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, 51, Avenue J. F. Kennedy, L-1855, Luksemburg. Datum zadnje revizije besedila: 11.12.2009
Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila.

Pfizer Luxembourg SARL, Grand Duchy of Luxembourg, 51, Avenue J.F. Kennedy, L-1 855, PFIZER, Podružnica za svetovanje s področja farmacevtske dejavnosti, Ljubljana, Letališka 3c, 1 000 Ljubljana, SLOVENIJA
ADIOLQGY
NEOLOGY	°1[
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Association of Radiology and Oncology Affiliated with
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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.
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December 2011 Vol. 45 No. 4 Pages 227-314 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM
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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 Miletić Rijeka, Croatia Maja Osmak Zagreb, Croatia Branko Palčič Vancouver, Canada Dušan Pavčnik Portland, USA 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 Teissié Toulouse, France Sandor Toth Oroshaza, Hungary Gillian M. Tozer Sheffield, UK Andrea Veronesi Aviano, Italy Branko Zakotnik Ljubljana, Slovenia
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contents
review
227 Titanium dioxide in our everyday life; is it safe?
Matej Skocaj, Metka Filipic, Jana Petkovic, Sasa Novak
radiology
248 Assessing renal function in children with hydronephrosis - additional feature of MR urography
George Hadjidekov, Savina Hadjidekova, Zahari Tonchev, Rumiana Bakalova, Ichio Aoki
experimental oncology
259 Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines
Matija Rojnik, Zala Jevnikar, Bojana Mirkovic, Damjan Janes, Nace Zidar, Danijel Kikelj, Janko Kos
clinical oncology
267 Sinonasal inverted papilloma associated with squamous cell carcinoma
Jasna But-Hadzic, Klemen Jenko, Mario Poljak, Bostjan J Kocjan, Nina Gale, Primoz Strojan
273 Comparison of survival of patients receiving laparoscopic and open radical resection for stage II colon cancer
Cui-Zhen Fan, Yu-Ping Chu, Ping Wei, Hong Dai, Wenming Chen
279 Expression of NF-kB p65 phosphorylated at serine-536 in rectal cancer with or without preoperative radiotherapy
Andreas Lewander, Jinfang Gao, Gunnar Adell, Hong Zhang, Xiao-Feng Sun
285 Efficacy of first-line systemic treatment in correlation with BRAF V600E and different KRAS mutations in metastatic colorectal cancer - a single institution retrospective analysis
Martina Rebersek, Marko Boc, Petra Cerkovnik, Jernej Benedik, Zvezdana Hlebanja, Neva Volk, Srdjan Novakovic, Janja Ocvirk
292 Hepatocellular carcinoma with subcutaneous metastasis of the scalp
Yilmaz Tezcan and Mehmet Koc
296 Genotyping of BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes in a male patient with secondary breast cancer
Ana Lina Vodusek, Srdjan Novakovic, Vida Stegel, Berta Jereb
300 Periampullary localized pancreatic intraepithelial neoplasia-3 (PanIN-3): evaluation with contrast-enhanced MR cholangiography (MRCP)
Oktay Algin, Evrim Ozmen, Pamir Eren Ersoy, Mustafa Karaoglanoglu
304 Brain meningioma invading and destructing the skull bone: replacement of the missing bone in vivo
Tomaz Velnar, Rado Pregelj, Clara Limbaeck-Stokin
radiophysics
310 Dosimetric verification of compensated beams using radiographic film
Slaven Jurkovic, Gordana Zauhar, Dario Faj, Deni Smilovic Radojcic, Manda Svabic, Mladen Kasabasic, Ana Diklic
Slovenian abstracts
vili authors Index 2011
X sub ject Index 2011
RADiOLOGY AND ONCOLOGY is covered in Science Citation Index Expanded (SciSearch®),Journal Citation Reports/Science Edition, Scopus, DOAJ, EMBASE/Excerpta Medica, Open J-gate, Chemical Abstracts, Biomedicina Slovenica
Titanium dioxide in our everyday life; is it safe?
Matej Skocaj1, Metka Filipic2, Jana Petkovic2, Sasa Novak1
1	Jožef Stefan Institute, Department for Nanostructured Materials, Ljubljana, Slovenia
2	National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia
Received 7 October 2011 Accepted 27 October 2011
Correspondence to: Saša Novak, Jožef Stefan Institute, Department for Nanostructured Materials, Jamova 39, SI-1000 Ljubljana, Slovenia. Phone: +386 1 4773 271; Fax: +386 1 4773 221; E-mail: sasa.novak@ijs.si
Disclosure: No potential conflicts were disclosed.
Background. Titanium dioxide (TiO2) is considered as an inert and safe material and has been used in many applications for decades. However, with the development of nanotechnologies TiO2 nanoparticles, with numerous novel and useful properties, are increasingly manufactured and used. Therefore increased human and environmental exposure can be expected, which has put TiO2 nanoparticles under toxicological scrutiny. Mechanistic toxicological studies show that TiO2 nanoparticles predominantly cause adverse effects via induction of oxidative stress resulting in cell damage, genotoxicity, inflammation, immune response etc. The extent and type of damage strongly depends on physical and chemical characteristics of TiO2 nanoparticles, which govern their bioavailability and reactivity. Based on the experimental evidence from animal inhalation studies TiO2 nanoparticles are classified as "possible carcinogenic to humans" by the International Agency for Research on Cancer and as occupational carcinogen by the National Institute for Occupational Safety and Health. The studies on dermal exposure to TiO2 nanoparticles, which is in humans substantial through the use of sunscreens, generally indicate negligible transdermal penetration; however data are needed on long-term exposure and potential adverse effects of photo-oxidation products. Although TiO2 is permitted as an additive (E1 71 ) in food and pharmaceutical products we do not have reliable data on its absorption, distribution, excretion and toxicity on oral exposure. TiO2 may also enter environment, and while it exerts low acute toxicity to aquatic organisms, upon long-term exposure it induces a range of sub-lethal effects.
Conclusions. Until relevant toxicological and human exposure data that would enable reliable risk assessment are obtained, TiO2 nanoparticles should be used with great care.
Key words: titanium dioxide; nanoparticles; toxicity; applications; safety
Introduction
Titanium dioxide (titania, TiO2) is chemically inert, semiconducting material that also exhibits photo-catalytic activity in the presence of light with an energy equal to or higher than its band-gap energy. These characteristics offer a wide range of applications. For these reasons, and because of the relatively low price of the raw material and its processing, titania has gained widespread attention over recent decades.
TiO2 has been classified in humans and animals as biologically inert12, and is widely considered to be a "natural" material, which at least partially contributes to its relatively positive acceptance by the public. In fact, most TiO2 has been synthesized from the mineral illmenite, FeTiO3, using the "sulphate" or "chloride" process for nearly 100 years.
The annual worldwide production of titania powder in 2005 has been estimated to be around 5 million tons3, provoking the question as to its abundance in the environment. The proportion of na-no-sized titania is estimated to have been approximately 2.5 % in 2009, increasing to 10 % by 20154, with an exponential increase over the past decade.
During recent decades, TiO2 powders have begun to appear in many applications, mainly due to their ability to confer whiteness and opacity on various products, such as paints, papers and cosmetics. Its high technological attractiveness originates from its light-scattering properties and very high refractive index, which mean that relatively low levels of the pigment are required to achieve a white, opaque coating. The range of light that is scattered depends on the particle size. Numerous technological improvements, based on nano-sized
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[A]
Anatase A Anatase B Rutile
FIGURE 1. Field emission electron micrographs of different TiO2 powders: A) Anatase A (Sigma 637254), B) Anatase B (Sigma T8141); C) Zeta-potential of these two powders, Rutile (Sigma 637262) and P25 (Degussa).
TiO2, have been introduced that enable its use for antifogging and self-cleaning coatings on glass, for building facades, in confectionary, in the plastics industry, and so on. Furthermore, TiO2 is accepted as a food and pharmaceutical additive.5 In the United States it is included in the Food and Drug Administration (FDA) Inactive Ingredients Guide for dental paste, oral capsules, suspensions, tablets, dermal preparations and in non-parenteral medicines.
The increasing production of nano-sized TiO2 powder has led to growing concerns about the consequences of exposure of humans and the environ-
ment.6 In the present paper we review and discuss the latest findings on potential hazard of exposure to nano-sized TiO2 for humans and environment, in regard to the particle size and the crystal structure of TiO2, the route of exposure as well as the effect of ultraviolet (UV) irradiation-induced pho-tocatalysis.
Chemical and physical properties of Tiö2 nanoparticles
Nanoparticles (NPs) are generally defined as particles having at least one dimension smaller than 100 nm. Accordingly, particles with different morphologies, from equi-axial shapes, whiskers, and nano-tubes to nanorods, need to be considered. Although micron-sized and nano-sized TiO2 powders are, in general, chemically identical, due to their significantly higher specific surface area, nano-powders may exhibit physical and chemical properties that differ from those of the coarser grades, and so should not be treated in the same way. In a recent paper7 the size-dependent properties of a variety of inorganic NPs were reviewed and it was suggested that they are likely to be of concern due to the appearance of unique properties when they have diameters of < 30 nm. In this size range, many particles undergo dramatic changes in behaviour that enhance their interfacial reactivity. While less than 20 % of the constituent atoms are at the surface of 30 nm NPs, approximately 35-40 % of the atoms are localized at the surface of a 10 nm particle.
In practice, it is difficult to draw a clear borderline between nano- and submicron-sized particles. Submicron-sized powders always contain a certain proportion of nano-sized particles and, conversely, NPs tend to associate to form relatively strongly bonded aggregates (Figure 1A) or soft agglomerates (Figure 1B). The latter can usually be disintegrated easily in a liquid; however, their dispersion depends strongly on the zeta-potential. As illustrated in Figure 1C, the zeta-potential of TiO2 powders may differ significantly over a wide range of pH values. The reported isoelectric points for TiO2 powders range from pH 3.5 to 8 8 which may greatly affect the bioavailability in the region of physiological pH values. The effective size of particles and their zeta-potential have been neglected almost completely in most of the studies of the interaction of TiO2 NPs with biological systems.
Crystalline TiO2 occurs naturally in three polymorphs - anatase, rutile and brookite - among which rutile is the most stable. A powder with an average particle size of 230 nm scatters visible light, while its counterpart, with an average size of 60 nm, scatters UV light and reflects visible light. Under UV, TiO2 exhibits photocatalytic activity, which is a consequence of the electronic structure of the titania, and is, to a large extent, more characteristic of anatase than of rutile and brookite. In the presence of light with energy equal to or higher than the TiO2 band-gap energy, an electron is promoted from the valence band to the conduction band, leaving behind a positive hole. The extrapolated optical absorption gaps of anatase and rutile are 3.2 and 3.0 eV at room temperature, which correspond to wavelengths of around 413 nm and 387 nm. Consequently, the photo-activation of nano-TiO2 can be achieved by irradiation with UV-A, B and C, visible, fluorescent light, and X-ray radiation. The photocatalytic activity results in formation of highly reactive radicals, that are capable of reacting with most of the surrounding organic substances.9-12
Mechanisms of TiO2 NPs toxicity
As already discussed, the physicochemical properties of particles depend on their size, so that, at the nanometre level, the material is chemically more reactive. This can be exploited as a desirable property, e.g., as a catalyst. However, at the same time, the material may possess biological activities that can be either desirable (e.g., carrier capacity
for therapeutics, penetration of cellular barriers for drug delivery) or undesirable (e.g., toxicity, induction of oxidative stress or cellular dysfunction), or a mix of the two.
Cellular uptake of TiO2 NPs
From a toxicological point of view the important characteristics of NPs are their size, surface area, surface chemistry and charge, crystallinity, shape, solubility and agglomeration/aggregation state. Surface groups may render NPs hydrophilic or hydrophobic, lipophilic or lipophobic, catalytical-ly active or passive. Cellular uptake, subcellular localization, and ability to cause toxic effects depend on these properties of NPs.13 The two main pathways of NP uptake in the cell are active uptake by endocytosis, and passive uptake by free diffusion. Phagocytosis is an actin-dependent, endocytic mechanism, typical of "professional" phagocytes like macrophages. Geiser et al.14 reported that, in rats exposed to TiO2 powders by inhalation, alveolar macrophages effectively cleared micron-sized (3-6 |jm) but not nano-sized (20 nm) TiO2 particles. This is important, since phagocytes generally remove particulate matter >500 nm 15 and, as they are unable to phagocytose smaller particles, the latter are retained in the tissue, leading to a sustained burden on other tissues and cells. It was demonstrated that the uptake of 50 nm nano-TiO2, by endocytosis with alveolar A549 epithelial cells, was limited to aggregated particles.16 After inhalation exposure of rats to TiO2 NPs, free particles were found within the cytoplasm of epithelial and endothelial cells and fibroblasts.17 Rothen-Rutishauser et al.18, used an in vitro airway wall model, and found membrane-bound aggregates (>200 nm) of TiO2 as well as smaller unbound aggregates within the cell cytoplasm. In an in vitro study Kocbek et al.19 demonstrated the endocytotic uptake of 25 nm-sized anatase TiO2 by human keratinocytes. They observed highly aggregated NPs within early and late endosomes and in amphisomes, confirming endocytotic uptake. Experiments with red blood cells, which lack phagocytic receptors18, revealed that TiO2 NP aggregates smaller than 200 nm are able to enter red blood cells, while larger particles were only found attached to the cell's surface. Xia et al.20 showed that fluorescence-labelled TiO2 NPs (11 nm) were taken up and localized in late endosomal and caveolar compartments in phagocytic RAW 246.7 and lung endothelial BEAS-2B cells.
Oxidative stress induced by TiO2 NPs
Oxidative stress is thought to be a key mechanism responsible for adverse biological effects exerted by NPs.2122 The role of oxidative stress in TiO2-induced adverse effects has been confirmed by evidence that it induces an increase in reactive oxygen species (ROS) production and oxidative products (i.e., lipid peroxidation), as well as the depletion of cellular antioxidants.23-29
TiO2 mediates oxidative stress under UV irradiation as well as without it. Uchino et al.30 showed that, under UV irradiation, the TiO2 NPs of different crystalline structures and sizes produces different amounts of hydroxyl radicals, and that cytotoxicity against Chinese hamster ovary cells correlates with the production of radicals. Dodd and Jha31 confirmed that hydroxyl radicals are the primary damaging species produced by UV irradiated nano-sized TiO2, and react to give carboxyl radicals. A number of studies have shown photo-activated anatase TiO2 to induce higher cytotoxic-ity and genotoxicity than similarly activated rutile TiO2. These differences could arise from the fact that anatase particles possess a wider absorption gap and a smaller electron effective mass, resulting in the higher mobility of the charge carriers and the greater generation of ROS. On the other hand, there is evidence that TiO2 also induces ROS formation and the associated adverse effects in the absence of photo-activation. For instance, Gurr et al.24 found that anatase TiO2 NPs and mixtures of anatase and rutile TiO2 NPs induced oxidative damage in human bronchial epithelial (BEAS-2B) cells, and Petković et al.32 reported that in human hepatoma cells (HepG2), non-irradiated anatase nano-TiO2 induced significantly higher levels of intracellular ROS than the corresponding rutile-TiO2, and only anatase nano-TiO2 caused oxidative DNA damage. Recently, Petković et al.33 compared cytotoxicity and genotoxicity of non-irradiated and UV pre-irradiated anatase TiO2 of two sizes (<25 nm and >100 nm). They showed that non-irradiated TiO2 particles did not affect survival of the cells; they caused slight increase in number of DNA strand breaks, while only TiO2 NPs caused increase in oxidative DNA damage. After pre-irradiation with UV both sizes of anatase TiO2 particles reduced cell viability, induced DNA strand breaks and oxidative DNA damage. This is an important finding that, for the first time, showed that photo-activated TiO2 particles retained higher cytotoxic and genotoxic potential also when UV irradiation was discontinued and that it was not particle size dependent.
ROS are also important signalling modulators, therefore exposure of cells to NPs may, via elevated ROS formation, affect cellular signalling cascades that control processes such as cell proliferation, inflammation and cell death.34 The role of oxidative stress in TiO2-induced inflammation has recently been confirmed by Kang et al.35 In the mouse peritoneal macrophage cell line RAW 246.7 exposed to nano-TiO2, ROS production was associated with the activation of pro-inflammatory cascade, as indicated by extracellular signalregulated kinases ERK1/2 phosphorylation, tumour necrosis factor TNFa production and macrophage inflammatory protein MIP-2 secretion.
Taken together, these studies indicate that the high level of oxidative stress that is related to an exposure to a high concentration of TiO2 NPs leads to cell damage-associated responses, whereas at moderate levels of oxidative stress, inflammatory responses may be stimulated by the activation of ROS-sensitive signalling pathways.
Genotoxicity of TiO2 NPs
Several studies show that nano-TiO2 induces ge-notoxic effects, including DNA damage, and mi-cronuclei formation that is indicative of chromo-soma! aberrations in different cell lines32, 35-38 The studies also showed that genotoxic effects elicited by TiO2 NPs strongly depended upon their size and form. For instance, Gurr et al.24 showed that anatase TiO2 NPs up to 20 nm in size induced an increase in micronuclei formation, while 200 nm anatase or 200 nm rutile TiO2 did not. Zhu et al.39 demonstrated clear differences in the cytotoxicity and the extent of DNA strand scission, together with the formation of 8-hydroxy-2-deoxyguano-sine (8-OHdG) adducts in isolated DNA, after a treatment with different types of TiO2 NPs in the order 10-20 nm anatase > 50-60 nm anatase > 5060 nm rutile. At the molecular level it has been shown that the exposure of peripheral human lymphocytes to TiO2 NPs caused the activation of DNA damage check points and the accumulation of tumour suppressor protein p53, the main regulator of the cellular response to DNA dam-age.40 Exposure of human hepatoma HepG2 cells under similar conditions led to the elevated expression of tumor suppressor p53 mRNA and its downstream regulated DNA damage response genes (cyclin-dependent kinase inhibitor p21, growth arrest and DNA damage-inducible gene GADD45a and the E3 ubiquitin ligase MDM2).32
On the other hand, TiO2 NPs were devoid of mutagenic activity in microbial mutation assays (with Salmonella typhimurium) and in chromosomal aberration the in Chinese hamster ovary cells.41 Similarly, Theogaraj et al.42 reported that nano-sized TiO2 (eight different anatase and rutile forms) at concentrations up to 5 mg/ml did not induce any increase in the chromosomal aberration frequency in Chinese hamster ovary cells, in either the absence or the presence of UV light. However, in this study only a short-term, 3-hour, and no continuous (i.e., 20 hours) exposure, was performed.
In an early study Driscoll et al.43 reported that in rats intratracheal instillation of TiO2 NPs (100 mg/kg BW) induced increased HPRT mutation frequency in alveolar cells. They also showed that mutagenicity in alveolar cells was associated with inflammation. Trouiller et al.44 recently reported that oral exposure of mice to TiO2 NPs through drinking water (50-500 mg/kg BW/day for 5 days) induced oxidative DNA damage, micronuclei formation and y-H2AX foci, the indicators of DNA double strand breaks. Since also high-level gene expression of pro-inflammatory cytokines was also observed, the authors suggested the inflammatory effects were responsible for the induction of geno-toxic effects.
The in vitro and in vivo genotoxicity studies using different experimental models indicate that nano-TiO2 may cause genotoxic effects via secondary mechanisms that include oxidative stress and inflammation.32,38 40 43 44 However, there is some evidence that nano-sized TiO2 can locate in nuclei 17, and recently Li et al.45 reported the presence of anatase nano-sized TiO2 in DNA extracted from the liver of mice exposed intraperitoneally to these NPs (5-150 mg/kg BW/day for 14 days). The authors showed that Ti inserted between DNA base pairs or bound to DNA nucleotides, in such a way that it altered the conformation of the DNA and, at higher doses, caused DNA cleavage. These findings indicate that TiO2 may also induce genetic damage by a direct interaction with the DNA.
Immunotoxic effects of TiO2 NPs
Depending on physicochemical properties of NPs, they are recognized and taken up by immune cells, such as macrophages, monocytes, platelets, leukocytes and dendritic cells, and can trigger an inflammatory response. In a human monoblastoid cell line (U937) exposure to TiO2 NPs induced apopto-sis and necrosis in concentrations corresponding to those found in blood, plasma, or in tissues sur-
rounding Ti implants 46. Palomäki et al.47 reported that rutile TiO2 NPs and silica-coated rutile TiO2 NPs induced the enhanced expression of a variety of proinflammatory cytokines in murine dendritic cells (bm-DC) and in murine macrophages (RAW 246.7). The particles were for dendritic cells more toxic than for macrophages. In dendritic cells nano-sized TiO2 led to an upregulation of maturation markers and activated the NLRP3 inflammas-ome, a multiprotein complex within the cytoplasm of antigen-presenting cells, leading to significant IL 1|3-secretion. It was demonstrated for neutrophils that the short-term exposure of neu-trophils to nano-anatase TiO2 induces changes in their morphology, indicating its potential to activate these cells, while longer exposure resulted in the inhibition of apoptosis and cytokine production, confirming that in vitro TiO2 exerts neutrophil agonist properties.
Immunomodulating effects after exposure to TiO2 NPs have been observed also in in vivo studies. Larsen et al.48 showed that in ovalbumin immunized mice intraperitoneal exposure to TiO2 NPs promoted a T-helper type 2 cells mediated dominant immune response with high levels of oval-bumin-specific immunoglobulins IgE and IgG1 in serum and influx of eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid. Airway inflammation and immune adjuvant activity in ovalbumin immunized mice was observed also after intranasal exposure to TiO2 NPs 48 49 indicating that airborne exposure to TiO2 NPs may induce respiratory allergy, where the possible mechanism could be an adjuvant-like activity of NPs on allergic sensitization. Associated with the impairment of the immune response, recently Moon et al.50 showed that the intraperitoneal exposure of mice to TiO2 NPs enhanced the growth of subcu-taneously implanted B16F10 melanoma through the immunomodulation of B- and T-lymphocytes, macrophages, and natural killer cells.
