehajo kaditi, saj so plazemske koncentracije erlotiniba pri kadilcih manjše kot pri nekadilcih. Nedrobnocelični rak pljuč: priporočeni dnevni odmerek zdravila Tarceva je 1 50 mg. Rak trebušne slinavke: Priporočeni dnevni odmerek zdravila Tarceva je 100 mg, v kombinaciji z gemcitabinom. Pri bolnikih, pri katerih se kožni izpuščaj v prvih 4 do 8 tednih zdravljenja ne pojavi, je treba ponovno pretehtati nadaljnje zdravljenje z zdravilom Tarceva. Kontraindikacije: Huda preobčutljivost za erlotinib ali katero koli pomožno snov.
Posebna opozorila in previdnostni ukrepi: Močni induktorji CYP3A4 lahko zmanjšajo učinkovitost erlotiniba, močni zaviralci CYP3A4 pa lahko povečajo toksičnost. Sočasnemu zdravljenju s temi zdravili se je treba izogibati. Bolnikom, ki kadijo, je treba svetovati, naj prenehajo kaditi, saj so plazemske koncentracije erlotiniba pri kadilcih zmanjšane v primerjavi s plazemskimi koncentracijami pri nekadilcih. Verjetno je, da je velikost zmanjšanja klinično pomembna. Pri bolnikih, pri katerih se akutno pojavijo novi in/ali poslabšajo nepojasnjeni pljučni simptomi, kot so dispneja, kašelj in vročina, je zdravljenje z zdravilom Tarceva treba prekiniti, dokler ni znana diagnoza. Bolnike, ki se sočasno zdravijo z erlotinibom in gemcitabinom, je treba skrbno spremljati zaradi možnosti pojava toksičnosti, podobni intersticijski pljučni bolezni. Če je ugotovljena inter-sticijska pljučna bolezen, zdravilo Tarceva ukinemo in uvedemo ustrezno zdravljenje. Pri približno polovici bolnikov, ki so se zdravili z zdravilom Tarceva, se je pojavila driska. Zmerno do hudo drisko zdravimo z loperamidom. V nekaterih primerih bo morda potrebno zmanjšanje odmerka. V primeru hude ali dolgotrajne driske, navzeje, anoreksije ali bruhanja, povezanih z dehidracijo, je zdravljenje z zdravilom Tarceva treba prekiniti in dehidracijo ustrezno zdraviti. O hipokaliemiji in ledvični odpovedi so poročali redko. Posebno pri bolnikih z dejavniki tveganja (sočasno jemanje drugih zdravil, simptomi, bolezni ali drugi dejavniki, vključno z visoko starostjo) moramo, če je driska huda ali dolgotrajna oziroma vodi v dehidracijo, zdravljenje z zdravilom Tarceva prekiniti in bolnikom zagotoviti intenzivno intravensko rehidracijo. Dodatno je treba pri bolnikih s prisotnim tveganjem za razvoj dehidracije spremljati ledvično delovanje in serumske elektrolite, vključno s kalijem. Pri uporabi zdravila Tarceva so poročali o redkih primerih jetrne odpovedi. K njenemu nastanku je lahko pripomogla predhodno obstoječa jetrna bolezen ali sočasno jemanje hepatotoksičnih zdravil. Pri teh bolnikih je treba zato premisliti o rednem spremljanju jetrnega delovanja. Dajanje zdravila Tarceva je treba prekiniti, če so spremembe jetrnega delovanja hude. Tablete vsebujejo laktozo in jih ne smemo dajati bolnikom z redkimi dednimi stanji: intoleranco za galaktozo, laponsko obliko zmanjšane aktivnosti laktaze ali malabsorpcijo glukoze/galaktoze.
Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Erlotinib se pri ljudeh presnavlja v jetrih z jetrnimi citokromi, primarno s CYP3A4 in v manjši meri s CYP1A2. Presnova erlotiniba zunaj jeter poteka s CYP3A4 v črevesju, CYP1A1 v pljučih in CYP1B1 v tumorskih tkivih. Z zdravilnimi učinkovinami, ki se presnavljajo s temi encimi, jih zavirajo ali pa so njihovi induktorji, lahko pride do interakcij. Erlotinib je srednje močan zaviralec CYP3A4 in CYP2C8, kot tudi močan zaviralec glukuronidacije z UGT1A1 in vitro. Pri kombinaciji ciprofloksacina ali močnega zaviralca CYP1A2 (npr. fluvoksamina) z erlotinibom je potrebna previdnost. V primeru pojava neželenih dogodkov, povezanih z erlotinibom, lahko odmerek erlotiniba zmanjšamo. Predhodno ali sočasno zdravljenje z zdravilom Tarceva ni spremenilo očistka prototipov substratovCYP3A4, midazolama in eritromicina. Inhibicija glukoronidacije lahko povzroči interakcije z zdravili, ki so substrati UGT1A1 in se izločajo samo po tej poti. Močni zaviralci aktivnosti CYP3A4 zmanjšajo presnovo erlotiniba in zvečajo koncentracije erlotiniba v plazmi. Pri sočasnem jemanju erlotiniba in močnih zaviralcev CYP3A4 je zato potrebna previdnost. Če je treba, odmerek erlotiniba zmanjšamo, še posebno pri pojavu toksičnosti. Močni spodbujevalci aktivnosti CYP3A4 zvečajo presnovo erlotiniba in pomembno zmanjšajo plazemske koncentracije erlotiniba. Sočasnemu dajanju zdravila Tarceva in induktorjev CYP3A4 se je treba izogibati. Pri bolnikih, ki potrebujejo sočasno zdravljenje z zdravilom Tarceva in močnim induktorjem CYP3A4 je treba premisliti o povečanju odmerka do 300 mg ob skrbnem spremljanju njihove varnosti. Zmanjšana izpostavljenost se lahko pojavi tudi z drugimi induktorji, kot so fenitoin, karbamazepin, barbiturati ali šentjanževka. Če te zdravilne učinkovine kombiniramo z erlotinibom, je potrebna previdnost. Kadar je mogoče, je treba razmisliti o drugih načinih zdravljenja, ki ne vključujejo močnega spodbujanja aktivnosti CYP3A4. Bolnikom, ki jemljejo varfarin ali druge kumarinske antikoagulante, je treba redno kontrolirati protrombinski čas ali INR. Sočasna uporaba zaviralcev P-glikoproteina, kot sta ciklosporin in verapamil, lahko vodi v spremenjeno porazdelitev in/ali spremenjeno izločanje erlotiniba. Za erlotinib je značilno zmanjšanje topnosti pri pH nad 5. Zdravila, ki spremenijo pH v zgornjem delu prebavil, lahko spremenijo topnost erlotiniba in posledično njegovo biološko uporabnost. Učinka antacidov na absorpcijo erlotiniba niso proučevali, vendar je ta lahko zmanjšana, kar vodi v nižje plazemske koncentracije. Kombinaciji erlotiniba in zaviralca protonske črpalke se je treba izogibati. Če menimo, da je uporaba antacidov med zdravljenjem z zdravilom Tarceva potrebna, jih je treba jemati najmanj 4 ure pred ali 2 uri po dnevnem odmerku zdravila Tarceva. če razmišljamo o uporabi ranitidina, moramo zdravili jemati ločeno: zdravilo Tarceva je treba vzeti najmanj 2 uri pred ali 10 ur po odmerku ranitidina. V študiji faze Ib ni bilo pomembnih učinkov gemcitabina na farmakokinetiko erlotiniba, prav tako ni bilo pomembnih učinkov erlotiniba na farmakokinetiko gemcitabina. Erlotinib poveča koncentracijo platine. Pomembnih učinkov karboplatina ali pakli-taksela na farmakokinetiko erlotiniba ni bilo. Kapecitabin lahko poveča koncentracijo erlotiniba. Pomembnih učinkov erlotiniba na farmakokinetiko kapecitabina ni bilo.
Neželeni učinki: Zelo pogosti neželeni u~inki so kožni izpuščaj in driska, kot tudi utrujenost, anoreksija, dispneja, kašelj, okužba, navzeja, bruhanje, stomatitis, bolečina v trebuhu, pruritus, suha koža, suhi keratokonjunktivitis, konjunktivitis, zmanjšanje telesne mase, depresija, glavobol, nevropatija, dispepsija, flatul enca, alopecija, okorelost, pireksija. Pogosti neželeni u~inki so gastrointestinalne krvavitve, krvavitev iz nosu, nenormalnosti testov jetrne funkcije, keratitis, zanohtnica. Redko so poročali o jetrni odpovedi. Ob~asno pa o poraščenosti moškega tipa pri ženskah, spremembah trepalnic/obrvi, krhkih nohtih, odstopanju nohtov od kože, resni intersticijski pljučni & bolezni, vključno s smrtnimi primeri.	AT
Režim izdaje zdravila: H/Rp.	cn
Imetnik dovoljenja za promet: Roche Registration Limited, ? 6 Falcon Way, Shire Park, Welwyn Garden City, AL7 1TW, Velika rg Britanija. Verzija: 1.0/09.