Neurotoxic effects of TiO2 NPs
It has been reported that inhaled NPs can translocate to the central nervous system through the olfactory pathway22 and by crossing the blood-brain barrier.5152 In vitro studies of non-irradiated TiO2 NPs (Degussa P25) showed that they cause oxidative stress in the brain microglia BV2 cell line 53 that was associated with the up-regulation of genes involved in the inflammation, apoptosis, and the cell cycle, and down-regulation of genes involved in energy metabolism.25 While Degussa
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FIGURE 2. Field emission electron micrographs of the powders from two commercial sunscreens: A-SPF 20 (A) and N-SPF 10 (B), and their XRD diffraction (C).
stem cells towards neurons. These results indicate that the responses may be cell-type dependent and oxidative stress-mediated.
Recently Scuri et al.56 reported that inhalation exposure of newborn (2-day-old) and weanling (2-week-old), but not adult, rats to TiO2 NPs (12 mg/m3; 5.6 h/day for 3 days) up-regulates the expression of lung neurotrophins, key regulatory elements of neuronal development and responsiveness that play a critical role in the pathophysiology of childhood asthma. The effect was associated with the development of airway hyperreactivity (AHR) and mild airway inflammation. These results suggest the presence of a critical window of vulnerability in the earlier stages of lung development, which may lead to a higher risk of developing asthma.
P25 NPs stimulated ROS formation in BV2 microglia, they were nontoxic to isolated N27 neurons. However, in complex brain cultures the Degussa P25 particles rapidly damaged neurons, plausibly through microglial generated ROS. In contrast, Liu et al.54 reported that, in the neuronal cell line PC12, exposure to nano-TiO2 induced dose-dependent oxidative stress and apoptosis that was partly prevented by pre-treatment with a ROS scavenger. Surprisingly, it was shown recently 55 that TiO2 NPs (rutile TiO2 coated with SiO2; 80-100 nm) might be an inducer of the differentiation of (mouse) neural
TiO2 NPs in everyday life
Nano-sized TiO2 in various forms is used widely in everyday life in a variety of products, such as anti-fouling paints, household products, plastic goods, medications, cosmetics, sunscreens, pharmaceutical additives and food colorants, and many new applications are under development or already in pilot production. In the following sections we consider the main entry ports of nano-sized TiO2 into the human body and potential adverse effects.
Dermal exposure to TiO2 NPs
TiO2 NPs as a component of the sunscreen-technology revolution
During recent decades, skin cancer has become the most frequent neoplastic disease among the Caucasian population in Europe, North America
and Australia, and its incidence has reached epidemic proportions.57 As a consequence, the trend in sun protection in daily cosmetics is towards increased use of organic and inorganic UV filters. It is estimated that worldwide use of nano-sized TiO2 in sunscreens is around 1000 tons per year.51
TiO2 has been used in sunscreens since 1952, however the Food and Drug Administration (FDA) approved the use of TiO2 in sunscreens in 1999.5859 Currently it is not required to label sunscreens as containing nano-TiO2 60 The situation could change if the European Union (EU) commission adopts a proposed new regulation within EU Cosmetic Directive, under which all cosmetics that contain more than 1 % w/w of NPs will have to declare it on the packaging. Since TiO2 is considered as low-irritating, it is the only inorganic UV filter allowed by European legislation in concentrations as high as 25 %.6162 There is also some confusion regarding the classification of sunscreens. In the EU they are classified as cosmetics, while in the USA, they are classified as over the counter (OTC) drugs.63
The average size of the TiO2 particles in sunscreens ranges between 10 and 100 nm, while some products contain particles down to 5 nm or up to 500 nm.64 TiO2 particles in the size range between 200 and 500 nm are opaque and act as a true sunblock when applied to the skin.61 6566 However, this opacity is lost when much finer particles are used. Such sunscreens are more transparent, less viscous, and blend into the skin more easily. Therefore, the optimum size of TiO2 particles was suggested to be around 50 nm, which provides good protection against UV light, while the dispersion of visible light is such that sunscreens do not appear white on the skin.67
Sunscreens typically, but not exclusively, contain rutile TiO2 powder, which is less photo-active than the anatase TiO2. Micrographs of the powders extracted from two commercial sunscreens from different producers are shown in Figures 2A and B. From the X-ray diffractograms (Figure 2C) it is evident that the TiO2 powder in the sunscreen "N-SPF10" is predominantly in the anatase form, with an estimated particle size of around 50 nm (Figure 2A, C), while the powder in the sunscreen "A-SPF20" contained rutile TiO2 with two size populations (Figure 2B, C). (The original commercial names of the products were adapted for this study.)
To minimize the harmful effects of photo-active nano-TiO2, various coatings such as magnesia, silica, alumina or zirconia68-71 were introduced. However, certain coating materials may have side effects, such as aluminium-based ones (Figure 3),
FIGURE 3. Transmission electron micrograph of an AlOOH-coated TiO2 NP (Courtesy of dr. G. Dražić).
and it is also not clear how stable the coatings are and what is the lifetime of the "inert" particle released from sunscreens.
Cytotoxic and genotoxic effects of TiO2 NPs in dermal cells and skin models
Different dermal cell types have been reported to differ in their sensitivity to nano-sized TiO2. Kiss et al.72 exposed human keratinocytes (HaCaT), human dermal fibroblast cells, sebaceous gland cells (SZ95) and primary human melanocytes to 9 nm-sized TiO2 particles at concentrations from 0.15 to 15 |jg/cm2 for up to 4 days. The particles were detected in the cytoplasm and perinuclear region in fibroblasts and melanocytes, but not in kerati-nocytes or sebaceous cells. The uptake was associated with an increase in the intracellular Ca2+ concentration. A dose- and time-dependent decrease in cell proliferation was evident in all cell types, whereas in fibroblasts an increase in cell death via apoptosis has also been observed. Anatase TiO2 in 20-100 nm-sized form has been shown to be cytotoxic in mouse L929 fibroblasts.73 The decrease in cell viability was associated with an increase in the production of ROS and the depletion of glutath-ione. The particles were internalized and detected within lysosomes. In human keratinocytes exposed for 24 h to non-illuminated, 7 nm-sized anatase TiO2 a cluster analysis of the gene expression revealed that genes involved in the "inflammatory response" and "cell adhesion", but not those involved in "oxidative stress" and "apoptosis", were up-regulated.73 The results suggest that non-illu-
minated TiO2 particles have no significant impact on ROS-associated oxidative damage, but affect the cell-matrix adhesion in keratinocytes in extracellular matrix remodelling. In human keratinocytes, Kocbek et al.19 investigated the adverse effects of 25 nm-sized anatase TiO2 (5 and 10 |Jg/ml) after 3 months of exposure and found no changes in the cell growth and morphology, mitochondrial function and cell cycle distribution. The only change was a larger number of nanotubular intracellular connections in TiO2-exposed cells compared to non-exposed cells. Although the authors proposed that this change may indicate a cellular transformation, the significance of this finding is not clear. On the other hand, Dunford et al.23 studied the ge-notoxicity of UV-irradiated TiO2 extracted from sunscreen lotions, and reported severe damage to plasmid and nuclear DNA in human fibroblasts. Manitol (antioxidant) prevented DNA damage, implying that the genotoxicity was mediated by ROS.
Recently, Yanagisawa et al.74 reported that the transdermal exposure (mimicking skin-barrier dysfunction or defect) of NC/Nga mice to TiO2 NPs (15, 50, or 100 nm), in combination with allergen, aggravated atopic dermatitis-like lesions through a T-helper type 2 (Th2) dominant immune response. The study also indicated that TiO2 NPs can play a role in the initiation and/or progression of skin diseases, since histamine was released, even in the absence of allergen.
the relevance of this study for human exposure is not conclusive because hairless mice skin has abnormal hair follicles, and mice stratum corneum has higher lipid content than human stratum cor-neum, which may contribute to different penetration. Recently Sadrieh et al.84 performed a 4 week dermal exposure to three different TiO2 particles (uncoated submicron-sized, uncoated nano-sized and coated nano-sized) in 5 % sunscreen formulation with minipigs. They found elevated titanium levels in epidermis, dermis and in inguinal lymph nodes, but not in precapsular and submandibular lymph nodes and in liver. With the energy dispersive X-ray spectrometry and transmission electron microscopy (TEM) analysis the authors confirmed presence of few TiO2 particles in dermis and calculated that uncoated nano-sized TiO2 particles observed in dermis represented only 0.00008 % of the total applied amount of TiO2 particles. Based on the same assumptions used by the authors in their calculations it can be calculated that the total number of particles applied was 1.8 x 1013 /cm2 and of these 1.4 x107/cm2 penetrated. The surface area of skin in humans is around 1.8 m2 85 and for sun protection the cream is applied over whole body, which would mean that 4 week usage of such cream with 5 % TiO2 would result in penetration of totally 2.6 x 1010 particles. Although Sadrieh et al.84 concluded that there was no significant penetration of TiO2 NPs through intact normal epidermis, the results are not completely confirmative.
Skin-penetration studies
The skin of an adult person is, in most places, covered with a relatively thick (~10 |om) barrier of keratinised dead cells. One of the main questions is still whether TiO2 NPs are able to penetrate into the deeper layers of the skin.75 The majority of studies suggest that TiO2 NPs, neither uncoated nor coated (SiO2, Al2O3 and SiO2/Al2O3) of different crystalline structures, penetrate normal animal or human skin.76 77-82 However, in most of these studies the exposures were short term (up to 48 h); only few long-term or repeated exposure studies have been published. Wu et al.83 have shown that dermal application of nano-TiO2 of different crystal structures and sizes (4-90 nm) to pig ears for 30 days did not result in penetration of NPs beyond deep epidermis. On the other hand, in the same study the authors reported dermal penetration of TiO2 NPs with subsequent appearance of lesions in multiple organs in hairless mice, that were dermal exposed to nano-TiO2 for 60 days. However,
TiO2 NPs intake by food
TiO2 has been well accepted in the food industry and can be found as the E171 additive in various food products, mainly for whitening and texture. It is present in some cottage and Mozzarella cheeses, horseradish cream and sauces, lemon curd, and in low-fat products such as skimmed milk and icecream. Even if the product is labelled as containing E171, no information is usually given about the quantity, particle size and particle structure. FDA claims that TiO2 may be safely used as a colour additive for colouring foods in quantities up to 1 % by weight of the food.86 Interestingly, TiO2 is frequently declared as a "natural colouring agent" and is therefore well accepted by consumers.
TiO2 is also used in oral pharmaceutical formu-lations5, and the Pharmaceutical Excipients handbook considers nano-sized TiO2 a non-irritant and non-toxic excipient. Despite the fact that TiO2 submicron- and nano-sized particles are widely used as food and pharmaceutical additives, information
on their toxicity and distribution upon oral exposure is very limited.
Potential hazards of oral exposure to TiO2 NPs
The gastrointestinal tract is a complex barrier/exchange system, and is the most important route by which macromolecules can enter the body. The main absorption takes place through villi and mi-crovilli of the epithelium of the small and large intestines, which have an overall surface of about 200 m2. Already in 1922, it was recognized by Kumagai87, that particles can translocate from the lumen of the intestinal tract via aggregation of intestinal lymphatic tissue (Peyer's patch , containing M-cells (phagocytic enterocytes)). Uptake can also occur via the normal intestinal enterocytes. Solid particles, once in the sub-mucosal tissue, are able to enter both the lymphatic and blood circulation.
In an early study Jani et al.88 administred rutile TiO2 (500 nm) as a 0.1 ml of 2.5 % w/v suspension (12.5 mg/kg BW) to female Sprague Dawley rats, by oral gavage daily for 10 days and detected presence of particles in all the major gut associated lymphoid tissue as well as in distant organs such as the liver, spleen, lung and peritoneal tissue, but not in heart and kidney. The distribution and tox-icity of nano- (25 nm, 80 nm) and submicron-sized (155 nm) TiO2 particles were evaluated in mice administered a large, single, oral dosing (5 g/kg BW) by gavage.89 In the animals that were sacrificed two weeks later, ICP-MS analysis showed that the particles were retained mainly in liver, spleen, kidney, and lung tissues, indicating that they can be transported to other tissues and organs after uptake by the gastrointestinal tract. Interestingly, although an extremely high dose was administrated, no acute toxicity was observed. In groups exposed to 80 nm and 155 nm particles, histopathological changes were observed in the liver, kidney and in the brain. The biochemical serum parameters also indicated liver, kidney and cardiovascular damage and were higher in mice treated with nano-sized (25 or 80 nm) TiO2 compared to submicron-sized (155 nm) TiO2. However, the main weaknesses of this study are the use of extremely high single dose and insufficient characterisation of the particles.
Duan et al.90 administered 125 mg/kg BW or 250 mg/kg BW of anatase TiO2 (5 nm) intragastrically to mice continuously for 30 days. The exposed mice lost body weight, whereas the relative liver, kidney, spleen and thymus weights increased. Particles seriously affected the haemostasis of the blood and the immune system. The decrease in the immune response could be the result of damage to
the spleen, which is the largest immune organ in animals and plays an important role in the immune response. Powel et al.91 demonstrated that TiO2 NPs may trigger immune reactions of the intestine after oral intake. They showed that TiO2 NPs conjugated with bacterial lipopolysaccharide, but not TiO2 NPs or lipopolysaccharide alone, trigger the immune response in human peripheral blood mononuclear cells and in isolated intestinal tissue. This indicates that TiO2 NPs may be important mediators in overcoming normal gut-cell hyporesponsiveness to endogenous luminal molecules, which may be particularly relevant to patients with inflammatory bowel disease, which is characterized by an abnormal intestinal permeability.
The National Cancer Institute tested TiO2 for possible carcinogenicity by the oral route of exposure by feeding rats and mice with TiO2 (size not specified) at doses 25,000 or 50,000 ppm TiO2 for 103 weeks. They concluded that TiO2 was not carcinogenic.92 Also, the study with rats fed diets containing up to 5 % TiO2 coated mica for 130 weeks showed no treatment-related carcinogenicity.93 Since the size and other TiO2 properties were not specified or determined, we cannot generalize this conclusion and we have to take into account other possible outcomes of this scenario in different exposure conditions (other size/crystalline structure of TiO2 etc.).
It should also be considered that due to the low pH in the stomach, the increased dissolution of the TiO2 particles may increase its bioavailability and may facilitate the entry of titanium ions into the blood circulation.94 Despite the relatively large consumption of TiO2 as a food additive, no studies on the effect of pH on its absorption and bioavailabil-ity have been found in the literature. This can be attributed to a general belief that TiO2 is completely insoluble. However, this is not completely true, as TiO2 particles show a certain degree of solubility.33
Exposure to TiO2 NPs by inhalation
Inhalation exposure to TiO2 particles occurs predominantly in occupational settings during production of TiO2 powders and manufacturing the products containing TiO2.95 The highest levels of exposure occur during packing, milling and site cleaning however, the empirical data regarding airborne TiO2 particle concentrations in occupational settings is very limited. Fryzek et al.96 reported that packers, micronizers and addbackes had the highest TiO2 exposure levels measuring 6.2±9.4 mg/m3, whereas ore handlers had lower TiO2 exposure lev-
el of 1.1±1.1 mg/m3. Boffetta et al.97 reported that the yearly averaged estimated exposure to TiO2 dust in EU factories varied from 0.1 to 1.0 mg/m3, and the average levels ranged up to 5 mg/m3 for individual job categories. However, in these studies the particle size distribution has not been determined. Nevertheless, the data indicate that in certain jobs categories the exposure exceed the values of time-weighted average (10 h TWA) concentrations of 2.4 mg/m3 for submicron-sized TiO2 and 0.3 mg/m3 for nano-sized TiO2, which are recommended as exposure limits by National Institute for Occupational Safety and Health (NIOSH).98
Potential hazards of inhalation exposure to TiO2 NPs
The lung consists of about 2300 km of airways and 300 million alveoli. The epithelium of airways is protected by a viscous layer of mucus, and is a relatively robust barrier. In alveoli, the barrier between the alveolar wall and the capillaries is very thin, about 0.5 |jm. Thus, the large surface area of the alveoli and the intense air-blood contact in this region makes the alveoli less protected against environmental damage than other parts of the respiratory system.75 The clearance of particles from the upper airways is achieved through the mucociliary escalator, while clearance from the deep lung is supposed to be achieved predominantly by macrophage phagocytosis. Deposited particles can lead to the activation of cytokine production and inflammation by macrophages and epithelial cells. It has been reported that besides the pulmonary and systemic inflammation, inhaled insoluble NPs can also accelerate atherosclerosis and alter the cardiac autonomic function.99-102
Following administration of nano-sized TiO2 to rats by inhalation the particles were detected in the cytoplasm of all lung-cell types in a non-membrane bound manner.17 Ferin et al.103 reported that 20 nm-sized TiO2 particles penetrate more easily into the pulmonary interstitial space of rats than 250 nm-sized TiO2 particles. Three-month inhalation exposure in rats demonstrated that the clearance of 20 nm TiO2 particles was significantly slower than that of 200 nm TiO2 particles, and more particles translocated to interstitial sites and regional lymph nodes.104 Geiser et al.14 confirmed that alveolar macrophages were not primarily responsible for the uptake and clearance of TiO2 NPs. These findings are in agreement with the known size limitations of uptake processes such as phagocytosis, which is thought to be restricted to particles that are 1 to
5 |om in size, while NPs might escape macrophage phagocytosis.101105
Inhaled TiO2 NPs can enter the alveoli of the lung and consequently the blood circulation106107 and can then translocate to other organs.102 1 08 1 09 In addition to several reports on the absence of toxic-ity following the inhalation of TiO2 NPs in rodents, the majority of lung-inhalation and instillation studies have pointed out obvious toxic effects, like inflammation and damage to pulmonary epithe-lium.110 The studies also showed that TiO2 NPs induced greater pulmonary inflammation and tissue damage than an equal dose of submicron-sized TiO2 particles. The greater toxicity of TiO2 NPs has been explained as being related to their larger surface area and their increased internalization.111 Multiple studies showed the reversibility of the inflammatory response after cessation of the exposure to TiO2 particles. After a single instillation exposure to different types of submicron- and nano-sized TiO2, acute inflammatory response returned to control levels within one week112 or 90 days113 after the instillation. In mice that were exposed to TiO2 NPs (2-5 nm) by whole body inhalation (0.77 or 7.22 mg/m3 4 h/day 10 days) the recovery was observed during the third week after exposure.114
Pulmonary toxicity studies suggest that, besides the particle size and surface area, crystal structure and surface treatment are also important parameters. Warheit et al.115 demonstrated higher pulmonary toxicity of anatase than rutile TiO2 NPs. These observations were confirmed in a recent study by Roursgaard et al.116 who showed that the intrat-racheal instillation of submicron- and nano-sized rutile, nano-sized anatase, or amorphous TiO2 to mice induced a dose-dependent acute inflammation, while subchronic inflammation was apparent only in mice exposed to nano-sized rutile and amorphous TiO2.
Recently, toxicogenomic studies were published that may contribute to a better understanding of the mechanisms of TiO2-mediated pulmonary tox-icity. In mice exposed to a single intratracheal dose (0.1 or 0.5 mg/kg BW) of TiO2 with an average particle size of 20 nm Chen et al.117 showed that changes in the morphology and histology of the lungs were associated with the differential expression of hundreds of genes, including those involved in cell cycle regulation, apoptosis, chemokines, and complement cascades. In particular, TiO2 NPs upregu-lated the expression of the placenta growth factor and other chemokines that are associated with pulmonary emphysema and alveolar epithelial cell ap-optosis. Park et al.118 showed that exposure of mice
to nano-sized TiO2 (5-50 mg /kg BW) by a single intratracheal instillation can, in addition to chronic inflammation, also trigger an autoimmune response. They found that many classes of genes related to antigen presentation and the induction of chemotaxis of immune cells were over-expressed.
The studies have shown that submicron-sized TiO2 119 and nano-sized TiO2 120121 induce lung tumors in chronically exposed rats. TiO2 NPs induced a significantly increased number of lung tumors during inhalation exposure to 10 mg/m3 (18 h/day, 2 years) , while submicron-sized TiO2 increased the number of lung tumors at exposure to 250 mg/m3 (6 h/day 2 years). In contrast, no tumours were observed in similarly exposed mice and ham-sters.121122 These apparent species differences suggest that the experimentally induced lung tumours may be a rat-specific, threshold phenomenon, depending on lung overloading accompanied by chronic inflammation to exert the observed tumori-genic response. Comparative toxicological studies of the development and possible progression of the lung response in rats, mice and hamsters exposed to a range of concentrations of submicron- or nano-sized TiO2 over a period of 90 days showed distinct species differences in the lung responses. Rats and mice had similar lung burdens and clearance rates, while hamsters showed higher clearance rates. At high lung-particle burdens, rats showed a marked progression of the histopathological lesions during the post-exposure period, while mice and hamsters showed minimal initial lesions with apparent recovery during the post-exposure period.123,124 It has been thus argued that the dose response data from inhalation studies in rats should not be used when extrapolating the cancer risk to humans.95 However, clearance of insoluble particles is in humans slower than in rats.125 In addition, it has been shown that the lung-tumour response to exposure to non-soluble particles can be predicted by the particle surface area dose without the need to account for overloading.98 Therefore, for workers with a high dust exposure the doses that cause overloading in rats may be relevant for estimating the health risk for humans.
Animal studies showed also other adverse effects after inhalation exposure to TiO2 particles. Nurkiewicz et al.109 showed that exposure to TiO2 particles may cause cardiovascular effects at concentrations below those causing adverse pulmonary effects. In rats exposed to submicron-sized TiO2 (<1 |om) or nano-sized TiO2 (21 nm) at airborne exposures aimed at achieving similar particle mass deposition in the lungs (nano-sized: 1.5-12 mg/m3,
240-720 min; submicron-sized: 3-15 mg/m3, 240-480 min) they observed systemic microvessel dysfunction in the absence of pulmonary inflammation or lung damage. The effect was related to the adherence of polymorphonuclear leukocytes to the microvessel walls and the production of ROS in the microvessels. As already described previously, inhalation exposure to TiO2 NPs may cause immune responses and neurotoxic effects that may lead to respiratory allergy and higher risk of developing asthma, respectively.
It has been reported that TiO2 NPs can translocate to the central nervous system following nasal instillation, potentially via the olfactory bulb, and accumulate mainly within the cerebral cortex, thalamus and hippocampus.2229126 The absorption appears to occur via neuronal transport, bypassing the blood-brain barrier.29126 The main target is the hippocampus, where TiO2 NPs caused morphological alteration and the loss of neurones. In addition, TiO2 induced oxidative stress and an inflammatory response within the whole brain, with anatase nano-TiO2 inducing a stronger inflammatory response than rutile. However, from these studies it is not clear to what extent large local doses during nasal instillation reflect inhalation exposure.
Human epidemiological studies
Several case reports described adverse health effects in workers with potential TiO2 exposure that later lead to epidemiological studies of a relationship between occupational exposure and observed cases.98 The lung particle analyses indicated that workers exposed to respirable TiO2 had particle retention in their lungs that included TiO2, silica, and other minerals, sometimes years after cessation of exposure. In most cases of tissue-deposited TiO2 was associated with a local macrophage response and fibrosis that was generally mild. In one case papillary adenocarcinoma and TiO2 associated pneumoconiosis was reported in the lung of a 53-year-old male who had been engaged in packing TiO2 for about 13 years and had 40-year smoking history.127 The cohort epidemiological studies undertaken in the USA 96128 did not report excess risks of lung cancer; nor did a Canadian population-based case-control study.129 The retrospective cohort lung cancer mortality study130, which included workers in the TiO2 production industry in six European countries, showed a small but significant elevation in lung cancer mortality among male TiO2 workers when compared to the general
population. However, the data did not suggest an exposure-response relation.
TiO2 has been classified by the International Agency for Research on Cancer (IARC) as an IARC Group 2B carcinogen, ''possibly carcinogenic to humans'' by inhalation.131 Although the IARC working group concluded that the epidemiologi-cal studies on TiO2 provide inadequate evidence of carcinogenicity, they considered that the results from animal studies of inhalation and intratracheal instillation provide sufficient evidence to classify TiO2 in Group 2B.132 Also NIOSH98 has recently classified TiO2 NPs as a potential occupational carcinogen but considered that there is insufficient evidence at this time to classify also submicron-sized TiO2 as a potential occupational carcinogen. NIOSH also recommended new exposure limits at 2.4 mg/m3 for submicron-sized TiO2 and 0.3 mg/m3 for nano-sized TiO2, as time-weighted average concentrations for up to 10 hours per day during a 40-hour work week.
Exposure to TiO2 NPs through body implants
A few-nanometres-thick layer of amorphous TiO2 is commonly formed on the surface of orthopaedic and dental implants made of titanium metal or its alloys. In non-moving implants (hip stems, plates, screws, etc.) this does not appear to represent the same kind of risk for the body as free TiO2 NPs discussed in previous sections. However, this is not the case for wear-exposed implants, such as hip and knee joints. There are many reports proving that under mechanical stress or altered physiological conditions, Ti-based implants can release biologically relevant amounts of debris, in both the micrometre and nanometre ranges, that can migrate to the surrounding tissues. During the wear process, a thin amorphous oxide layer is continuously being created and removed, resulting in large numbers of titanium particles. It is increasingly being suggested that they are associated with major inflammation and systemic diseases.133 Furthermore, increasing numbers of reports indicate that the delayed hypersensitivity to titanium and its oxides may constitute a health risk for individuals with higher susceptibility.134-136
The effects of the TiO2 particles released from implants were investigated by Wang et al.137 in rats by intra-articular injection of 0.2 to 20 mg of anatase nano-TiO2 per kg BW. Their results demonstrate that particles can potentially affect major organs like the heart, lung and liver.
Generally, the maximum diameter of particles that move across the synovial capillary wall was suggested to be 50 nm. The released TiO2 NPs resulted in synovial hypotrophy, lymphocyte and plasma infiltration, and fibroblast proliferation in the knee joint. Oxidative stress and lipid peroxidation was detected in exposed synovial fluid. Seven days after the initial exposure a brown particulate deposit was observed in vascular endothelial cells and in alveolar macrophages. Similar results have been reported by Urban et al.138, who found TiO2 particles in the liver and in the spleen of the patients with implants. TiO2 NPs were observed in joint simulators and in joint periprosthetic tissues. Margevicius et al.139 characterized the debris around the total hip joint prosthesis and found up to 140.109 particles/g dry weight, in diameters ranging from 0.58 to 100 |jm. Agins et al.140 found concentration of wear particles in the tissue adjacent to a prosthesis in the range between 56 |Jg/g and 3.7 mg/g dry weight. Thus, due to the natural tendency of titanium to oxidise, Ti-based implants should not be neglected as a possible source of TiO2 exposure.
On the other hand, the man-made (crystalline) TiO2 coatings on the surfaces of pure Ti or Ti alloys are reported to be able to modulate protein absorption, cell adhesion, osseointegration and bone mineralization at the bone-biomaterial interface, both in vivo and in vitro.141,1442 For this reason, the development of a more stable crystalline titania coating on Ti-based implants is in progress.143
Environmental pollution by TiO2 NPs
Toxic effects of TiO2 NPs on aquatic organisms
The trend in the production of NPs is likely to lead to increasing amounts of nano-powders in the air, water and soil, which will consequently affect living organisms. Labielle et al.68 demonstrated that 25 % of Al(OH)3-coated TiO2 particles from sunscreens are dispersed as a stable colloid and become available to microorganisms and filterfeeders, while the remaining 75 % are probably incorporated into geogenic sediments, where they could become available to benthic fauna. Solar UV iradiation may penetrate as far as 20 m in the water column 144 and therefore photo-activate the dispersed particles, which may have an adverse effect on various aquatic organisms.
Freshwater algae show low-to-moderate susceptibility to TiO2 exposure, with more pronounced
toxic effects in the presence of UV irradiation. It has also been shown that nano-sized TiO2 is significantly more toxic to algae Pseudokirchneriella sub-capitata than submicron-sized TiO2.145 Hund-Rinke and Simon 146 reported that UV irradiated 25 nm TiO2 NPs are more toxic to green freshwater algae Desmodesmus subspicatus than UV irradiated 50 nm particles, which is in agreement with Hartmann et al.147 UV irradiated TiO2 NPs also inactivated other algae species such as Anabaena, Microcystis, Melsoira148 and Chroococcus.149 It was demonstrated that smaller particles have a greater potential to penetrate the cell interior than submicron-sized particles and larger aggregates. Studies have shown that the amount of TiO2 adsorbed on algal cells can be up to 2.3 times their own weight.142
Nano-sized TiO2 generally shows low or no acute toxicity in both invertebrates146 and verte-brates.150 However, exposure of Daphnia magna to 20 ppm TiO2 for 8 consecutive days was found to cause 40 % mortality.151 Zhu et al.152 showed minimal toxicity to D. magna after 48 h exposure, while upon chronic exposure for 21 days, D. magna suffered severe growth retardation and mortality. A significant amount of nano-sized TiO2 was found also accumulated in the body of the animals. Similar findings with coated nano-sized TiO2 (T-Lite™ SF, T-Lite™ SF-S and T-Lite™ MAX; BASF SE) were reported by Wiench et al.153 Biochemical measurements showed that exposure to TiO2 NPs induces significant concentration-dependent antioxidant enzyme activities in D. magna154. Lee et al.155 showed that 7 and 20 nm-sized TiO2 induced no genotoxic effect in D. magna and in the larva of the aquatic midge Chironomus riparius.
No acute effects of nano-sized TiO2 were observed in Danio rerio (zebrafish) embryos.156 Exposure of rainbow trout to TiO2 NPs triggered lipid peroxidation, influence on the respiratory tract, disturbance in the metabolism of Cu and Zn, induction of intestinal erosion 157 and accumulation in kidney tissue.158 Linhua et al.159 exposed juvenile carp to 100 and 200 mg/ml of TiO2 particles and observed no mortality. However, the fish suffered from oxidative stress and pathological changes in gill and liver. In the infaunal species Arenicola marina, exposure to TiO2 NPs in sediment caused sub-lethal effects including decrease in casting rate and increase in cellular and DNA damage.160 Aggregated particles were visible in the lumen of the gut, but no uptake through the gut or the skin was observed.
Zhu et al.161 were the first to provide evidence that TiO2 NPs (21 nm) can transfer from daphnia to
zebrafish by dietary exposure. Hence, dietary intake could be a major route of exposure to NPs for high trophic level aquatic organisms. Ecological research should therefore focus, not only on the concentration of NPs in the environment, but also on its bioconcentration, bioaccumulation and biomag-nification. In addition it has been shown that TiO2 NPs can increase accumulation of other environmental toxicants: enhanced accumulation of cadmium (Cd) and arsenic (As) was found in carp in the presence of TiO2 NPs.162 163 The strong adsorption capacity for Cd and As was explained by the large specific surface area and strong electrostatic attraction of TiO2 NPs that contribute to facilitated transport into different organs.
In vitro, in the hemocytes of the marine mussel Mytilus hemocytes, suspension of TiO2 NPs (Degussa P25, 10 |jg/ml) stimulated immune and inflammatory responses, such as lysozyme release, oxidative burst and nitric oxide production.164 Vevers and Jha165 demonstrated the intrinsic genotoxic and cytotoxic potential of TiO2 NPs on a fish-cell line derived from rainbow-trout gonadal tissue (RTG-2 cells) after 24 h of exposure to 50 |jg/ml. Reeves et al.166 demonstrated a significant increase in the level of oxidative DNA damage in goldfish cells, and suggested that damage could not repaired by DNA repair mechanisms. Another suggestion from the mentioned study was that hydroxyl radicals are generated also in the absence of UV light. It has been shown that fish cells are generally more susceptible to toxic/oxidative injury than mammalian cells.
Toxic effects of TiO2 NPs on soil organisms
Drobne et al.167 used the terrestrial arthropod Porcellio scaber as a test organism for determining the cytotoxic effect of TiO2 NPs (anatase). The animals were exposed to TiO2 NPs of two different sizes (25 nm and 75 nm) in the concentration range 10-1000 |jg TiO2/g dry food for 3 to 14 days. No adverse effects, such as mortality, body weight changes or reduced feeding, were observed. In fact, quite the opposite, an enhanced feeding rate, food absorption efficiency and increase in catalase activity were observed. The intensity of these responses appeared to be time- but not dose-dependent. It should also be noted that the concentrations tested in this study were much higher than the predicted concentration (4.8 |Jg/g soil) at high emission scenario of nano-sized TiO2.168 Using the same test organism another group169 showed that exposure to
TiO2 NPs induced destabilization of cell membrane in the epithelium of digestive glands isolated from exposed animals. They also showed that this effect can be observed after just 30 minutes of exposure.
TiO2 NPs appeared to be more toxic to nematode Caenorhabditis elegans than submicron-sized TiO2.170 At a concentration of 1 mg/l, 7 nm particles affected its fertility and survival rate and were more toxic than 20 nm anatase particles.171 Similarly, Hu et al.172 showed that rutile particles (10-20 nm), at concentrations above 1 g/kg soil, can be bio-accumulated in earthworms, where they induce oxidative stress, inhibit the activity of cellulase and induce DNA and mitochondrial damage.
The effects of TiO2 NPs in plants
In addition to the toxic effects of TiO2 NPs, discussed in previous chapters, these NPs have been also shown to promote photosynthesis and nitrogen metabolism, resulting in the enhanced growth of spinach.173-175 It increases the absorption of light and accelerates the transfer and transformation of the light energy.176 It was also found that treatment with nano-sized TiO2 significantly increased the level of antioxidant enzymes, and decreased the ROS accumulation and malonyldialdehyde content in spinach chloroplasts under visible and UV irradiation.177 TiO2 NPs also increased the superoxide dismutase activity of germinating soybean, enhanced its antioxidant ability, and promoted seed germination and seedling growth.178
Potential desirable effects of TiO2 NPs
The same properties of nano-sized TiO2 that are associated with undesirable, harmful effects can be exploited for certain useful applications. The antimicrobial effect of photo-activated TiO2 NPs has been known since 1985179 and since then numerous reports have described its potential antimicrobial activity against numerous microorganisms.180 As expected, the antimicrobial effect increases with smaller particle sizes181; however, powder agglomeration may obscure this effect.151 When submitted to UV-C irradiation, TiO2 depresses the photo-activation and dark repair of DNA in bacteria, which increases the bactericidal efficiency of UV-C irra-
diation.182
TiO2 NPs have potential application in removing or minimizing the effect of the red tides183 that are associated with the harmful algae K.brevis that
produces neurotoxic brevetoxin (PbTxs). Further, it can be used for disinfecting water, air and surfaces, with possible applications of TiO2 in form of solid films or free particles. Given its use for eradicating toxins, pollutants and spores from water and air, it can be classified as a broad-spectrum oxidizing/cleaning substance. However, an informed balance between the benefits of such a cleaning system and its potential adverse effects needs to be maintained.
NPs are offering new possibilities for in medicine either for diagnostic or therapeutic purposes. For instance recent studies indicate that magnetic NPs may be used in cancer treatment for targeted drug delivery.184 Several recent studies indicated that also cultured cancer cells are more sensitive to TiO2 NPs than normal cells.. Photo-activated TiO2 exhibited selective cytotoxicity against highly malignant breast-cancer cells MDA-MB-468, in comparison with non-malignant MCF-7 cells.185 Similarly, UV-irradiated Degussa P25 TiO2 NPs reduced viability of sarcoma cells but were not toxic to cultured fibroblasts MCR-5.186 In addition, UV-C photo-activated TiO2 particles inhibited aggregation of sarcoma cells with human platelets, thus preventing the formation of metastases. Cai et al.187 found that photo-activated (50 |jg/ml), but not non-irradiated nano-sized TiO2, was lethal for HeLa cells in vitro and suppressed the growth of HeLa tumours in nude mice. Photo-activated TiO2 also showed antitumour activity in vivo against murine skin tumours.188 The potential usefulness of nano-sized TiO2 in cancer cell therapy has also been reported by other research groups.189-192 Cytotoxicity against different cancer cell lines appears to depend on the cell type, the particle concentration and the surface chemistry.
The appearance of multidrug-resistant tumour cells is a major obstacle to the success of chemotherapy. Song et al.193 reported an enhanced effect of nano-sized TiO2 on drug uptake by drug-resistant leukaemia cells under UV irradiation. Very promising is also the finding that cancer cells can be effectively destroyed by the use of X-ray irradiated nano-sized TiO2.194 A combination of monoclonal antibody conjugated nano-sized TiO2 with photoinduction195 and electroporation196 have also been proposed for selective cancer treatment. The monoclonal antibodies would enable selective targeting of cancer cells, photoinduction would trigger local generation of radicals and electroporation would accelerate the delivery of nano-sized TiO2 into the cancer cells. A novel possibility of cancer treatment was recently suggested197, in which TiO2
NPs and folic acid were coupled and shown to be internalized by HeLa cells via the folate receptor.
Where we are and where to go
The mechanistic toxicological studies showed that TiO2 NPs induced adverse effects are predominantly mediated by oxidative stress, which may lead to cell damage, genotoxic effects, inflammatory responses and changes in cell signalling. The studies also showed that these effects strongly depend on numerous chemical and physical characteristics of the TiO2 particles: size, crystal structure, specific surface area, particle shape, purity, surface charge, solubility, agglomeration rate, photo-activation, etc. TiO2 particles are without doubt associated with the hazardous properties, and the risk for human health and environment depends on the route and extent of exposure.
Based on the widespread use of creams with SPF based on nano-sized TiO2, human exposure to TiO2 NPs by dermal applications is apparently enormous. In vitro studies with skin models showed that TiO2 NPs are taken up by keratinocytes, fi-broblasts, and melanocytes, in which they cause toxic effects that are not different from the effects observed in other cell types. Current experimental evidence indicates that TiO2 NPs do not penetrate through healthy skin and thus do not reach viable skin cells and distribute to other organs and tissues. However, the data on TiO2 NPs skin penetration during long-term or repeated exposure and in the presence of UV, which is actually characteristic for real life exposure, are insufficient. Therefore, there is no simple answer to the question regarding safety of the use of TiO2 NPs in sunscreens. The safety of the use of TiO2 in cosmetics is often argu-mented by the claim, that it has been used for decades without observing any adverse effects on human health. This, however, is not completely true, as no monitoring and post market health surveillance has been conducted, neither for submicron-sized nor for nano-sized TiO2 in sunscreens. Such surveillance is currently impossible, since current legislation does not require labelling whether the products contain nano-sized TiO2, which is also incorrect to customers who have no possibility to make a choice whether to use or not the sunscreen containing nano-sized TiO2. In our opinion dermal applications of TiO2 NPs as sunscreen should be limited until appropriate long-term experimental studies confirm their harmlessness. It is undeniable that long-term sun exposure can induce skin
cancer. It is questionable, however, whether people are, by using sunscreens, actually encouraged to expose themselves to the sun instead of avoiding it, and if the benefit provided by TiO2 as a protection from UV compensates for the potential harm.
The available data on absorption, distribution, elimination or any consequent adverse effects after oral exposure to specific TiO2 NPs are extremely limited. TiO2 NPs have been shown to be absorbed from gastrointestinal tract and distributed to other organs, however this was observed at extremely high, for human exposure, irrelevant doses. On the other hand, it has been shown that at lower concentrations TiO2 may induce different adverse effects. TiO2 is an approved food additive with the limit set at 1 % by weight of the food; however, neither the size nor the structure is defined. It has been estimated that the average daily exposure to TiO2 from food, medicines and toothpaste is around 5 mg/in-dividual (i.e., about 0.07 mg/kg BW)198, which is a much lower dose than those that showed adverse effects in experimental animals. Currently there is no data if, and what proportion of TiO2 NPs is absorbed at doses relevant for human exposure, and how different food matrices affect behaviour and absorption of TiO2 NPs. However, even if very small portion of consumed nano-sized TiO2 is absorbed from gastrointestinal tract and distributed to distant organs, this brings into question accumulation of TiO2 NPs that may, through a constant lifetime oral exposure, reach concentrations that would trigger adverse effects. Another important question, which should not be neglected is, whether low exposure may trigger symptoms in subjects with an underlying susceptibility. Before in vivo toxicokinetic data for nano-sized TiO2 are available, no conclusion about the risk of nano-sized TiO2 by oral exposure is possible. Therefore, it should be seriously reconsidered if the use of TiO2 NPs in nutrition and pharmacy just to shade or stabilise the products is justified at all.
Inhalation seems to be the most vulnerable entrance point of the TiO2 NPs and the toxic effects of inhalation exposure are therefore by far the most studied. Animal studies showed that on inhalation exposure the particles deposit in the lung, where they may cause chronic inflammation and lung-tissue damage, which can lead to lung-tumour development. The important finding is that inhalation exposure to nano-sized TiO2 represents a higher health risk than exposure to submicron-sized TiO2 particles. Experimental data indicate that on inhalation exposure nano-sized TiO2 may translocate to distant organs and tissues, which may be associ-
ated with systemic effects, such as allergy, asthma and cardiovascular effects, however further studies are needed to confirm these observations and to clarify if they are associated with increased risk for humans. In the scientific community there is still a debate whether the data from in vivo rodent toxicity studies are reliable enough to predict the effects in humans in particular regarding mode of exposure (instillation vs. inhalation exposure) and the differences in susceptibility between different experimental species. Nevertheless, the experimental evidence, although not clearly supported by human epidemiological data, was considered to be sufficient to classify TiO2 (unrespect-able to particle size and form) as "possible human carcinogen" upon inhalation exposure by IARC. Recently also NIOSH classified nano-sized, but not submicron-sized TiO2 as occupational carcinogen, and accordingly established different limit values for occupational inhalation exposure for nano-sized (0.3 mg/m3) and submicron-sized (2.4 mg/m3) TiO2. At present, through environmental air pollution general population is probably not at risk. However, occupational exposure should be controlled and protective measures applied, not only in TiO2 production industries, but also in certain areas of TiO2 applications; for instance when removing paints or destroying TiO2 containing materials the workers may be exposed to high concentrations of TiO2. Thus, accurate, portable, and cost effective measurement techniques should be developed and applied for effective exposure control and protection.
TiO2 can also be released within the human body as a result of the wear of Ti-based implants. The released particles cause local inflammation, but even more importantly they distribute over the body and can potentially cause systemic effects. Generally the benefit provided by the implant compensates for the potential harm, in particular in the cases where there is no better alternative to the Ti-based implants available. However, although there is no direct experimental evidence that released TiO2 can be deposited in the body or can cause systemic effects, it can be postulated from other exposure studies and mechanistic data that at least for individuals with hypersensitivity to titanium such exposure may represent a permanent health threat. Thus, it should be obligatory to test the patients for titanium hypersensitivity prior to implantation of titanium based implants.
Due to the widespread use TiO2 can enter aquatic and terrestrial environment and potentially affect the indigenous organisms. Although data from
acute ecotoxicity tests in crustaceans, fish and algae indicate a low toxic potential of TiO2 NPs for aquatic species, when chronic exposure was applied TiO2 NPs induced a range of sub-lethal adverse effects. In addition it has been shown that na-no-sized TiO2 can enter the freshwater food chain, which means that it can be transferred from lower to higher trophic organisms, including humans.