Informacija pripravljena: marec 2009.
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Radiology and Oncology
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Dent RAG, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95.
Chapman S, Nakielny R. A guide to radiological procedures. London: Bailliere Tindall; 1986.
Evans R, Alexander P. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology of macrophage. New York: Academic Press; 1976. p. 45-74.
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BISTVENE INFORMACIJE IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA
AROMASIN® 25 mg obložene tablete
Sestava in oblika zdravila: obložena tableta vsebuje 25 mg eksemestana. Indikacije: Adjuvantno zdravljenje žensk po menopavzi, ki imajo invazivnega zgodnjega raka dojke s pozitivnimi estrogenskimi receptorji in so se uvodoma vsaj 2 do 3 leta zdravile s tamoksifenom. Zdravljenje napredovalega raka dojke pri ženskah z naravno ali umetno povzročeno menopavzo, pri katerih je bolezen napredovala po antiestrogenskem zdravljenju. Učinkovitost še ni bila dokazana pri bolnicah, pri katerih tumorske celice nimajo estrogenskih receptorjev. Odmerjanje in način uporabe: 25 mg enkrat na dan, najbolje po jedi. Pri bolnicah z zgodnjim rakom dojke je treba zdravljenje nadaljevati do dopolnjenega petega leta adjuvantnega hormonskega zdravljenja oz. do recidiva tumorja. Pri bolnicah z napredovalim rakom dojke je treba zdravljenje nadaljevati, dokler ni razvidno napredovanje tumorja. Kontraindikacije: znana preobčutljivost na učinkovino zdravila ali na katero od pomožnih snovi, ženske pred menopavzo, nosečnice in doječe matere. Posebna opozorila in previdnostni ukrepi: predmenopavzni endokrini status, jetrna ali ledvična okvara, bolniki z redko dedno intoleranco za fruktozo, malabsorpcijo glukoze/galaktoze ali pomanjkanjem saharoza-izomaltaze. Lahko povzroči alergijske reakcije ali zmanjšanje mineralne gostote kosti ter večjo pogostnost zlomov. Ženskam z osteoporozo ali tveganjem zanjo je treba na začetku zdravljenja izmeriti mineralno kostno gostoto s kostno denzitometrijo. Čeprav še ni dovolj podatkov, kako učinkujejo zdravila za zdravljenje zmanjšane mineralne kostne gostote, ki jo povzroča Aromasin, je treba pri bolnicah s tveganjem uvesti zdravljenje ali profilakso osteoporoze ter bolnice natančno spremljati. Medsebojno delovanje z drugimi zdravili: Sočasna uporaba zdravil - npr. rifampicina, antiepileptikov (npr. fenitoina ali karbamazepina) ali zdravil rastlinskega izvora s šentjaževko - ki inducirajo CYP 3A4, lahko zmanjša učinkovitost Aromasina. Uporabljati ga je treba previdno z zdravili, ki se presnavljajo s pomočjo CYP 3A4 in ki imajo ozek terapevtski interval. Kliničnih izkušenj s sočasno uporabo zdravila Aromasin in drugih zdravil proti raku ni. Ne sme se jemati sočasno z zdravili, ki vsebujejo estrogen, saj bi ta izničila njegovo farmakološko delovanje. Vpliv na sposobnost vožnje in upravljanja s stroji: Po uporabi zdravila je lahko psihofizična sposobnost za upravljanje s stroji ali vožnjo avtomobila zmanjšana. Neželeni učinki: neželeni učinki so bili v študijah, v katerih so uporabljali standardni odmerek 25 mg, ponavadi blagi do zmerni. Zelo pogosti (> 10 %): vročinski oblivi, bolečine v sklepih, mišicah in kosteh, utrujenost, navzea, nespečnost, glavobol, močnejše znojenje, ginekološke motnje. Način in režim izdajanja: zdravilo se izdaja le na recept, uporablja pa se po navodilu in pod posebnim nadzorom zdravnika specialista ali od njega pooblaščenega zdravnika. Imetnik dovoljenja za promet: Pfizer Luksembourg SARL, 283, route d'Arlon, L-8011 Strassen, Luksemburg. Datum zadnje revizije besedila: 11.4.2008 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila.
Pfizer Luxembourg SARL, Grand Duchyof Luxembourg, 51, Avenue J.F. Kennedy, L-1855, PFIZER, Podružnica za svetovanje s področja farmacevtske dejavnosti, Ljubljana, Letališka 3c, 1000 Ljubljana, SLOVENIJA