Taken together, the overall exposure of an average individual TiO2 NPs is not known; there are still opened questions regarding toxicokinetics and specific organ toxicity of TiO2 NPs, in particular at oral and dermal exposure, and thus it is impossible to make a reliable quantitative risk assessment. One of the main observations of this review is that, due to the versatility of the TiO2 NPs in terms of particle size, shape, crystal structure, dispersion in biological surroundings (bioavailability) and UV-induced photocatalytic activity, no single conclusion can be drawn, since different forms of TiO2 may act very differently. Until we know more, in our opinion TiO2 NPs should be used with great care, in particular in food and cosmetics. The least that should be done for the consumer is that a declaration of nano-sized TiO2 in these products is obligatory, so that we will have the choice whether to use it or not.
Acknowledgement
The study was supported financially by the Slovenian Research Agency within the research programmes "Nanostructured Materials" (P2-0084) and "Ecotoxicology, Toxicological Genomics and Carcinogenesis (P1-0245). We thank Prof. Roger Pain and Assist. Prof. Paul McGuiness for critical reading of the manuscript. Authors would also like to thank Dr. Zoran Samardžija for field-emission-gun scanning electron microscopy observations of the TiO2 powders.
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Assessing renal function in children with hydronephrosis - additional feature of MR urography
George Hadjidekov12, Savina Hadjidekova3, Zahari Tonchev12, Rumiana Bakalova24, Ichio Aoki4
1	Department of Radiology, University Hospital "Lozenets", Sofia, Bulgaria
2	Department of Physics, Biophysics & Radiology, Medical Faculty, University Sofia University "St. Kl. Ohridski", Sofia, Bulgaria
3	Department of Medical Genetics, Medical University, Sofia, Bulgaria
4	Molecular Imaging Center, National Institute of Radiological Sciences (NIRS), Chiba, Japan.
Received 21 April 2011 Accepted 5 October 2011
Correspondence to: Assist. Prof George V. Hadjidekov, MD, Department of Radiology, University Hospital "Lozenetz" -1, Koziak str. Sofia 1407, Bulgaria. Tel.: +359 898 797612; Fax: +359 2 962 4771; E-mail: jordiman76@yahoo.com
Disclosure: No potential conflicts of interest were disclosed.
Background. Magnetic resonance urography (MRU) is one of the most attractive imaging modalities in paediatric urology, providing largest diagnostic information in a single protocol. Therefore, the aim of our study was to assess the diagnostic value of MRU in children with urogenital anomalies (especially anomalies of the renal pelvis and ureter) and the renal function using different post-processing functional software.
Patients and methods. Ninety six children (7 days - 18 years old) were examined. In 54 patients of them, a static T2 MRU was completed by excretory T1 MRU after gadolinium administration and functional analysis has been performed using two functional analysis softwares "CHOP-fMRU" and "ImageJ" software.
Results. MRU showed suspicious renal and the whole urinary tract anomalies with excellent image quality in all children. In ureteropelvic obstruction, MRU was confirmatory to the other imaging techniques, but it was superior modality concerning the evaluation of end-ureteral anomalies. There was an excellent correlation between the MRU data and diagnosis, determined by surgery. The renal transit times, renal volumes and volumetric differential renal function were assessed separately by "CHOP-fMRU" and "ImageJ" with excellent agreement with 99mTc-DTPA and among them. Conclusions. MRU overcomes a lot of limitations of conventional imaging modalities and has a potential to become a leading modality in paediatric uroradiology. Synthesis of both anatomical and functional criteria in MR urography enables to select the best candidates for surgical treatment. Even small kidney dysfunction can be detected by functional analysis software.
Key words: MR urography; children; functional analysis; urinary tract
Introduction
The imaging of urinary tract is important clue in paediatrics. Different methods for evaluation of the genitourinary system are routinely used in the clinic. However, there is no single method providing the whole information, necessary for the diagnostic. The conventional methods have many limitations. For example: ultrasound examination is operator-dependant, with sometimes difficult visualization of the end-ureter; in intravenous urogra-phy, there is a risk of contrast media and ionizing
radiation; retrograde methods are invasive with limited application; scintigraphy has a poor anatomical resolution.1
Novel methods have developed to overcome the limitations of the conventional methods and MR urography (MRU) is one of the most attractive. MRU is a promising method for early diagnosis, having an impact on the management of congenital malformations and other urogenital anomalies in children.1 This diagnostic modality provides a detailed visualization of various morphologic abnormalities of the genitourinary system and avoids
radiation, which is mutagenic.12 To avoid ionizing radiation is one of the most important diagnostic approaches in children.3
Currently, MRI is used in paediatrics for assessment of the congenital abnormalities of the genitourinary system, different cases of obstruction of the excretory system and evaluation of renal tumours, which are prevalent solid tumours in infants.14 In addition to the morphological imaging, MRI can be used to quantify the renal function. Following contrast administration and using appropriate software, time-intensity curves can be generated and other parameters (e.g., renal transit times, renal volumes and differential renal function) can be quantified.1 This is the reason some authors to define MRI as a potential "one-stop-shop" imaging technique for a variety of renal diseases.6-8 In the present study, we assess the diagnostic value of MRU in a cohort of paediatric patients with various urogenital anomalies (especially with anomalies of the renal pelvis and ureter) using two post-processing functional software "CHOP-fMRU" and ImageJ and in comparison to 99mTc-DTPA scintigraphy.
Materials and methods
Patient population
We retrospectively reviewed all 96 children (age: between 7 days and 18 years) referred from the Department of Urology and Paediatrics, between 2006 and 2010 with suspected congenital urinary tract anomalies, controversial findings from the conventional imaging studies and difficulties to establish the final diagnosis. In 54 of them an excretory, T1 MRU after contrast administration of gadolinium has been performed for renal function assessment in addition to T2 MR urography. In the remaining 42 patients, static T2 MR urography has been employed in order to confirm conditions affecting the urinary tract without impact on the renal function, co-existing renal pathology or due to contraindications for gadolinium (Gd) injection in cases of renal failure. The frequency of age distribution in the patient population was as follows: 0 day - 1 month: 7 patients (7.3% from the whole study group of 96 patients); 1 month - 1 year: 29 patients (30.2%); 1 year - 6 years: 18 patients (18.8%); 6 years - 14 years: 15 patients (15.6%); 14 years - 18 years: 27 patients (28.1%).
Cross-sectional sequences, MR angiography in the arterial and venous phase, serial evaluation of the renal parenchymal perfusion and contrast-en-
hanced MRU were combined in one imaging session instead of lining up several different imaging modalities. Time-intensity curves were generated, based on the dynamic 3D post-contrast sequences. "CHOP-fMRU" and ImageJ analysis software was used for calculation the functional curves, plots and maps, renal transit times, renal volumes and differential renal function.510 In all cases, an informed consent was obtained after the procedure was fully explained to the parents and older children and the study was approved by the Ethics Committee of the University Hospital "Lozenets", Sofia, Bulgaria.
Ultrasonography was conducted in all patients prior to MRU examination. Voiding cystourethrog-raphy (VCUG) was performed in 10 children with suspicion for dilatation of urinary tract in accordance to vesicoureteral reflux (VUR). In 8 children intravenous urography (IVU) has been previously done and in 19 cases 99mTc-DTPA scintigraphy as a part of the uroradiological work-up has been done with a delay prior or after the MRI exam no longer than 1 month, in another institution. The 99mTc-DTPA protocol was similar to our MRU protocol in terms of hydration with intravenous administration of 10 ml/kg sodium chloride solution 30 min prior to the scan. The amount furosemide (1 mg/ kg, i.v.) was the same as in our examination, although diuretics have been given when maximum pelvicalyceal distension was observed (usually 1015 min after administration of 99mTc-DTPA).
Patient preparation
The adequate preparation is a prerequisite for a good image quality.5-11 We didn't place routinely a bladder catheter, although catheterisation of small children is recommended in case of megaureter (with or without reflux). We used catheter in few patients with suspected VUR, but due to technical problems we abandoned this procedure. Then we started to scan without catheterization and we were happy with cooperative, toilet-trained children, without cases of severe discomfort or inability to conduct the examination. The intravenous hydration and administration of furosemide are crucial for reducing the concentrations of Gd.10 Diuretics are recommended in both static urog-raphy and dynamic urography before contrast administration. In this context, we administered standardised hydration (10-15 ml/kg sodium chloride or Ringer's solution) and diuretics (furosemide - 1 mg/kg, max. dose 20 mg) 15 min prior to Gd injection, in order to reduce artefacts, to distend
Coronal T2 native image
(without contrast probe)
MIP image in arterial phase MIP image during excretion
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FIGURE 1A-C. Normal MR urogram in 5-year-old boy. A. Coronal T2 native image of both kidneys. B,C. MIP images from two separated time-points of the excretory post-contrast MRU in arterial phase (B) and during excretion (C).
the urinary tract, to maintain the linearity between signal intensity and concentration of Gd and to shorten the examination time, adopting the F-15 protocol, proposed by Grattan-Smith.12 In children younger than 6-year-old and non-cooperative for breath-hold techniques, successful sedation was performed with ketamine (Ketalar) and midazolam (Dormicum) according to the department's standard sedation protocol with no serious adverse effects. In 15 patients intravenous sedation (Ketalar - 1 mg/kg or Thiopental 4-5 mg/kg) was successfully performed with minor motion artefacts in 2 infants without any impact on the diagnostic value of the image quality. Oral sedation using midazolam (Dormicum - 0.5 mg/kg) was sufficient to perform MRU with excellent diagnostic image quality in 32 patients and there was no major complaint of nausea and vomiting that could be related to an-tiemetic effects of midazolam.13-14 The blood pressure, respiration, heart rate, and oxygen saturation were continuously monitored throughout MR imaging in all patients.
MRU protocol
High-field strength tomographs (1.5 Tesla) (Signa, General Electric Medical Systems and Magnetom Essenza, Siemens Medical Solutions) were used with large field of view (FOV) above diaphragm to avoid artefacts from aliasing or post-contrast signal intensity decline in the upper renal poles and obtain an oblique coronal plan angled parallel to the long axis of the kidneys, including ureters and bladder. Our MRU protocol consisted of
native MR examination with T2 coronal, T1 and T2 axial sequences, followed by dynamic study with Gd injection, administration of furosemide prior to the dynamic acquisitions and 3D reconstructions. Following the coronal T2 plan, we performed axial T2 and T1 sequences. Fat-suppression techniques were used in T1 and T2 hyperintense findings and in cases of suspicion of tumour formation - In/Out phase dual-echo sequences for contour delineation. The most important pre-contrast sequence was 3D T2 urogram with fat-suppression and respiratory-triggering. T1-weighted gradient-echo sequence with fat-saturation (3D Dyn SPGR for GE 1.5 T Signa and 3D VIBE Dynamic for Siemens Essenza 1,5T) was used for the post-contrast scan. The dynamic scan was repeated within 15 min, following Gd injection with increasing intervals between acquisitions, for the need of post-processing. Our sequences were compatible on both MR units and the software used for post-processing has been properly validated for correctness and applicability in our MR protocols. We employed a standard dose of 0.1 mmol/kg of Gd in the majority of our studies, however in some occasions low-dose Gd opacification - 0.01 mmol/kg has been employed, especially in small infants and in cases of glomer-ular filtration between 30 and 60 ml/min/1.73 m2. In all our patients, serum creatinine levels were strictly observed and we estimated individually the glomerular filtration rate according to the Schwartz's formula.15 New-borns and small infants were scanned with a head-coil and the older children were scanned with a phased-array torso coil. Normal MR urogram is shown in Figure 1.
Axial T2 FSE images
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FIGURE 2A-I. MR imaging of horseshoe kidney in 12-year-old boy. A-D. Axial T2 FSE images - clearly dilated pyelocalyceal system and ureters in horseshoe kidney. E. Coronal volume MIP image from arterial phase of 3D Gd-enhanced MR angiography - the main left and right renal arteries extending from the anterior aspect of the aorta. F. Coronal volume MIP image from venous phase - both renal veins in their expected locations. G. Coronal volume MIP image from parenchymal phase - lower poles of the kidneys without any parenchymal abnormalities. H. Coronal volume MIP image from excretory phase - marked dilatation of both pyelocalyceal systems and ureters. I. Intraoperative findings prove the diagnosis of bilateral megaureters in horseshoe kidney with dysplastic changes in their distal thirds.
B
Statistical analysis and ethical consideration
Groups were compared with Mann-Whitney U-test, P-values >0.05 were taken as indicators of no statistically significant differences. SPSS 13.0 (SPSS Inc., Chicago, Illinois, USA) was used.
The investigators strictly followed recommendations of the Helsinki Declaration (1964, with later amendments) and of the European Council Convention on Protection of Human Rights in Bio-Medicine (Oviedo 1997).
Results
MR urography for visualization of morphological renal anomalies in children
Static, T2 MR urography was successfully performed in 96 children with 99 exams, totally 197 kidneys (in three children follow-up MRI exams after surgery have been done and in one patient left nephrectomy was performed). T2 images for anatomic evaluation were helpful in the assess-
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FIGURE 3A-C. MR imaging of bilateral ureterocystoneostomies in 10-months old boy. A. Coronal post-contrast MIP image - persistent bilateral hydronephrosis and hydroureters following surgery. B. Coronal T2 SS-FSE image - a bladder diverticula. C,D. Post-contrast coronal 3D Dyn SPGR image on the kidney level (C) and on the level of the bladder (D) - re-implanted dilated hydroureters.
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ment of children with severe hydronephrosis and poorly functioning systems. The majority of the population (91 cases) presented with congenital anomalies of the renal pelvis and ureter, including megacalycosis, ureteropelvic (UPJ) obstruction and primary megaureters. We also detected 36 cases of congenital anomalies of the kidney, including: renal agenesis - 6; renal hypoplasia - 5; cystic anomalies of the kidneys - 8; anomalies of renal rotation, horseshoe kidney - 6 (Figure 2); renal dystopia - 3; abnormal renal vessels - 6; Fraley's syndrome -2. Static, T2 MRU allows us also to find the following anomalies: (i) bladder anomalies - in 3 children; (ii) encountered lower urinary tract anomalies of urogenital sinus - in 7 children, including disorders of sex development with ambiguous genitalia (her-maphroditism) (n=3), anorectal and vaginal malformations (n=4); (iii) renal infections - in 18 children. 11 cases of renal neoplasms were confirmed or detected on MRU. In 13 cases, no abnormalities were detected on the static, T2 MR urography.
MR urography for assessment of renal function in children
In 54 children (from the whole population), T1 excretory MR urography with injection of Gd has been performed in addition to static, T2 MR urography for the main purpose of our study - to assess the renal function. The majority of them had anomalies of the renal pelvis and ureter: ureteropelvic (UPJ) obstruction (hydronephrosis) - 43 (bilateral - 10, right side - 14, left side - 19); primary meg-aureter and anomalies of vesicoureteral segment (UVJ) - 30 (bilateral - 8, right side - 8, left side -14) including 7 patients with vesicoureteric reflux
(VUR), diagnosed by VCUG, ureter duplication -2; ureterocele - 2. We observed obstructed systems on MR urography morphologically by the presence of narrowed ureter with proximal dilatation and we were able to distinguish obstructed from non-obstructed systems functionally by the presence of delayed contrast excretion into the collecting system and ureter on the basis of the functional analysis in particular by the calculation of renal transit times (RTT). In 40 children MR functional analysis proved the presence of obstructive systems and the remaining 14 children were classified as nonobstructive and they have been followed-up. Both static and excretory MR urography was helpful in differentiating the causes of hydronephrosis in these patients. Typical images of a child with several bilateral ureterocystoneostomies and persistent bilateral hydronephrosis and hydroureters following surgery are shown in Figure 3.
We consider images quality of the kidney and the collecting system to be superior with MR urog-raphy in comparison to ultrasound and DTPA re-nogram in all 96 cases. The agreement of grading of hydronephrosis was equal in MR urography and ultrasound (US), however MR provides a detailed visualization of the entire ureters and presents ure-teric pathology clearly US.
A correlation between MRU data and final diagnosis determined by surgery or observation was excellent in all 96 patients. 40 children benefits from surgical interventions for obstructive systems. Pyeloplasty has been performed in 11 with MR findings of ureteropelvic junction (UPJ) obstruction (Figure 4). In 29 children with UVJ obstruction and primary megaureter, reimplantation of the ureters - ureterocystoneostomy (UCNS) has been
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FIGuRE 4A-G. Imaging of UPJ obstruction in 9-month-old boy. A-D. Consecutive coronal Tl-weighted MR images (Gd-enhanced) - successively filling of the right renal pelvis with preservation of the right kidney function. E. Coronal T2-weighted MIP image - on the left side an outflow tract obstruction with marked dilatation of the left pyelocalyceal system; F. Dynamic 99mTc-DTPA presenting urinary obstruction of the left kidney; G. Postoperative pathologic specimen in th same child following pyeloplasty a modo Anderson-Hynes.
performed (Figure 3). Other surgical interventions (74 in total for the whole study population), such as nephrectomies, partial or atypical kidney resection, nephrostomies, external genitalia corrections, masculinizing surgical procedures, retroperitoneal tumours resections etc. were also confirmed at MR urography.
VCUG was performed in 10 patients. Vesico-ureteric reflux (VUR) in 7 patients and in one case an ureterocele was identified. The vesico-ureteric reflux was classified as grade III in 4 children, grade IV in 2 and grade V in 1; in both cases pre-
senting dilatation of the ureter and the pyelocalyc-eal system were clearly visible on MR urograms. In two cases VCUG present normal findings.
A comparison of the results from the functional analysis has been done by two different softwares - "CHOP-fMRU" and "ImageJ", as well as by the data from the 99mTc-DTPA. The results from the functional analysis of transit times, volumes and volumetric differential renal function are presented on Table 1. No statistically significant differences (P>0.05) were found between the calyceal and renal transit times and the parenchymal kidney
TABLE 1. Calculated transit times, parenchymal volumes and volumetric differential renal function
Transit times
Parenchymal volumes
Volumetric differential renal function
CHOP-fMRU/ ImageJ
CHOP-CTT-R
ImageJ-CTT-R
CHOP-CTT-L
ImageJ-CTT-L
CHOP-RTT-R
ImageJ-RTT-R
CHOP-RTT-L
ImageJ-RTT-L
Time (range)
313 sec. 279 sec. 267 sec. 243 sec.
150-476)
151-407) 141-393) 126-361)
CHOP-fMRU/ ImageJ
CHOP-Volume-R ImageJ-Volume-R CHOP-Volume-L ImageJ-Volume-L
534 sec. (287-780) 550 sec. (306-793) 476 sec. (290-663) 475 sec. (277-673)5
Volume (range)
134,9 ml (14,3-255,6) 129,2 ml (19,5-238,9)
147.2	ml (12,4-282,0)
150.3	ml (15,7-284,9)
CHOP-fMRU/ ImageJ/NucMed
CHOP-vDRF-R
CHOP-vDRF-L
ImageJ-DRF-R
ImageJ-DRF-L
NucMed-DRF-R
NucMed-DRF-L
Percent (range)	SE	SD
54,15% (44,18-64,11)	3,88	9,50
48,85% (35,88-55,82)	3,88	9,50
52,40% (44,60-60,20)	3,04	7,44
47,60% (39,80-55,40)	3,04	7,44
51,92% (47,27-56,56)	1,81	4,43
48,08% (43,44-52,73)	1,81	4,43
Legend: R = right kidney, L = left kidney; CHOP-CTT = mean calycial transit time measured with CHOP-fMRU; CHOP-RTT = mean renal transit time measured with CHOP-fMRU;, ImageJ-CTT - mean calycial transit time measured with ImageJ; ImageJ-RTT = mean renal transit time measured with ImageJ; CHOP-Volume and ImageJ-Volume = parenchymal volumes, measured with CHOP-fMRU and ImageJ; CHOP-vDRF, ImageJ-vDRF and NucMed-DRF = volumetric differential renal function, measured respir. with CHOP-fMRU, ImageJ and Nuclear Medicine; SE = standard error; SD = standard deviation.
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volumes, measured by CHOP-fMRU and ImageJ (Figure 5A,B). The values for the volumetric differential kidney function assessed by CHOP-fMRU and ImageJ measured separately for each kidney were not statistically different to those derived from the Tc-DTPA study (P>0.05) (Figure 5C12,3). MR urography and renal scintigraphy showed confirmatory results in the diagnosis of obstruction both UPJ and UVJ in terms of volumetric differential renal function values.
Discussion
MRU is a feasible method for evaluation of urinary tract pathology in neonates and infants.1,16 It overcomes the limitations of the conventional imaging techniques and is superior tool in many aspects, especially in the evaluation of parenchymal kidney diseases and poorly functioning systems, assessment of ureteral anatomy and renal vasculature as shown in our study. The method is particularly helpful for further therapeutic decisions, planning of surgical intervention and future diagnostic work-up.
The most common MRU techniques, used to visualize the urinary tract, are the static (T2) MRU and excretory (T1) MRU.17-19 Three-dimensional (3D) sequences are used to obtain thin-section data sets that can be further post-processed to create volume-rendered (VR) or maximum-intensity-projection (MIP) images of the entire urinary tract (Figure 6). Similar observations have been as reported by Roy et al. and O'Malley et al., using MRU.20-21 Excretory
(T1) MRU is similar to CT urography and intravenous urography. The use of dose of Gd (0.1 mmol/ kg) and in some occasions low-dose Gd opacifica-tion (0.01 mmol/kg) allowed us to maintain the linearity between signal and Gd concentration, which is essential for quantitative measurements and analysis. Administration of diuretics improved the quality of MRU by increasing the quantity of the urine and therefore, leads to better dilution and appropriate distribution of Gd in the urinary tract.22-23 The most important sequence of excretory MRU in our study was 3D gradient-echo. Fat-suppression is recommended for better demonstration of the ureters. Modern MR-units scan simultaneously in one volume the kidneys, the ureters and the bladder, using 3D gradient-echo sequences in one breath-hold.1924 Sometimes segmental scanning of the kidneys or bladder separately for visualization of image details is recommended.
Currently, there are two major MRU processing software available free of charge, which we have verified, compared each other and used in our practice routinely.59 Post-processing algorithms permits us to evaluate and compare to scintigra-phy several parameters - (i) calycial (cTT) and renal transit times (rTT); (ii) parenchymal volumes; (iii) differentiated renal function (vDRF) and (iv) the time-intensity curves representative for the renal function.
Our results demonstrate that MRU should be a method of choice for visualization of the upper urinary tract in children as it is difficult to assess by US or scintigraphy. In some cases, such as UPJ obstruction, MRU was confirmatory to ultrasound,
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FIGURE 7A-E. Automated functional analysis of MRU data in bilateral normal kidney with vesicoureteral reflux(VUR) - grade 1 on the right side using "CHOP-fMRU". A. Enhancement curves. B. Excretion curves. C. Patlak plots. D. Calculation of renal transit times and differential renal function. E. Enhancement curves, generated on "ImageJ".
but superior concerning the evaluation of end-ureteral anomalies. US provides initial information concerning renal parenchyma, bladder morphology, presence and degree of dilated systems, but failed in visualization of non-accessible ureters, hidden in the retroperitoneum and is pretty week in information about renal excretion.
The graphic presentation of time-intensity curves, obtained by the dynamic MRU studies, was in accordance with the renal curves, obtained by scintigraphy. Moreover, the calculated values of the volumetric differential renal function, using "CHOP-fMRU" software were similar to those calculated on the basis of ImageJ software; both corresponded to the values from the dynamic 99mTc-DTPA scintigraphy. Comparable results regarding parenchymal volumes and transit times were observed using the two different software programs. Scintigraphy also supplies information about the renal function and morphology; however it is time-consuming ionizing imaging method with low spatial resolution.625 In our study, the basic parameters of the curves (amplitude, washout) were assessed, as well as the presence of certain characteristic features of the curve. The data from the "signal-intensity versus time-curve" analysis were combined with the other parameters, derived from the MRU analysis, including estimation of the renal transit times, parenchymal volumes and differential renal function. The resulted data-set provided a powerful tool, of high importance for the diagnosis of obstruction.
In the data processing, several parameters were also calculated, using "CHOP-fMRU" and "ImageJ": CTT - calyceal transit time; RTsT - renal transit time; TTP - time to peak; parenchymal volume; vDRF - volumetric differential renal function; pDRF - Patlak differential renal function etc. Typical example of data processing is shown in Figure 7 - a child with recurrent renal infections and a low-grade vesicoureteral reflux (VUR) on the left side. On non-contrast MRU images dilatation of the distal part of the left ureter was observed, the resulting enhancement curves were non-obstructive and the patient was referred to ultrasonogra-phy follow-up. No significant difference concerning the listed parameters was found whatever functional analysis software has been used.
Our results as well as the presented case (Figure 7) showed that both renal and calycial transit times, parenchymal volumes and differential renal function are indicators for kidney dysfunction.
Even small functional disorders can be detected using MRU and analysing these parameters. The complex software functional analysis of the whole patient population confirmed this assumption.
In addition to the advantages of MRU, mentioned above, it is necessary to note that this technique has also some limitations. Sometimes it requires a placement of bladder catheter, administration of furosemide and Gd, sedation and even anaesthesia (for newborns and younger children), as a complementary risk. Breath-hold techniques could not be applied in neonates and small infants and motion artefacts should be at a minimum. Patient preparation and examination itself are time-consuming; post-processing and calculation of functional curves and differential renal function requires additional time.
In 2006, it was demonstrated that some Gd-based contrast agents may provoke the development of nephrogenic systemic fibrosis (NSF) and/ or a generalized fibrotic disorder in renal failure patients.26 Gd-ions, released from Gd-based MR contrast agents, are the likely etiologic agent of NSF.27 The ESUR guidelines suggest a very careful administration of Gd in children with renal failure. Absolute contraindications are high levels of creatinine and a glomerular filtration under 30 ml/min/1.73 m2. Individual assessment for the indications and the need of contrast-enhanced MR examination was performed after discussions with paediatric nephrologists in cases of glomerular filtration between 30 and 60 ml/min/1.73 m2. Written consent should be obtained in spite of the fact that most cases of NSF occurs in adults and the reported cases of NSF without Gd administration. In all patients with high risk for development of NSF and in the paediatric group, we used cyclic Gd-helators due to their higher stability.28 We didn't observe any adverse effects or cases of NSF, following contrast administration in our study-group.
In conclusion, MR urography is useful, non-ionizing method for assessment of obstructive uropathies and facilitates surgical decisions. There is growing number of publications concerning the criteria for assessment of the renal function in children by dynamic MRU, but the achievement of consensus requires more and deeper investigations. The advances of molecular imaging techniques provide new insights about the nature of hereditary diseases in paediatric nephrology and urology.
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Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines
Matija Rojnik1, Zala Jevnikar1, Bojana Mirkovic1, Damjan Janes1, Nace Zidar2, Danijel Kikelj2, Janko Kos1
1	Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
2	Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
Received 15 July 2011 Accepted 13 September 2011
Correspondence to: Prof. Dr. Janko Kos, University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, SI-1000 Ljubljana, Slovenia. Phone: +386 1 4769 604; Fax: +386 1 4258 031; E-mail: janko.kos@ffa.uni-lj.si
Disclosure: No potential conflicts of interest were disclosed.
Background. Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines.
Materials and methods. BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing.
Results. In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity.
Conclusions. Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression.
Key words: bone morphogenetic protein 4; cancer; cathepsin H; human cell lines; proteolytic enzymes
Introduction
Cathepsin H (CTSH) (EC.4.22.16), a cysteine protease, is ubiquitous in cells and tissues, but its physiological role is poorly understood.12 CTSH acts mainly as an aminopeptidase but also exhibits limited endopeptidase activity.3 In addition to heavy and light chains, which are typical of a number of mammalian papain-like cysteine proteases, mature CTSH also contains an octapeptide EPQNCSAT, termed the mini-chain, that originates from the propeptide and is bound to the
mature form by a disulphide bond.4 The mini-chain is essential for the aminopeptidase activity of CTSH.5
CTSH is synthesized as a preproenzyme of 41 kDa which is proteolytically activated through a multistep process to a 30 kDa intermediate form and finally to the single chain mature form of 28 kDa.6 This form can be further processed to a 22 kDa heavy chain and a 5-6 kDa light chain.237
CTSH was identified to play an important role in the establishment and development of a functional tumor vasculature and increases the meta-
static potential of human hepatoma cell lines.8-10 Expression of CTSH differs in breast carcinoma11, colorectal cancer12, melanoma13, head and neck carcinoma14,15, glioma16 and prostate cancer17 and from that in normal tissue. CTSH is associated with physiological and pathological processes of the lung.18-20 Furthermore, it is involved in the N-terminal processing step of surfactant protein C in type II pneumocytes and pro-granzyme B in cytotoxic lymphocytes.21,22 Bone morphogenetic protein 4 (BMP-4) is a potential target for CTSH endopeptidase activity during the differentiation of mouse lungs, and lower activity was proposed to lead to marked accumulation of BMP-4 protein and disruption of branching morphogenesis.23
BMP-4, as a member of the transforming growth factor ß family is involved in the development of many organs and tissues and was shown to play a role in cancer progression.24-26 BMP-4 is synthesized as a large inactive precursor which is proteo-lytically cleaved to the mature protein in a multi-step process.27 Non-processed BMP-4 is targeted to the lysosomes for degradation which can lead to severe loss of BMP-4 activity in specific tissues.2829 Lü et al.23 presented evidence that CTSH and BMP-4 expression coincides during branching morphogenesis in mouse models. They showed that inhibition of CTSH leads to accumulation of the mature BMP-4 in embryonic mouse lungs. However, they failed to demonstrate the cleavage of BMP-4 by mature 28 kDa CTSH in vitro.
In this study we have evaluated the role of human CTSH in BMP-4 processing and regulation at the mRNA and protein level, using various human cell lines.
Materials and methods
Cell culture
Human U937 pro-monocytic (CRL-1593.2; ATCC, Manassas, VA, USA) and WEHI231 mouse lymphoma cells (CRL-1702 cell line; ATCC, Manassas, VA, USA) were maintained in advanced RPMI 1640 (Gibco, Invitrogen, Scotland) supplemented with 2mM glutamine (Sigma, St. Louis; MO, USA), antibiotics (Penicillin-Streptomycin, Sigma, St. Louis, USA) and 10% fetal bovine serum (FBS) (HyClone, Logan, USA). U937 cells were differentiated with PMA (50 nM) (Sigma, St. Louis; MO, USA) for 24 h to achieve attachment. Human osteosarcoma HOS cells (CRL-1543 cell line, ATCC, Manassas, VA, USA) were grown in minimal essential medium (MEM) (Sigma, St. Louis, MO, USA) supple-
mented with 2.2 g/L sodium bicarbonate (Riedel de Haën, St. Louis, MO, USA), 2 mM glutamine, antibiotics and 10% FBS. Human prostate cancer PC-3 cells (cell line CRL-1435, ATCC, Manassas, VA) were cultured in DMEM/F12 (1:1) medium (Gibco, Invitrogen, Scotland) supplemented with antibiotics, 2 mM glutamine and 10% FBS.
Western blot analysis
Lysates of HOS, PC-3 and PMA differentiated U937 cells were prepared in 50 |jl of 0.05 M sodium acetate buffer (pH 6) with added 1mM EDTA, 0.1 M NaCl, 0.25% Triton X-100 (lysis buffer). Complete lysis of the cells was achieved by three 5 to 7 s sonication cycles. Clear supernatants were obtained after centrifugation at 4°C and 16200 g for 15 min. Total protein concentration was determined by the Bradford method using Coomassie Plus Protein Assay reagent (Pierce, Thermo Fischer Scientific) with BSA (Sigma, St. Louis; MO, USA) as standard. Samples containing 100 |jg of proteins were heated at 100°C in reducing sample buffer for 10 min, separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The molecular weight of the proteins was determined using SeeBlue® Plus2 Pre-Stained Standard (Invitrogen, USA). The membrane was blocked in 5% skimmed milk in Tween-PBS for 30 min and incubated with sheep polyclonal anti-cathepsin H (5 ^g/ml)30 or goat polyclonal anti-BMP4 (dilution 1:400, sc-6896, Santa Cruz Biotechnology, CA, USA) antibodies overnight at 4°C. After washing with Tween-PBS the membrane was incubated with secondary HRP conjugated rabbit anti-sheep (1:10000, sc-2770, Santa Cruz Biotechnology, CA, USA) or donkey anti-goat (1:10000, Santa Cruz Biotechnology, CA, USA) antibodies for 2 h at room temperature.
Real Time PCR analysis
PCR-arrays (Common Cytokine PCR Array; PAHS-021, SABiosciences, MD, USA) were used according to the manufacturer's protocol. RNA was isolated from U937 cells treated with 0.5 |jM native human liver CTSH (nCTSH) and compared to RNA from control cells. Data was analyzed using RT2 Profiler PCR Array Data Analysis (SABiosciences, MD, USA). Quantitative Real Time PCR (qPCR) was performed as reported.31 Total RNA was isolated from U937, HOS and PC-3 cells using RNeasy Mini kit (Qiagen, Hilden, Germany) according to manufacturer's protocol.
For cDNA synthesis 1 pg of total mRNA was reverse transcribed using OmniscriptRT Kit (Qiagen, Hilden, Germany). qPCR was carried out on an ABI PRISM 7000 apparatus (Applied Biosystems, Life Technologies Corporation, CA, USA) in a total reaction volume of 25 pl containing 5 pl cDNA of different concentrations, BMP4 QuantiTect Primer Assay (Qiagen, Hilden, Germany) and Maxima™ SYBR Green/ROX qPCR Master Mix (2x) (Fermentas International Inc, Ontario, Canada). The cycling program was 2 min at 50°C, 10 min at 95°C, followed by 40 cycles (15 s at 95°C and 60 s at 60°C). Multiple housekeeping genes were checked (the primer sequences were found in the Real Time PCR Primer and Probe Data Base) for their stability using geNorm normalization. The data was normalized to the endogenous controls HPRT and GAPD for U937, HPRT and YWHAZ for HOS and PC-3. A melting curve of PCR products (60-95°C) was also performed to ensure the absence of artefacts. All assays were performed in parallel and in three biological repetitions.
Confocal immunofluorescence microscopy
HOS and PC-3 cells were grown on glass coverslides in 24-well plates for 24 h prior to the experiment; U937 cells were differentiated with PMA (50 nM) for 24 h. WEHI231 were seeded on slides and cytospinned for 6 min at 2500 g. Before labeling, cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min and permeabilized with 0.1% Triton X-100 in PBS (pH 7.4) for 10 min. Non-specific staining was blocked with 3% BSA in PBS (pH 7.4). CTSH was labeled with primary mouse monoclonal anti-CTSH 1D10 antibody (10 pg/ml of 3%BSA in PBS).30 Goat polyclonal antihuman BMP4-N16 antibody was used for BMP-4 labeling (Santa Cruz Biotechnology, CA, USA). After 2 h of incubation, cells were washed three times with PBS and treated with Alexa 488-la-beled rabbit anti-mouse and Alexa 555-labeled donkey anti-goat (2:1000, Molecular Probes, Invitrogen, USA) antibodies for 2 h. After washing with PBS, ProLong Antifade kit (Molecular Probes, Invitrogen, USA) was mounted on dried coverslides and allowed to dry overnight at 4°C. Cells were studied by fluorescence microscopy at room temperature using a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Jena, Germany); immersion oil was used as imaging medium. Images were analyzed using Carl Zeiss LSM image software 3.0.
Synthesis of specific
synthetic irreversible inhibitor of
CTSH - H2N-Ser(OBzl)-CHN2 (CTSHi)
(S)-(9H-Fluoren-9-yl)methyl (1-(benzyloxy)-4-dia-zo-3-oxobutan-2-yl)carbamate (A). Triethylamine (0.175 mL, 1.258 mmol) in THF (3 mL) was added to a stirred solution of Fmoc-L-Ser(Bzl)-OH (1, 500 mg, 1.198 mmol) in THF (6 mL) at -20°C under argon, followed by the addition of ethyl chloroformate (0.120 mL, 1.258 mmol) in THF (3 mL). The mixture was stirred for 30 minutes at -5°C, after which the precipitated Et3NH+Cl- was filtered off. Acetonitrile (5 mL) and trimethylsilyldiazomethane (2.0 M sol. in hexane, 1.198 mL, 2.395 mmol) were added to the filtrate and the mixture was stirred overnight at +4°C. Ethyl acetate (50 mL) was added and organic phase washed successively with 10% aq. citric acid (2 X 20 mL), sat. aq. NaHCO3 (2 * 20 mL) and brine (2 * 20 mL). The organic phase was dried over Na,SO4, filtered and the solvent evaporated under reduced pressure. The crude product was purified with flash column chromatography using ethyl acetate/petroleum ether (1:4) as eluent to afford 2 as a pale yellow solid (378 mg, 0.856 mmol). Yield: 71%; Rf = 0.43 (EtOAc/petroleum ether = 1:1); IR (KBr): v = 3552, 3414, 3311, 3076, 2859, 2106 (C=N=N), 1800, 1696, 1630, 1534, 1450, 1385, 1293, 1266, 1103, 1029, 736 cm-1. 1H NMR (DMSO-d6): 5 3.60-3.69 (m, 2H, CH2), 4.21-4.37 (m, 4H, CH, CH, CH2), 4.49 (s, 2H, CH2Ph), 6.07 (s, 1H, CHN2), 7.26-7.44 (m, 9H, Ar-H), 7.74 (d, 2H, J = 7.2 Hz, Ar-H), 7.84 (d, 1H, J = 8.1 Hz, NH), 7.90 (d, 2H, J = 7.5 Hz, Ar-H). MS (ESI): m/z (%) = 464 ([M+Na]+, 33), 414 ([MH-N2]+, 35). (S)-3-Amino-4-(benzyloxy)-1-diazobutan-2-one (B). To a solution of A (150 mg, 0.340 mmol) in acetonitrile (10 mL), diethylamine (10 mL) was added and the mixture stirred at room temperature. After 20 min the mixture was concentrated under reduced pressure and purified with flash column chromatog-raphy using dichloromethane/methanol (20:1) as eluent, to afford 3 as an yellow oil (20 mg, 0.091 mmol). Yield: 27%; Rf = 0.23 (CH2Cl2/MeOH = 10:1); 1H NMR (CDCl3): 5 1.73 (br s, 22H, 2NH2), 3.59-3.70 (m, 3H, CH, CH2), 4.55 (s, 2H, CH2Ph), 5.79 (s, 1H, CHN2), 7.31-7.40 (m, 5H, Ph).
Analytical TLC was performed on silica gel Merck 60 F254 plates (0.25 mm), using visualization with UV light and ninhydrin. Column chro-matography was carried out on silica gel 60 (particle size 240-400 mesh). 1H NMR spectra were recorded at 300 MHz on a Bruker AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6 solution, with TMS as the internal standard. IR spectra were re-
®
U937
HOS
pc-3
nCTSH
30 k Da 28 KD a
actin
41 kDa
28 kDa 22 kDä
CTSH activity buffer (pH 6.8) using the same protocol as Obermajer et al.33 nCTSH was pretreated with 10 |jM CTSHi or DMSO for controls. Samples were analyzed using 12% SDS-PAGE followed by Western blot or reverse-phase HPLC (Shimadzu Coorporation, Japan) using a Discovery BIO Wide Pore C5 column (Sigma, St.Louis, MO, USA) with UV-VIS detector.
FIGURE 1. CTSH processing forms. (A) Different forms of CTSH were detected in human cell lines and (B) in the sample of nCTSH using Western blot. CTSH was visualized with sheep anti-CTSH primary pAb and rabbit anti-sheep horse radish peroxidise (HRP) labeled secondary antibody. The intermediate 30 kDa CTSH form is present in all of the selected cell lines, while the mature 28 kDa CTSH form is missing in HOS cells. A procathepsin H (41 kDa) and single chain form (22 kDa) could be detected in smaller amounts compared to the mature forms. nCTSH contains the procathepsin H, the mature and heavy chain forms.
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FIGURE 2. CTSH dependent cytokine mRNA expression. (A) The effect of CTSH on the mRNA expression of cytokines from BMP family. Differentiated U937 cells were incubated with 0.5 |jM nCTSH and the mRNA levels were screened by PCR-arrays enabling simultaneous screen of the of 84 cytokine genes. The expression of BMP-2 is not presented, while it was probably an artifact, as shown by the analysis of the melting curve. (B) The influence of CTSH on BMP-4 mRNA expression was further evaluated with quantitative real time PCR analysis in U937, HOS and PC-3 cell lines. Cells were treated with 0.5 jM nCTSH for 24 h. The mRNA levels obtained from control samples were normalized to 1. Each bar represents the mean±SD. Ns, non-significant, *P<0.05.
corded on a Perkin-Elmer 1600 FT-IR spectrometer. Mass spectra were obtained using a VGAnalytical Autospec Q mass spectrometer.
Inhibition constants for CTSH (k2 2938.3 s-1M-1) and CTSB (k2 5.1 s-1M-1) were similar as referred in the literature.32
Cleavage of mature human recombinant BMP-4 protein
nCTSH was tested for its ability to degrade human recombinant BMP-4 protein (GenwayBio, San Diego, CA, USA). BMP-4 (7.5 |jM) was incubated with nCTSH (0.75 |jM) for 1.5 h at 37°C in
Statistical analysis
SPSS PC software (Release 13.0) was used for statistical analysis. Statistical significance was evaluated by Student's t test. P values of less than 0.05 were considered to be statistically significant.
Results
Different processing forms of CTSH
The presence of different processing forms of CTSH was determined by Western blot in U937, HOS and PC-3 cell lines (Figure 1A). A 30 kDa intermediate form was detected in all selected cell lines, while the mature single chain 28 kDa form was absent in HOS cells. Only small quantities of the 22 kDa heavy chain (from the two-chain form) were detected in human cell lines (data not shown). The sample of CTSH isolated from human liver (nCTSH)30 contains predominantly mature 28 kDa form (Figure 1B), with a small amount of 22 kDa heavy chain. It is likely that the 30 kDa form was further processed to mature and heavy chain forms.
CTSH regulates the expression of BMP family genes
The effect of CTSH on human cytokine mRNA levels was screened by PCR-array, enabling simultaneous expression of 84 cytokine genes. Incubation of differentiated U937 cells with nCTSH (0.5 |jM) induced significant changes in the expression of several BMP family genes. BMP-3, 6 and 7 were significantly up-regulated, while BMP-4, 5 and 8 were down-regulated (Figure 2A). The expression of BMP-1 was not altered. CTSH dependent regulation of BMP-4 mRNA expression in human cell lines was further confirmed by specific quantitative real time PCR analysis (Figure 2B). After the addition of nCTSH, BMP4 mRNA levels in HOS increased 2.33 ± 0.08 fold, while BMP-4 mRNA levels in U937 and PC-3 were decreased 0.55 ± 0.01 and 0.45 ± 0.03 fold.
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FIGURE 3. Co-localization of CTSH (green fluorescence) and BMP-4 (red fluorescence) in U937 (A), HOS (B), PC-3 (C) and WEHI231 (D) cells. Weak or no co-localization was found in selected human cells (A, B, C), while clear co-localization can be seen in mouse WEHI231 cell line (D). CTSH was labeled with 1D10 monoclonal primary antibody and anti-mouse AlexaFluor™488 secondary antibody. BMP-4 was labeled with primary goat polyclonal anti-BMP-4 antibody (Santa Cruz) and anti-goat AlexaFluor™ 555 secondary antibody. The sites of co-localization are shown in white (frame 4) and correspond to the pixels that are over the threshold in both channels (frame 5). Scale bars represent 5 pm (A and D) and 20 pm (B and C).
CTSH is not involved in the proteolytic processing of BMP-4
No significant co-localization of CTSH and BMP-4 was observed in human U937, HOS, PC-3 cells under confocal microscopy. To present the difference between the co-localization of CTSH and BMP-4 in human and in mouse cells mouse WEHI231 cells were used revealing strong co-localization of both proteins (Figure 3). Furthermore, the role of CTSH in the intracellular degradation of mature BMP-4 was determined using a specific synthetic irreversible inhibitor of CTSH - H2N-Ser(OBzl)-CHN2 (CTSHi; 5 |jM).32 The inhibition of CTSH was fol2
lowed by SDS-PAGE and Western blot analysis (Figure 4A). No increase of mature BMP-4 was observed in U937, HOS and PC-3 cells treated with CTSHi, indicating that CTSH inhibition does not have a direct impact on BMP-4 processing or degradation in human cells. Intramolecular cleavage of BMP-4 by CTSH endopeptidase activity was excluded in vitro by incubation of recombinant BMP-4 and nCTSH. No additional protein bands appeared on Western blots (Figure 4B). The products of the proteolytic cleavage were also analyzed by reverse phase HPLC to detect possible N-terminal cleavage of BMP-4 by CTSH aminopeptidase activity. No changes in the height or area of the BMP-4
®
U937
HOS
PC-3
cant
CTSHi cont.
CTSHi
m m
CTSHi
buffer
36 kDa
BMP-4
precursor
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Mature BMP-4
®
+
BMP-4 nCTSH CTSHi
13 kDa
FIGuRE 4. CTSH processing of mature BMP-4 protein. (A) Human cell lines were treated with 5 pM CTSHi for 24 h and then cell lysates were analyzed using Western blot. Proteins (50 pg) from cell lysates were separated on SDS-PAGE and transferred on to PVDF membrane. BMP-4 was detected with anti-BMP-4 N16 antibody (Santa Cruz) and then with secondary antibody labelled with HRP. The molecular mass in kDa is indicated on the left hand side of the blots. Molecular mass of the mature BMP-4 is detected to be approximately 18 kDa. (B) Using Western blot we analyzed the products of the reaction between mature human recombinant BMP-4 and nCTSH. Mature human recombinant BMP-4 was incubated for 1.5 h at 37°C in CTSH activity buffer (lane 1), with 60 ng of nCTSH in CTSH activity buffer (lane 2) and 60 ng of nCTSH in CTSH activity buffer that was pre-treated for 10 min with 10 pM CTSHi (lane 3). Mature human recombinant BMP-4 has a molecular mass of 13 kDa. (C) Using reverse phase HPLC we analyzed CTSH activity buffer (buffer), mature human recombinant BMP-4 in CTSH activity buffer (BMP-4) and the products of the reaction between mature human recombinant BMP-4 and nCTSH in CTSH activity buffer (BMP-4 + nCTSH). BMP-4 was eluted in the fraction around 17.0 min.
peak were observed after incubation with nCTSH, excluding significant amounts of aminopeptidase processing (Figure 4C).
Discussion
BMP-4 regulates cell proliferation, differentiation, apoptosis and cell fate throughout mammalian development.34 CTSH has been suggested to regulate its recycling or degradation in the developing lung of mice.23 In the current study, we identified CTSH as a regulator of BMP-4 mRNA expression in human cell lines, however, we excluded direct pro-teolytic processing of BMP-4 by CTSH amino- or endopeptidase activity.
CTSH expression is ubiquitous, with very high levels in the kidney.35 There is growing evidence that its expression changes under various pathological conditions, the most extensively studied
being its role in cancer.173637 However, its natural substrates and mechanism of action are not known. Using Western blot we have detected different processing forms of CTSH in human prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) cell lines. Whereas the 41 kDa proenzyme and 30 kDa intermediate were present in all cell lines, the mature 28 kDa form was missing in human osteoblasts HOS. The 28 kDa single chain is believed to be the most important form of active CTSH in exerting specific aminopeptidase activ-ity.5 However, del Re et al.36 demonstrated that in colorectal carcinoma the expression of the 30 kDa form is decreased, while the expression of the mature 28 kDa form is increased in tumor comparing to normal tissue, thus showing the importance of different CTSH processing forms.
We attempted to identify potential targets of CTSH proteolytic activity in human cells, using PCR array technology. In U937 cells a strong as-
sociation between CTSH and mRNA expression of BMP family members was observed. The results of quantitative real time PCR demonstrate that CTSH affects BMP-4 mRNA expression differently in selected cell lines. In U937 and PC-3 cells the addition of nCTSH decreased the expression of BMP-4, whereas in HOS cells the level of BMP-4 mRNA was increased. Interestingly, in HOS cells, where the 28 kDa single chain form of CTSH is missing, the trend of BMP-4 mRNA regulation by nCTSH is opposite to that in U937 and PC-3 cells, where this form is present. This implies that CTSH dependent regulation of BMP-4 mRNA expression is probably controlled by the mature chain form of CTSH, possibly interfering with the promoter of BMP-4 biosynthesis.
Proteolytic processing by BMP-4 is dependent on the proteolytic activity at the two different sites,
51	and S2, in BMP-4 pro-domain.38 Cleavage of the
52	site is enhanced in slightly acidic conditions, as occurs in subcellular organelles like endosomes and lysosomes. Other studies also stressed the need of lysosomal (and proteasomal) function for processing of BMP-4.28 Therefore, CTSH as a lysosomal protease could be involved in BMP-4 pro-teolytic cleavage and, indeed, the endopeptidase activity of the 22 kDa form of CTSH was proposed to cleave BMP-4.23 Confocal microscopy was used to determine whether CTSH protein is co-localized with BMP-4, thus being capable of its proteolytic degradation. No co-localization between the two proteins was found in human cell lines, indicating that CTSH and BMP-4 are not present in the same subcellular organelles, so it is unlikely that CTSH is involved directly in the processing or degradation of BMP-4. Furthermore, inhibition of CTSH did not alter the processing of BMP-4 or increase the levels of mature BMP-4 in human cells. These results strongly suggest that CTSH has no direct role in intracellular BMP-4 proteolytic cleavage in the selected human cells. On the other hand, in mouse WEHI231 cells, the significant co-localization of CTSH and BMP-4 indicates the possible involvement of CTSH in BMP-4 protein processing and showing presumably different processing of BMP-4 in mice compared to humans
To confirm a possible indirect action of nCTSH on human recombinant BMP-4 by either endopepti-dase or aminopeptidase activity in vitro, Western blot analysis was performed, clearly showing that nCTSH, which contains both the mature 28 kDa and heavy chain 22 kDa, does not cleave mature BMP-4 as an endopeptidase. Moreover, using reverse phase HPLC analysis of BMP-4 following
incubation with nCTSH, we demonstrated that the N-terminal of human recombinant BMP-4 is also not cleaved by its aminopeptidase activity. The latter is consistent with the fact that CTSH is not able to hydrolyze substrates by its aminopeptidase activity if proline is at the S1' position39, as is the case at the N-terminal of mature human BMP-4, which starts with the Ser-Pro-Lys-His-His- sequence.40
BMP-4 plays an important role in the differentiation and proliferation of neural41, and colorec-tal cancer stem cells42 and is a critical component in regulating hematopoietic stem cell function.43 The involvement of cysteine cathepsins in the migratory potential and differentiation of stem cells was studied before.44 Our results imply that CTSH might be important in the processes of stem cell differentiation by regulating the expression of BMP-4.
In conclusion, we have demonstrated that CTSH activity is not directly involved in proteolytic processing of BMP-4 in human cells but can regulate mRNA expression of BMP family members, depending on the presence of different processing forms of CTSH. However, the mechanisms of regulation of its mRNA expression, as well as the impact of CTSH on members of BMP family other than BMP-4, remain to be elucidated.
Acknowledgements
The work was supported by Research Agency of the Republic of Slovenia (grant P4-127 J.K.) and partially by 7th EU project Nanophoto.
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Sinonasal inverted papilloma associated with squamous cell carcinoma
Jasna But-Hadzic1, Klemen Jenko2, Mario Poljak3, Bostjan J Kocjan3, Nina Gale4, Primoz Strojan1
1	Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia
2	University Department of Otorhinolaryngology and Cervicofacial Surgery, University Clinical Centre Ljubljana, Ljubljana, Slovenia
3	Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia
4	Institute of Pathology, Medical Faculty University of Ljubljana, Ljubljana, Slovenia
Received 21 August 2011 Accepted 1 September 2011
Correspondence to: Prof. Primož Strojan, M.D., Ph.D., Department of Radiation Oncology, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 110, Fax: +386 1 5879 400; E-mail: pstrojan@onko-i.si
Disclosure: No potential conflicts of interest were disclosed.
Background. The aims of the study were to review single-institution experiences with sinonasal inverted papilloma associated with squamous cell carcinoma (IP/SCC), to analyze the presence of human papillomavirus (HPV) and to evaluate the role of radiotherapy.
Patients and methods. Five patients with IP/SCC were identified in the prospective institutional databases (19952005) and HPV status was determined in all five tumors.
Results. Four out of five patients had T3-4 tumors; no nodal involvement was seen in any of them. Four patients had curative surgery, supplemented in three of them with radiotherapy. Debulking surgery was performed in the patient with a non-resectable tumor followed by radical radiotherapy. Tumor was controlled locally in three patients at 8, 46 and 58 months post-surgery. Local failure occurred in two patients: after endoscopic resection of a T1 tumor (the recurrent tumor was successfully salvaged with additional surgery) and in a patient with an inoperable tumor. No regional or distant metastases occurred. HPV status was determined in all five tumors and three of them were found positive for HPV type 11.
Conclusions. In operable sinonasal IP/SCC, upfront surgery and postoperative radiotherapy to the tumor bed with dose levels comparable to those used for invasive SCC are recommended. For non-resectable disease, radical radiotherapy to a dose of 66-70 Gy could be of benefit.
Key words: inverted papilloma; squamous cell carcinoma; radiotherapy; human papillomavirus infection; outcome
Introduction
Inverted papilloma (IP, one of three types of Schneiderian papilloma) of the nasal cavity and paranasal sinuses is a benign epithelial tumor of unknown etiology, first described by Ward in 1854.1 IP represents 0.5-4% of all sinonasal tumor and arises from the mucosa of the lateral wall of the nasal cavity, almost always unilaterally. IP is best characterized by the male-to-female ratio of 3:1 with the peak incidence between 5th and 6th decade of life, destructive pattern of local growth, tendency to recur and, occasionally, associated malig-
nancy.23 The most common presenting symptom is unilateral nasal obstruction; the duration of symptoms is variable, with an average of >5 years but even up to >45 years.2-4
The frequency of carcinoma in patients with sinonasal IP is around 11%. In two thirds of cases, carcinoma occurs synchronously with IP, but in some patients carcinoma develops at a later time, after previous resection of IP (metachronous carcinoma). The associated malignancy is predominantly squamous cell carcinoma (SSC), which may arise within the papilloma or is merely associated with a histologically bland IP.2 This group of patients is
FIGuRE 1. Inverted squamous papillomas are seen on the right side, synchronous moderately differentiated non-keratinizing invasive squamous cell carcinoma on the left side.
characterized by older age and male preponderance compared with those without associated malignancy.23 An etiological role for human papillomavirus (HPV) and the mutation of the p53 tumor suppressor gene in malignant transformation of IP has been suggested.5-7
Literature reports on IP associated with SCC (IP/ SCC) are scarce, describing only small cohorts of patents treated with varying degree of success. In the present report, we describe a group of five patients treated for IP/SCC. The presence of HPV was analyzed and the role of adjuvant radiotherapy was discussed.
Patients and methods
The databases of the University Department of Otorhinolaryngology and Cervicofacial Surgery, Clinical Center Ljubljana and the Institute of Pathology, Medical Faculty University of Ljubljana for the years 1995-2005 were used for identification of patients with a diagnosis of IP of the nasal cavity and paranasal sinuses. Out of 89 patients with this diagnosis, 5 patients (5.62%) were found to have IP/SCC. Pathology specimens of all 5 patients were re-examined by an experienced head and neck pa-
thologist (N.G.) (Figure 1) and the medical records of identified patients were reviewed for clinical characteristics, treatment and outcome.
Detection of HPV DNA
Tissue processing, DNA extraction and quantification as well as HPV detection and genotyping were described elsewhere.7 Briefly, total DNA was extracted from two 10-|om sections of paraffin blocks using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. For detection of HPV, PCR amplification was performed on all samples using HotStarTaq® Plus DNA Polymerase kit (Qiagen) and consensus GP5+ and GP6+ primers targeting approximately 150-bp fragments of alpha-HPV L1 gene. The PCR products that appeared as visible bands of the expected size were purified by QIAquick PCR Purification Kit (Qiagen) and sequenced directly using BigDye Terminator v1.1 Cycle Sequencing Kit (PE Applied Biosystems, Foster City, USA) with GP5+/GP6+ primers. A comparison of the HPV-DNA sequences obtained with those of officially designated alpha-HPV genotypes was carried out using the Blast server.8 Results from the Blast comparison software were confirmed additionally by pairwise alignment using the sequence of interest and full L1 gene of a reference HPV genotype.9
Results
Patients and tumors
Detailed information on the clinical characteristic of patients and their tumors, treatment and outcome is given in Table 1. There were four males and one female, from 45 to 77 years old (median 73 years). Unilateral nasal obstruction was the most frequent presenting symptom reported by four patients. The duration of symptoms before a diagnosis of IP with or without associated SCC was confirmed ranged from 2-6 months (median: 5 months).
In all but one patient, the disease extended from the nasal cavity to neighboring structures and no nodal or distant metastases were presented at the time of diagnosis in any of them. According to the UICC TNM staging system (7th edition, 2009) for malignant tumors of the nasal cavity and ethmoid sinus, four patients had locally advanced T3-4 disease, whereas using the Krouse staging system,10 all tumors were categorized as stage T4 (i.e. due to associated malignancy).
TABLE 1. Sinonasal inverted papilloma associated with squamous cell carcinoma: clinical and tumor characteristics, treatment and outcome
Parameter	Patients				
	1	2	3	4	5
M/45
M/77
M/75
Unilateral nasal obstruction
Unilateral nasal obstruction, nasal dis- Nasal discharge charge, anosmia, pain
6 mos.
5 mos.
2 mos.
Sex/Age (yrs.)
Presenting symptoms
Duration of symptoms
Extent of disease
TNM stage*
Histology at 1st biopsy
No. of recurrences SCC type
Histpathological grade of SCC
Surgery HPV status Radiotherapy Follow-Up
Lt nasal cavity
T1N0M0 P
1
M n.s.
Endoscopic resection; R0
Positive (type 11)
Not irradiated
Local recurrence at 8 mos., NED 62 mos. after salvage surgery
Rt nasal cavity, nasopharynx
T4bN0M0 IP 0 S
G II-III
Endoscopic resection; R0
Positive (type 11)
60 Gy , 30#, 5MV, 3 fields, continuous course; Tu site & Rt region II
DOC at 46 mos., no evidence of IP/SCC
Rt nasal cavity, ethmoid, orbit
T3N0M0 P 1
M
G II-III
Lateral rhinotomy; R0
Positive (type 11)
60 Gy, 30#, 6MV, 3 fields, continuous course; Tu site
NED at 58 mos.
F/73
Unilateral nasal obstruction, headache, diplopia
2 mos.
Rt nasal cavity, ethmoid, maxillary sinus, orbit
T4aN0M0 IP 6 M
G II
Sublabial and external supraciliar approach; R0
Negative
60Gy, 24#, 5MV, 3 fields, continuous course; Tu site & Rt neck
DOC at 8 mos., no evidence of IP/SCC
M/62
Unilateral nasal obstruction blurred vision, headache, anopsia
6 mos.
Rt nasal cavity, ethmoid, fossa pterygopa-latina, maxillary sinus, sphenoid, orbit
T4bN0M0
IP
0
S
n.s.
Sublabial approach, explorative and de-bulking procedure; R2
Negative
70 Gy, 35#, Co-60, 3 fields, continuous course; Tu site
DOD at 14 months, locally progressive disease
M - Male; F - Female; Rt - Right; Lt - Left; P - Papilloma; IP - Inverted papilloma; M - Metachronous; S - Synchronous; n.s. - Not specified; SCC - Squamous cell carcinoma; # - No. of fractions; Tu - Tumor. NED - No evidence of disease; DOD - Died of disease; DOC - Died of other cause.
*TNM clinical classification of malignant tumors of the nasal cavity and ethmoid sinuses.
The presence of SCC was confirmed by his-topathological examination of resected specimen (i.e. after a biopsy proved negative for the presence of SCC) in two patients (pts. 2 and 5). The other three patients had metachronous SCC found in recurrent IP at 3, 10 and 24 years after first surgery (in pt. 4 at the time of resection of the sixth recurrence of IP). An invasive SCC component was found in all cases.
Treatment
Four patients were operated on with curative intent. The surgical technique was dictated by the extent of the disease: a transnasal endoscopic technique and an external approach were used in two patients each. Surgery was declared as radical, without microscopic residual disease left behind,
in all four patients, and all but one were irradiated postoperatively. The patient with extensive local disease, extending to the right ethmoid complex, sphenoid, pterygopalatinal fossa and orbita, had only debulking surgery followed by radiotherapy as a definitive treatment. None of the patients received systemic chemotherapy.
Postoperatively, continuous-course radiotherapy of 5 fractions per week was delivered in three patients, using megavoltage 5-MV or 6-MV linear accelerator photon beams, to a total tumor dose of 60 Gy. In two patients, the daily dose was 2 Gy and in one patient 2.5 Gy per fraction. The patient with gross residual disease after surgical debulking received definitive radiotherapy using megavolt-age Co-60 photons in daily fractions of 2 Gy to a total dose of 70 Gy. Two-dimensional computer-based planning was used to cover the postopera-
tive tumor bed or gross tumor volume including sinuses at risk for containing microscopic disease with a >95% isodose curve. A conventional three-field technique employing a heavily weighted anterior field and two opposed lateral wedged fields to achieve dose homogeneity in the range of ±5% inside the treated volume, head holders with thermoplastic casts and individual shielding blocks were used. When appropriate, optic structures were shielded after a dose of 54 Gy. The ipsilateral regional lymphatics (regions II-V) were irradiated postoperatively in one patient (pt. 5), through an anterior field and with 2.5 daily fractions to a total dose of 40 Gy, whereas in another patient (pt. 2), region II was covered ipsilaterally to a dose of 50 Gy in 2-Gy daily fractions. In both patients, indication for neck irradiation was extensive local disease.
Outcome
After diagnosis of IP/SCC, two patients were alive at 58 and 70 months. In the patient treated solely with endoscopic resection (patient 1), isolated local recurrence, histologically confirmed as IP/SCC, developed on the nasal septum 8 moths later; an endoscopic salvage procedure resulted in a permanent local control of 62 months. Two patients died of disease-unrelated causes without disease reappearance at 8 and 46 months after diagnosis of IP/ SCC; at the time of death, they were 74 and 81 years old, respectively. The patient who underwent definitive radiotherapy died due to progression of residual disease at 14 months from biopsy.
No severe or unexpected complications of treatment were documented during therapy and none of the patients had severe late therapy-related sequelae that would demand surgical intervention or hospitalization.
Human papillomavirus
Three out of five tumors analyzed for the presence of HPV were found positive, with type 11 being present in all positive cases.
Discussion
In IP, whether or not associated with SCC, complete surgical removal of the tumor is advocated as the treatment of choice. Endoscopic treatment is preferred, whereas for lesions less accessible endoscopically, or in those with peripheral extension, open surgery is indicated.1112 When complete
resection is not possible, or for tumors with associated malignancy, radiotherapy is recommended as an adjunct to surgery.1314
While the experiences with IP are extensive410-12, series describing IP/SCC are limited and feature a limited number of cases. Tanvetyanon et al.15 collected survival data of 76 patients from his series and ten additional series published during the last 30 years, covering a recruitment period of almost six decades.413-22 The corresponding pooled median overall survival was 126 months with 3-year survival estimate of 63%, which is in the range reported for invasive SCC of the nasal cavity and paranasal sinuses23 but much lower compared to figures appearing in IP series.41112
Recommendations for the use of radiotherapy are based on clinical observations rather than scientific analyses. More than three quarters of 76 patients from pooled group reported by Tanvetyanon et al. had one or another form (i.e. pre- or postoperative, definitive, palliative) of irradiation but no analysis on the value of radiotherapy versus surgery alone was carried out in their study.15 After detailed review of the studies analyzed in the paper mentioned above, we conclude that the low quality of information on the tumor extent and treatment, including completeness of surgical resection, in some publications must be the reason.
According to Hug et al.13 and Gomez et al.14, the probability of regional or systemic dissemination of IP/SCC is low. Consequently, they proposed elective irradiation of regional lymphatics only in patients with extensive involvement of the nasopharynx or clinically or radiologically apparent neck metastases. Our limited experience corroborates their recommendations.
Because of generally accepted opinion that radiotherapy should not be used as an adjunct to surgery in bland IP, in IP/SCC cases the choice of irradiation dose level is usually dictated by the recommendations for invasive SCC. This is obviously the case when series with sufficiently described radiotherapy details are reviewed.14151720 In 19 pre- or postoperatively irradiated patients, radiotherapy doses ranged from 45 Gy to 70.4 Gy (median 60 Gy); all but four patients received 58.4-66.8 Gy. Furthermore, in a group of 13 patients with IP/ SCC reported by Hugh et al.13 only one local failure occurred after gross total resection and adjuvant hyperfractionated or conventionally fractionated radiotherapy to a mean dose of 59 Gy or 60 Gy, respectively. Following subtotal resection, one out of three tumors failed locally after hyperfractionated irradiation to a mean dose of 66 Gy.13 In the present
series, all three tumors postoperatively irradiated were locally controlled at 8, 46 and 58 months post-diagnosis; an irradiation dose of 60 Gy was adjusted to the tumor stage (T3-T4) and the presence of the SCC component.
Experience with chemotherapy in IP/SCC is very scarce. In the majority of cases, chemotherapy was aimed at palliating symptoms of unresectable disease, locally or at distant sites,15,20 or has exceptionally been used for reducing tumor size before sur-gery15,21, or in a postoperative setting, usually with irradiation.1519-21 The chemotherapeutics used were platinum compounds, 5-fluorouracil, paclitaxel, etoposide, and methotrexate.1521 No conclusions could be made on the effectiveness of chemotherapy in SCC/IP; however, none of the three patients with distant metastases reported by Tanvetyanon et al. 15 responded to any of the chemotherapy regimens used.
Presence of HPV type 11 was confirmed in three out of five tumors from the present series. Similarly, Cheung et al.5 demonstrated the presence of HPV in four out of seven IP/SCC cases, one of them being type 11 (typing was not done in other cases because of inadequate HPV DNA content). Simultaneously decreased expression of p16 found in above cited and other studies indicates that the role of HPV in the oncogenesis of IP/SCC differs from that in cervical SCC. It seems that HPV infection occurs as an early event in the multistep process of malignant transformation from IP to SCC.5624 However, others suggested that HPV infection may represent incidental colonization rather than being and important etiological factor.7
Rather high prevalence of HPV infection in IP but also IP/SCC specimens poses clinically relevant question on the potential prognostic significance of HPV status. Patients with HPV-positive SCCs of the head and neck, oropharynx in particular, have superior outcome, attributed to enhanced radiation and chemo-sensitivity due to an intact apoptotic mechanism in response to radiation and chemo-therapy.2526 Because in oropharyngeal SCCs HPV types 16 and 18 rather than 11 are usually found, the question of radio/chemo-sensitivity of HPV-positive IPs and IP/SCCs at this point remains to be elucidated. However, unexpectedly favorable responses after radiotherapy have also been reported in extensive IPs and SCC/IPs. Myers et al.17 described a case of IP/SCC destroying the bony walls of the antrum with orbital invasion; no residual IP or SCC was found in the surgical specimen after 60 Gy of preoperative radiotherapy. A similar experience with preoperative irradiation was reported by
Gomez et al.14, whereas in the patient with non-re-sectable IP with bilateral involvement of the nasal cavity and paranasal sinuses, radiotherapy alone with 65 Gy was deemed curative (no recurrence at 7 years). The authors draw attention to the rather long interval after irradiation, from 3 to 6 months, for gross disease to disappear.14 Also, after surgical debulking of locally recurrent IP associated with carcinoma in situ, affecting the zygomatic area and with extension into the infratemporal fossa, Levendag et al.27 found irradiation to 64 Gy highly effective, resulting in complete regression of the lesion for almost one year.
Conclusions
According to clinical experiences, combination of surgery and postoperative irradiation with radiotherapy dose levels in a range used for invasive SCC are recommended for operable IP/SCC. Elective neck irradiation should be considered only for patients with extensive nasopharyngeal involvement or apparent regional metastases. For non-resectable disease, radical radiotherapy to a dose of 66-70 Gy could be of benefit with potential for long-lasting remission or even cure.
Acknowledgement
A study was supported by Slovenian Research Agency Grant P3-0307.
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Comparison of survival of patients receiving laparoscopic and open radical resection for stage II colon cancer
Cui-Zhen Fan1, Yu-Ping Chu1, Ping Wei2, Hong Dai2, Wenming Chen3
1	Department of Oncology, Beijing Chaoyang Hospital, Capital University of Medical Science, Beijing, China
2	Department of Pathology, Beijing Chao yang Hospital, Capital University of Medical Science, Beijing, China
3	Department of Hematologic Neoplasms and Oncology, Beijing Chaoyang Hospital, Capital University of Medical Science, Beijing, China
Received 28 March 2011 Accepted 10 July 2011
Correspondence to: Wenming Chen, Departement of Hematologic Neoplasms & Oncology, Beijing Chaoyang Hospital, No.8 Baijazhuang Road Beijing 100020, China. Phone: +86 10 85231240; Fax: +86 10 85231244; Email: chenq52000@163.com
Disclosure: No potential conflicts of interest were disclosed.
Background. The aim of the study was to compare the survival of patients receiving laparoscopic vs. open radical resection for stage II colon cancer.
Patients and methods. Two hundred and twenty patients with stage II colon cancer were enrolled from Beijing Chaoyang Hospital of Capital Medical University from January 2000 to December 2009, including 61 patients in the laparoscopic radical resection group and 159 patients in the open radical resection group. The survival data in both groups were compared using the log rank test based on Kaplan-Meier survival curves.
Results. There was no statistically significant difference in the 3-year survival (88.5% vs. 80.5%; X2=1.98, P=0.159) and the 5-year survival (81.9% vs. 69.2%; X2=1.98, P=0.159) between both groups. However, statistically significant difference was found in median overall survival (mOS), which was 102.6 (95% CI: 76.8-122.7) months in the laparoscopic group and 90.0 (95% CI: 70.4-109.6) months in the open radical resection group (X2=4.183, P=0.041). mOS was 96 (95% CI: 68.6-111.4) months and 92.6 (95% CI: 56.8-107.2) months in those with and without postoperative chemotherapy, respectively (X2=6.389, P=0.011). For patients older than 75 years the mOS was 90.0 (95% CI: 25.3-105.0) months and 83.4 (95% CI: 13.1-96.9) months in the laparoscopic and open group, respectively. The difference between the both groups was statistically significant (X2=6.191, P=0.013).
Conclusions. The mOS of patients receiving laparoscopic radical resection was better than open radical resection for stage II colon cancer, especially for patients over 75 years old.
Key words: stage II colon cancer; laparoscopy; chemotherapy; prognosis
Introduction
The incidence of colorectal cancer is 3.6-59.1 per 100,000 people worldwide.1 It is one of the most common malignancies in the world. Its incidence is still increasing as people's lifestyle changes; especially in developing countries.23 Surgical resection is still the only approach for curing colorectal cancer. The gold criterion of successful removal is that the cancer margins and lymph nodes in relative regions are completely resected. Currently there are
many reports available on laparoscopic radical resection for colorectal cancer. Laparoscopic radical resection achieves rapid recovery and few postoperative complications with recognized short-term outcomes better than open radical resection.4-7 Latest follow-up data of laparoscopic radical resection also confirm the long-term outcomes of lapar-oscopic radical resection for colorectal cancer; the 1-year, 3-year, and 5-year survival following lapar-oscopic radical resection is similar to that following open radical resection.5-7 However, the survival might depended on post-treatment surveillance of
FIGURE 1. Overall survival curves for 220 patients undergoing FIGURE 2. Overall survival curves for 220 patients with or without laparoscopic and open radical resection.	adjuvant chemotherapy.
patients.8 There is also report on better efficacy of laparoscopic radical resection than open radical resection as laparoscopic radical resection reduces cancer recurrence, cancer-related mortality and other risks.9 In the present study, the survival of patients receiving laparoscopic and open radical resection for stage II colon cancer in 220 patients with stage II colon cancer enrolled from Beijing Chaoyang Hospital of Capital Medical University between January 2000 and September 2009 were retrospectively compared.
Patients and methods
Two hundred and forty-nine patients with stage II colon cancer were treated in Beijing Chaoyang Hospital of Capital Medical University from January 2000 to December 2009. Twenty-nine patients were lost during the follow up. Two hundred and twenty of them were included into the present study according to the inclusion criteria. There were 61 in the laparoscopic radical resection group and 159 in the open radical resection group. The inclusion criteria were: (a) complete medical records with definitive pathology diagnosis of colon cancer treated with radical resection; (b) stage II in the TNM staging system and neoadjuvant chemotherapy not practiced. Exclusion criteria were: (a) synchronous or metachronous colorectal carcinoma, or familial adenomatous polyposis; (b) multiple primary malignant tumours; (c) surgery complication related death; and (d) laparoscopic radical resection replaced by open radical resection. Informed consent was obtained from all these
patients. Sixty-four patients underwent postoperative 5-fluorouracil based chemotherapy, while 156 patients were only underwent radical resection.
Preparations prior to laparoscopic and open radical resection were similar. Tracheal catheteri-zation and general anaesthesia were administered. Surgical procedure was performed according to instructions for tumour-free surgery.
Among 64 patients treated with chemotherapy, fifty-tree patients underwent 5-fluorouracil based chemotherapy, complemented by calcium folinate, cis-platinum and oxaliplatin for 4-6 courses, and 11 patients were treated by xeloda alone or combination of xeloda and oxaliplatin for 6-8 courses.
Evaluation of recurrence of colon cancer comprised physical examination, chest X-ray, abdominal CT, and colonoscopy (once a year). The patients were followed up through telephone, outpatient visits and inpatient records. The follow up started from the day of surgery and ended on December 31, 2010. The end-point-data would be 3, 5-year survival and median overall survival (mOS).
Statistical analyses were done using SPSS 15.0. X2 test was performed for general data including age and gender. The Kaplan-Meier survival curves and the log rank test were used to analyse the survival data with the selection of operation and treatment. For all analyses, the level of significance was set at P <0.05.
Results
There was no statistically significant differences in the gender, age, cancer site, histological classifica-
FIGuRE 3. Survival curves for 220 patients undergoing laparoscopic or open radical resection in different age periods: A: less than 60 years old; B, 60-75 years old; C=A+B; D, 75 years old.
tion, differentiation, vascular thrombus, nerve invasion, lymph nodes revealed by postoperative pathology, or postoperative chemotherapy between the laparoscopic and open radical resection groups (P>0.05) (Table 1).
Twenty-nine patients were lost to the follow up with a loss rate of 11.6%. The follow up period ranged from 3 to 128 months with an average of 52.5 months. There was no statistically significant difference in the 3-year survival (88.5% vs. 80.5%; X2=1.98, P=0.159) and the 5-year survival (81.9% vs. 69.2%; X2=1.98, P=0.159) between both groups. However, statistically significant difference was found in mOS, which was 102.6 (95% CI: 76.8-122.7) months in the laparoscopic group and 90.0 (95% CI: 70.4-109.6) months in the open radical resection group (X2=4.183, P=0.041) (Figure 1). mOS was 96 (95% CI: 68.6-111.4) months and 92.6 (95% CI: 56.8107.2) months in those with or without postoperative chemotherapy, respectively (X2=6.389, P=0.011) (Figure 2).
For patients below 75 years old, the mOS was 108 (95% CI: 68.9~173.0) months and 120.8 (95% CI: 69.5~172.5)90.0 months in the laparoscopic and open radical resection groups, without statistically significant difference (X2=1.0136.191, P=0.314). For patients older than 75 years the mOS was 90.0 (95% CI 25.3 - 105.0) months and 83.4 (95% CI: 13.1 - 96.9) months in the laparoscopic and open group, respectively. The difference between these two groups was statistically significant (X2=6.191, P=0.013) (Figure 3).
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TABLE 1. General information of 220 patients with stage II colon cancer
Laparoscopic radical resection
Open radical resection
X2
Total
Sex
Male Female Age
~60 years ~75 years >75 years
Tumor site
Ascending colon Transverse colon Descending colon Sigmoid colon Pathological classification Highly differentiated adenocarcinoma Moderately differentiated adenocarcinoma Mucous adenocarcinoma Lowly differentiated adenocarcinoma with signet ring cells Chemotherapy Yes No
Vascular thrombus
Yes No
Nerve invasion
Yes
No
Lymph node metastasis
>12 <12
61
28 33
19 24
20 6 10 25
50 6 4
23 38
54
7
54 6
24 37
159
86 73
59 73 27
75 14 22 48
4
123 20 12
41
145 14
146 13
90 69
183
4.255
3.910
2.485
0.364
0.1829
.418
0.276
0.119
0.418
0.608	0.962
0.115
0.546
0.669
0.492
P
Discussion
Since Jacobs et al. reported the initial use of laparo-scopic radical resection of sigmoid colon, laparo-scopic radical resection has been increasingly used for colorectal cancer.10 However, questions are raised regarding whether the long-term outcomes of laparoscopic radical resection are comparative to that of open radical resection and whether it leads to tumour metastasis.
This study showed that the 3-year survival was 88.5% and 80.5% in the laparoscopic and open radical resection groups in 220 patients with stage II colon cancer. Bonjer et al.6 reported that the 3-year survival was 82.2% and 83.5% respectively for both groups in stage II colon cancer. Kitano et al.11 found that the 5-year survival was 94.8% for laparoscopic radical resection, comparable to open radical resection. Fleshman et al.7 reported that the 5-year survival was 74.6% and 76.4% respectively for laparo-scopic and open radical resection group in a multi-centre study in 872 patients with colon cancer and concluded that there was no statistically significant difference in the overall survival and the disease-free survival between two groups, suggesting
that the long-term efficacy is similar for two procedures. This current study found similar results in stage II colon cancer patients, again confirming that laparoscopic radical resection can achieve favourable outcomes for early-stage colon cancer.
In our study the mOS was 102.6 months and 90.0 months in the laparoscopic and open radical resection groups with a statistically significant difference, further demonstrating laparoscopic radical resection has better survival outcomes than open radical resection. Bilimoria et al.12 reported that the 5-year survival was apparently better for laparoscopic radical resection in patients with stages I and II colon cancer. Lacy et al.9 revealed that laparoscopic radical resection reduced cancer recurrence, risks of mortality from cancer, and other risks, and that the tumour-bearing survival was better for laparoscopic radical resection than open radical resection in a long-term follow up of 218 cases of colon cancer. These results may be attributable to minimal invasion of the surgery and rapid rehabilitation of immune function following laparoscopic radical resection.13-15
The 5-year survival is 75% -80% for stage II colon cancer following radical resection and 20%-25% pa-
tients die of recurrence or distant metastasis.16 As there are no large-scale clinical trials that conclude that stage II colon cancer patients can benefit from postoperative adjuvant chemotherapy, postoperative chemotherapy is thus controversial for stage II colon cancer.1718 The National Surgical Adjuvant Breast and Bowel Project (NSABP) thought that stage II colon cancer patients could benefit from adjuvant chemotherapy as stage III patients.19 In this study, the mOS was 96 months for patients with postoperative chemotherapy and 92.6 months for those without chemotherapy with a statistically significant difference, showing that chemotherapy is advantageous whatever surgical technique is adopted.
Although meta-analyses could not substitute large randomised clinical studies20, we cannot neglected that a pooled analysis of five randomized trials did not show the radical resection with adjuvant chemotherapy was better than radical resection alone.21 Therefore, the National Comprehensive Cancer Network (NCCN) guidelines recommend adjuvant chemotherapy for stage II colorectal cancer patients with risks for poor prognosis (high risks), such as poor histological differentiation, stage T4, invasion to blood vessels or lymph vessels, intestinal obstruction or perforation, tumours too near resection margins, and less than 12 lymph nodes for pathology examination.22 Moreover, some proteins are accepted as predictors for adjuvant chemotherapy for high-risk stage II colorectal cancer.23 Though this study indicates that chemotherapy was beneficial for patients like in the metastatic diseasse24, multi-centre trials with a large sample size and different chemotherapy regimens are required to demonstrate the effect of adjuvant chemotherapy for stage II colon cancer. Additionally, there was no statistically significant difference in the cancer site, histological classification, nerve invasion, lymph nodes, or postoperative chemotherapy between the laparoscopic and open radical resection groups.
About 50% colorectal cancer patients are over 70 years old and colorectal cancer thus becomes a common disease for patients over 70 years old.25 According to the Colorectal Cancer Collaborative Group in UK, the risk of surgery for the old increases with age; the mortality was 1.3%-5.2% for patients of 65 years old or above, and 7.1%-8.9% for patients of 85 years or above.26 But another study demonstrates that radical resection is safe in old patients with colorectal cancer and high risks of radical resection are mainly correlated with complications and emergency treatment instead of age.27 In
this study, there were 18 patients over 75 years old in the laparoscopic radical resection group and 27 patients over 75 years old in the open radical resection group. For patients of over 75 years old, it is suggested that the survival of laparoscopic radical resection is superior over open radical resection for stage II colon cancer. The advantage in survival is probably related to less invasive nature of laparo-scopic procedure, which can be of greatest benefit in the patients older than 75 years
Conclusions
The survival of patients receiving laparoscopic radical resection was better than that of open radical resection for stage II colon cancer, especially for patients over 75 years old. Thus laparoscopic radical resection should be selected for these stage II colon cancer patients as well as postoperative adjuvant chemotherapy for better survival.
References
1.	Center MM, Jemal A, Smith RA, Ward E. Worldwide variations in colorectal cancer. CA Cancer J Clin 2009; 59: 366-8.
2.	Hendon SE,DiPalma JA. U.S. practices for colon cancer screening. Keio J Med 2005; 54: 179-83.
3.	Horvat M, Stabuc B. Microsatellite instability in colorectal cancer. Radiol Oncol 2011; 45: 75-81.
4.	Hewett PJ, Allardyce RA, Bagshaw PF, Frampton CM, Frizelle FA, Rieger NA, et al. Short-term outcomes of the Australasian randomized clinical study comparing laparoscopic and conventional open surgical treatments for colon cancer: the ALCCaS trial. Ann Surg 2008; 248: 728-38.
5.	Bonjer HJ, Hop WC, Nelson H, Sargent DJ, Lacy AM, Castells A, et al. Laparoscopically assisted vs open colectomy for colon cancer: a metaanalysis. Arch Surg 2007; 142: 298-303.
6.	Buunen M, Veldkamp R, Hop WC, Kuhry E, Jeekel J, Haglind E, et al. Survival after laparoscopic surgery versus open surgery for colon cancer: long-term outcome of a randomised clinical trial. Lancet Oncol 2009; 10: 44-52.
7.	Fleshman J, Sargent DJ, Green E, Anvari M, Stryker SJ, Beart RW Jr, et al. Laparoscopic colectomy for cancer is not inferior to open surgery based on 5-year data from the COST Study Group trial. Ann Surg 2007; 246: 655-62.
8.	Velenik V. Post-treatment surveillance in colorectal cancer. Radiol Oncol 2010; 44: 135-141.
9.	Lacy AM, Delgado S, Castells A, Prins HA, Arroyo V, Ibarzabal A, et al. The long-term results of a randomized clinical trial of laparoscopy-assisted versus open surgery for colon cancer. Ann Surg 2008; 248: 1-7.
10.	Jacobs M, Verdeja JC, Goldstein HS. Minimally invasive colon resection (laparoscopic colectomy). Surg Laparosc Endosc 1991; 1: 144-50.
11.	Kitano S, Kitajima M, Konishi F, Kondo H, Satomi S, Shimizu N; Japanese Laparoscopic Surgery Study Group. A multicenter study on laparoscopic surgery for colorectal cancer in Japan. Surg Endosc 2006; 20: 1348-52.
12.	Bilimoria KY, Bentrem DJ, Nelson H, Stryker SJ, Stewart AK, Soper NJ, et al. Use and outcomes of laparoscopic-assisted colectomy for cancer in the United States. Arch Surg 2008; 143: 832-9.
13.	Milsom JW, Jerby BL, Kessler H, Hale JC, Herts BR, O'Malley CM. Prospective, blinded comparison of laparoscopic ultrasonography vs. Contrast-enhanced computerized tomography for liver assessments undergoing colorectal carcinoma asurgery. Dis Colon Rectum 2000; 43: 44-9.
14.	Tang CL, Eu KW, Tai BC, Soh JG, MacHin D, Seow-Choen F. Randomized clinical trial of the effect of open versus laparoscopically assisted colectomy on systemic immunity in patients with colorectal cancer. Br J Surg 2001; 88: 801-7.
15.	O'Connell J, Maggard M, Ko C. Colon cancer survival rates with the new American Joint Committee On Cancer sixth edition staging. J Natl Cancer inst 2004; 96: 1420-5.
16.	Braga M, Vignali A, Zuliani W, Radaelli G, Gianotti L, Martani C, et al. Metabolic and functional results after laparoscopic colorectal surgery: a randomized, controlled trial. Dis Colon Rectum 2002; 45: 1070-7.
17.	Ota D, Nelson H. The surgeon and adjuvant therapy for stage colon cancer. Anna Surg Oncol 2007; 14: 272-3.
18.	Brain MW, Jeffreg AM, Harvey JM, Robert JM. Adjuvant treatment of colorectal cancer. CA Cancer J Clin 2007; 57: 168-85.
19.	Mamounas E, Wieand S, Wolmark N, Bear HD, Atkins JN, Song K, et al. Comparative efficacy of adjuvant chemotherapy in patients with Dukes'B versus Dukes'C colon cancer: results form fournational surgical adjuvant breast and bowel project adjuvant studies(C-01, C-02, C-03, and C-04). J Clin Oncol 1999; 17: 1349-55.
20.	Kovač V, Smrdel U. Meta-analyses of clinical trials in patients with non-small cell lung cancer. Neoplasma 2004; 51: 334-40.
21.	Efficacy of adjuvant fluorouracil and folinic acid in B2 colon cancer. International Multicentre Pooled Analysis of B2 Colon Cancer Trials (IMPACT B2) Investigators. J Clin Oncol 1999; 17: 1356-63.
22.	Benson AB 3rd, Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol 2004; 22: 3408-19.
23.	Strzelczyk B, Szulc A, Rzepko R, Kitowska A, Skokowski J, Szutowicz A, et al. Identification of high-risk stage colorectal tumors by combined analysis of the NDRG1 gene expression and the depth of tumor invasion. Ann Surg Oncol 2009; 16: 1287-94.
24.	Ocvirk J. Advances in the treatment of metastatic colorectal carcinoma. Radiol Oncol 2009; 43: 1-8.
25.	Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55: 74-108.
26.	Colorectal Cancer Collaborative Group. Surgery for colorectal cancer in the elderly: a systematic review. Lancet 2000; 16: 968-74.
27.	Kruschewski M, Germer CT, Rieger H, Buhr HJ. Radical resection of colorectal carcinoma in the oldest old. Chirurg 2002; 73: 241-4.
Expression of NF-kB p65 phosphorylated at serine-536 in rectal cancer with or without preoperative radiotherapy
Andreas Lewander1, Jinfang Gao1, Gunnar Adell2, Hong Zhang3, Xiao-Feng Sun1
1	Department of Oncology, Institute of Clinical and Experimental Medicine, University of Linköping, Linköping, Sweden
2	Department of Oncology, Karolinska University Hospital, Stockholm, Sweden
3	Department of Biomedicine, University of Skövde, Sweden
Received 16 July 2011 Accepted 8 August 2011
Correspondence to: Prof. Xiao-Feng Sun, M.D., Ph.D., Department of Oncology, Institute of Clinical and Experimental Medicine, University of Linköping, SE-581 85 Linköping, Sweden. Phone: +46-10-10-32066; Fax: +46-10-10-33090; Email: xiao-feng.sun@liu.se
Disclosure: No potential conflicts of interest were disclosed.
Background. In the present study, we investigated NF-kB p65 phosphorylated at Serine-536 (phosphor-Ser536-p65) in rectal cancer and its relationship to preoperative radiotherapy (RT), clinicopathological variables and biological factors.
Patients and methods. Expression of phosphor-Ser536-p65 was examined by using immunohistochemistry in 141 primary rectal cancers, 149 normal mucosa specimens and 48 metastases in the lymph nodes, from rectal cancer patients who participated in a Swedish clinical trial of preoperative RT.
Results. The expression of phosphor-Ser536-p65 in the cytoplasm increased from normal mucosa to primary tumour (p<0.0001, for both the group that did and the group that did not received RT). The expression did not further increase from primary tumour to metastasis in either group (p>0.05). Expression of phosphor-Ser536-p65 was positively related to, or tended to be related to, the expression of tumour endothelium marker 1 (TEM1, p=0.02), FXYD-3 (p=0.001), phosphatase of regenerating liver (PRL, p=0.02), p73 (p=0.048) and meningioma associated protein (MAC30, p=0.05) in the group that received RT but there were no such relationships in the group that did not received RT (p>0.05). The expression of phosphor-Ser536-p65 was not related to clinicopathological factors including survival (p>0.05). Conclusions. The increased expression of phosphor-Ser536-p65 may be involved in rectal cancer development. After RT, phosphor-Ser536-p65 seems to be positively related to the biological factors, which associated with more malignant features of tumours. However, phosphor-Ser536-p65 was not directly related to the response of RT based on recurrence and survival.
Key words: NF-kB, serine-536; radiotherapy; rectal cancer; immunohistochemistry; recurrence; prognosis
Introduction
Nuclear factor-kappaB (NF-kB) is responsible for expression by regulating many genes for immune response, cell adhesion, differentiation, proliferation, angiogenesis and apoptosis. The function of NF-kB is inhibited by binding to NF-kB inhibitory proteins, and imbalance of NF-kB and its inhibitors has been associated with development of tumours and other diseases.1-3 Five members of the NF-kB family have been found in human cells, RelA (p65), p105/p50, p100/p52, RelB and c-Rel. The most common form in human cells is p65/p50 heterodimer.
The regulation of the NF-kB protein family is very important. Upon activating signals the inhibitory proteins are degraded and the protein translocates into the nucleus where it exert its effect. The regulation also occurs at the posttranslational level, where protein phosphorylation of the different subunit is one very important mechanism of regulation. Several different phosphorylation sites on the subunits have been discovered. An important site of phosphorylation of p65 subunit is at Serine-536 (phospho-Ser536-p65), and this phosphorylation is involved in regulation of transcriptional activity, nuclear localisation and protein stability.1245
TABLE 1. Characteristics of patients and rectal cancers
Characteristics	Non-Radiotherapy		Radiotherapy
	No. (%)		No. (%)
Gender
Male Female
Age (years) <66 >66
Stage
IIA IIIA IIIB IIIC IV
Differentiation
Well
Moderately Poorly
Numbers of tumours
Single Multiple* Unknown Surgical type
Rectal amputation Anterior resection Resection margin Tumour free
Tumour involved margin Distance to anal verge (cm) Mean
45 34
29 50
22 18 9 12 14 4
5 56
68 9 2
42 37
75 4
7.3
(57) (43)
(37) (63)
(28) (23) (11) (15) (18) (5)
(6) (71) (23)
(86) (11) (3)
(53) (47)
(95) (5)
39 23
23 39
16 22 0 16 3 5
4 40
51 11 0
22 40
59 3
(63) (37)
(37) (63)
(25) (36)
(25) (5) (8)
(6) (65) (29)
(82) (18)
(35) (65)
(95) (5)
*Other colorectal cancer or other type of tumour besides the present rectal cancer.
It has also been shown that in some tumours NF-kB activation can enhance radiosensitivity.67 Preoperative radiotherapy (RT) is today a standard treatment for rectal cancer patients in Sweden and other countries.8 It has been shown to increase survival of the patients.910 In the present study, we investigated whether the phospho-Ser536-p65 was related to response of RT in rectal cancer patients who received or did not receive RT, and whether there were any relationships of the phospho-Ser536-p65 with clinicopathological variables and biological factors.
Patients and methods
Patients
This study included the patients with rectal ad-enocarcinoma from the Southeast Swedish Health Care region that participated in a Swedish clinical trial of preoperative RT between 1987 and 1990.9 Surgical specimens were obtained by either rectal amputation or anterior resection from 141 patients.
The mean age at diagnosis was 66 years (range 36-85). The mean follow-up time was 83 months (range 0-193). Seventy-nine patients had surgery alone. Sixty-two patients were randomised to pr-eoperative radiotherapy, receiving 25 Gy in 5 fractions over a median of 6 days (range 5-12). Surgery was performed after a median of 3 days (range 1-13) after radiotherapy. The characteristics of the patients and tumours are given in Table 1.
The data regarding expression of tumour en-dothelium marker 1 (TEM1, unpublished data), FXYD-3 (also known as MAT-8), phosphatase of regenerating liver (PRL, also known as PTP4A3, pro-tein-tyrosine phosphatase), p73 and meningioma associated protein (MAC30) on the same material used as in the present study, determined by immu-nohistochemistry, were taken from previous studies performed at our laboratory.11-14 The number of the patients listed in Table 2 was less than the number of the patients mentioned in the materials of the present study due to available numbers of the previous cases11-14, which matched, with the present study. The immunohistochemical staining
TABLE 2. Expression of NF-kB phosphorylated at Serine-536 in relation to biological factors expressed in rectal cancer
Non-radiotherapy
Weak (%)
Strong (%)
p-value
Radiotherapy
Weak (%)
Strong (%)
p-value
TEM1
Weak Strong
FXYD3
Weak Strong
PRL
Weak Strong
p73
Weak Strong MAC30
Weak Strong
9 (39) 13 (30)
13 (45) 10 (25)
(34) (33)
(29) (36)
(31) (35)
14 (61) 31 (70)
16 (55) 30 (75)
23 (66) 18 (67)
17 (71) 28 (64)
20 (69) 24 (65)
0.43
0.08
1.00
0.55
0.73
10	(59)
8	(25)
13	(68)
7	(21)
11	(50) 5	(19)
9	(56)
8	(27)
9	(53) 8	(25)
7 (41) 24 (75)
6 (32) 27 (79)
21
(50) (81)
7	(44) 22	(73)
8	(47) 24	(75)
0.02
0.001
0.024
0.048
0.05
for those factors was performed on the normal mu-cosa, primary tumour and metastasis in the lymph nodes from both the non-RT and RT groups.
Immunohistochemistry
Five-micrometer sections were deparaffinised in xylene and rehydrated in graded ethanol. As the method for antigen retrieval we used was high-pressure cooking in 0.01 M Tris-EDTA buffer (pH 9.0). The sections were heated to 125°C for 30 sec and then cooled to 90°C for 10 sec, the sections were then kept in the buffer till room temperature. The sections were incubated with 3% H2O2-methanol for 20 min and washed with phosphate-buffered saline (PBS, pH 7.4). After that the sections were incubated with rabbit anti-phospho-Ser536-p65 antibody (phospho S536, ab28856, Abcam, Cambridge, MA) at 20 mg/ml in antibody diluent (Dako, Carpinteria, CA) overnight, followed by rinsing with PBS. The antibody binds specifically to the Ser536-phosphorylated form of p65 and does not cross-react with non-phosphorylated p65 or any other members of the NF-kB family. Subsequently, the sections were incubated with a goat anti-rabbit/ mouse, coupled with peroxidase provided by the Dako ChemMate EnVision Detection Kit (Dako) for 25 min, and washed with PBS. The peroxidase reaction, using 3,3'-diaminobenzidine tetrahydro-chloride, was performed (Dako) for 8 min. Sections known to stain positively were included as positive controls. The negative controls used PBS instead of the primary antibody. In all staining procedures, the positive controls showed clear staining, and there was no staining in the negative controls.
The sections were microscopically examined and scored independently by Lewander A and Gao J without any information on the clinicopatho-logical data. The slides were initially classified as weak including negative (<5% of positive cells) and strong staining in the cytoplasm of normal epithelial cells, and tumour cells and metastasis irrespectively of the percentage of positive cells. To avoid artificial effects, cells in areas with necrosis, with poor morphology, or in the margins of sections were excluded from the analysis.
Statistical analysis
The significance of the difference in phospho-Ser536-p65 expression between normal mucosa, primary tumour and metastasis was tested by Chi-square and McNemar methods. The relationships between phospho-Ser536-p65 expression and clin-icopathological/biological variables were examined by Chi-square method, and the relationships to survival were tested by using Cox's proportional hazard model. Survival curves were calculated by using the Kaplan-Meier method. Two-sided p values of <0.05 were considered statistically significant.
Results
Phospho-Ser536-p65 expression in the cytoplasm of normal mucosa, primary tumour and metastasis in the lymph node
When we compared staining intensity of phospho-Ser536-p65 expression in the cytoplasm of normal
IS
C
O
M
£ a
X
LU
CT C
o Sö
80 70 60 50 40 30 20 10 0
p
m
n
m
Non-RT
RT
FIGuRE 1. Frequency of strong phospho-Ser536-p65 expression in normal mucosa (N), primary tumour (P) and metastasis in the lymph nodes (M) in non-radiotherapy (non-RT) and radiotherapy (RT).
mucosa, primary tumour and metastasis in the lymph node we found significantly more samples with the strong staining of phospho-Ser536-p65 expression in primary tumour than in normal muco-sa in the both non-RT and RT groups (p<0.0001 for both Chi-square and McNemar tests for both non-RT and RT groups, Figure 1). There was no significant difference between primary tumour and metastases in either non-RT or RT groups (p>0.05, Figure 1).
We compared phospho-Ser536-p65 expression before and after RT and found there were no differences in normal mucosa (p=0.06), primary tumour (p=0.30) as well as metastases (p=0.81) with chi-square test.
Figure 2 shows phospho-Ser536-p65 expression in normal mucosa, primary tumour and surgical specimens) and metastases in the lymph node. There was weak phospho-Ser536-p65 expression in normal mucosa, while strong expression in the cytoplasm of primary surgical and metastatic tu-
Phospho-Ser536-p65expression in the cytoplasm in relation to clinicopathological and biological factors
We compared the expression of phospho-Ser536-p65 expression with the expression of TEM1, FXYD3, PRL, p73 and MAC30 (Table 2). Phospho-Ser536-p65 expression was positively related to or tended to be positively related to TEM1 (p=0.02), FXYD-3 (p=0.001), PRL (p=0.02) and p73 (p=0.048) and MAC30 (p=0.05) in the RT group. However
in the non-RT group, there were no such relationships (p>0.05, Table 2).
We analysed the relationship of phospho-Ser536-p65 expression in the cytoplasm of primary tumour with clinicopathological variables and did not find any statistically significant relationship of phospho-Ser536-p65 expression with gender, age, differentiation, stage, local/distant recurrence and survival in the two sub-groups of non-RT and RT, or in the whole group of the patients (p>0.05, data not shown).
Discussion
In this study we examined materials from rectal cancer patients included in the Swedish rectal cancer trial of preoperative RT9, i.e., the patients divided into two groups, one that received and one that did not received preoperative RT.
When we compared staining intensity of phos-pho-Ser536-p65 expression in the cytoplasm of normal mucosa, primary tumour and metastasis in the lymph node we found significantly more samples with strong staining in primary tumour than in normal mucosa in either the non-RT or RT group. There was no significant difference between primary tumour and metastases in either the non-RT or RT group. Others have found similar results, that NF-kB is upregulated in tumour cells compared with the corresponding normal cells in previous studies. Lind et al. used electrophoretic mobility shift assay (EMSA) technique and demonstrated that NF-kB in primary tumour was greatly increased compared with adjacent normal tissue from the same patients.15 Yu et al. examined the expression of NF-kB p65 by using a monoclonal antibody against NF-kB p65 in normal colorectal mucosa, colorectal adenomas and colorectal adenocarcinomas, and showed that NF-kB p65 expression was significantly increased from normal mucosa to adenoma and to adenocarcinoma, furthermore the expression was increased with the transition from low to moderate and to high dysplasia of adenoma.16 Our previous study in colorectal cancer by immunohistochemis-try using the same antibody, showed primary tumour had stronger phospho-Ser536-p65 expression than normal mucosa but had no difference between primary tumours and metastases in the lymph node (unpublished data). Taken together, these results indicate that the NF-kB p65 may play a role in earlier development of colorectal cancer.
In the same materials used here we have previously studied expression of TEM1 (unpublished
n
p
FIGURE 2. The expression of phospho-Ser536-p65 was weak in normal mucosa (A) and strong expression in primary tumour (B) and metastases in the lymph node (C).
data), FXYD3 (9), PRL (11), p73 (10) and MAC30 (12). We found that phospho-Ser536-p65 expression was positively related to TEM1, FXYD-3, PRL, p73 and MAC30 in tumours that received RT, however there were no such relationships in the non-RT group. TEM1 was expressed on periendothelial mural cells (i.e., pericytes) and activated tumour fibroblasts, probably played a role in the tumour vasculature.17-19 In our previous study we found TEM1 expression in the stroma increased from normal mucosa to primary tumour both in the non-RT and RT group. In the RT group, TEM1 expression in the stroma significantly increased from Dukes' A to B-D. FXYD-3 is an 8-kDa trans-membrane protein and acts as a chloride channel or chloride channel regulator.20 FXYD-3 is overexpressed in several types of cancers including colorectal cancer.112021 In our previous study, we found that FXYD-3 expression in the primary tumours was, or tended to be increased compared with normal mucosa regardless of RT. Furthermore in the RT group, strong FXYD-3 expression alone or combined with PRL was related to an unfavourable prognosis independent of both, the TNM stage and tumour differentiation, which are important prognostic factors.22 In tumours with strong FXYD-3 expression, there were less tumour necrosis and a trend of increased incidence of distant metastasis after RT. None of these effects was seen in the non-RT group.11 PRL was identified as an important protein in the meta-static process of colorectal cancer. The PRL family consists of three members, PRL-1, -2, and -3. PRL-3, as a tyrosine phosphatase, may play critical roles in the regulation of cellular growth and cell cycle.2324 We earlier found that PRL expression was increased from normal mucosa to primary tumour. In the RT group, strong PRL expression was related to distant recurrence and poor survival,
independent of both stage and differentiation, but not in the non-RT group. Overexpression of p73 protein has also been correlated with a poor prognosis in colorectal, hepatocellular and breast can-cers.2526 In the same material, we earlier found that p73 was overexpressed in rectal cancer compared with normal mucosa. The patients with p73-over-expressing tumours tended to have a higher local recurrence after RT compared to non-RT cases.12 MAC30 mRNA is expressed in the foetal liver, but not in the adult liver, suggesting a possible role in growth and differentiation of liver.2728 The expression of MAC30 is stronger in breast, stomach and colorectal cancers than the corresponding normal tissues142930, indicating that MAC30 may act as an oncogene in the cancers and might play a role in tumour development and aggressiveness. Why the relationship of phospho-Ser536-p65 expression with TEM1, FXYD-3, PRL, p73 and MAC30 in the RT cases but not in the non-RT cases in this study? One possible speculation is due to the effect of RT, namely, RT resulted in these proteins being more active, temporarily or permanently, and the cells tried to survive. The results may raise a notion that one should consider the targets of RT and the checkpoints controlling the pathways which those factors were involved in. The impact of RT on phospho-Ser536-p65 protein needs to be further investigated in a larger number of patients.
In conclusion, the positive expression of phos-pho-Ser536-p65 may be involved in rectal cancer development. After RT, the expression of phospho-Ser536-p65 was positively related to the biological factors which associated with more malignant features of tumours. However, we did not find that the NF-kB protein was directly related to the response of RT based on local/distant recurrence and survival.
Acknowledgement
The study was supported by grants from the Swedish Cancer Foundation, Swedish Research Council and the Health Research Council in the South-East of Sweden. The authors thank Helen Richard, Cecilia Bergenwald, Gertrud Stridh, Gunnel Lindell and Kerstin Ingels, Department of Pathology, Linköping Hospital, Sweden, for kindly preparing tissue sections. We are grateful for the linguist revision by Dr. David Hinselwood.
References
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2.	Moynagh PN. The NF-kappaB pathway. J Cell Sci 2005; 118: 4589-92.
3.	Sun XF, Zhang H. NFKB and NFKBI polymorphisms in relation to susceptibility of tumour and other diseases. Histol Histopathol 2007; 22: 1387-98.
4.	Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W. IkappaB kinases phos-phorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain. J Biol Chem 1999; 274: 30353-6.
5.	Viatour P, Merville MP, Bours V, Chariot Al. Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci 2005; 30: 43-52.
6.	Miyakoshi J, Yagi K, Inhibition of IkB-œ phosphorylation at serine and tyro-sine acts independently on sensitization to DNA damaging agents in human glioma cells. Br J Cancer 2000; 82: 28-33.
7.	Yamagashi N, Miyakoshi J, Takebe H. Enhanced radiosensitivity by inhibition of nuclear factor kappa B activation in human malignant glioma cells. Int J Radiat Biol 1997; 72: 157-62.
8.	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.
9.	Improved survival with preoperative radiotherapy in resectable rectal cancer. Swedish Rectal Cancer Trial. New Eng J Med 1997; 336: 980-7.
10.	Velenik V. Locally recurrent rectal cancer: treatment options. Radiol Oncol 2009; 43: 144-51.
11.	Loftâs P, Önnesjö S, Widegren E, Adell G, Kayed H, Kleeff J, et al. Expression of FXYD-3 is an independent prognostic factor in rectal cancer patients with preoperative radiotherapy. int J Radiat Oncol Biol Phys 2009; 75: 137-42.
12.	Pfeifer D, Gao J, Adell G, Sun X-F. Expression of the p73 protein in rectal cancers with or without preoperative radiotherapy. int J Radiat Oncol Biol Phys 2006; 65: 1143-8.
13.	Wallin Â, Svanvik J, Adell G, Sun X-F. Expression of PRL proteins at invasive margin of rectal cancers in relation to preoperative radiotherapy. int J Radiat Oncol Biol Phys 2006; 65: 452-8.
14.	Zhang ZY, Zhao ZR, Adell G, Jarlstelt I, Cul YX, Kayed H, et al. MAC30 in rectal cancers with or without preoperative radiotherapy. Oncology 2006; 71: 259-65.
15.	Lind DS, Hochwald SN, Malaty J, Rekkas S, Hebig P, Mishra G, et al. Nuclear factor-KB is upregulated in colorectal cancer. Surgery 2001; 130: 363-9.
16.	Yu HG, Yu LL, Yang Y, LuoH-S, Yu J-P, MeierJJ, et al. Increased expression of RelA/nuclear factor-kappa B protein correlates with colorectal tumorigen-esis. Oncology 2003; 65: 37-45.
17.	Christian S, Winkler R, Helfrich I, Boos AM,Besemfelder E, Schadendorf D, et al. Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. Am J Pathol 2008; 172: 486-94.
18.	MacFadyen J, Savage K, Wienke D, Isacke CM. Endosialin is expressed on stromal fibroblasts and CNS pericytes in mouse embryos and is downregu-lated during development. Gene Expr Patterns 2007; 7: 363-69.
19.	Tomkowicz B, Rybinski K, Foley B, Ebel W, Kline B, Routhier E, et al. Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proc Natl Acad Sci (USA) 2007; 104: 17965-70.
20.	Morrison BW, Moorman JR, Kowdley GC, Kobayashi YM, Jones LR, Leder P. Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes. J Biol Chem 1995; 270: 2176-82.
21.	Kayed H, Kleeff J, Kolb A, Ketterer K, Keleg S, Felix K, et al. FXYD3 is over-expressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth. int J Cancer 2006; 118: 43-54.
22.	Ocvirk J. Advances in the treatment of metastatic colorectal carcinoma. Radiol Oncol 2009; 43: 1-8.
23.	Bardelli A, Saha S, Sager JA, Romans KE, Xin B, Markowitz SD, et al. PRL-3 expression in metastatic cancers. Clin Cancer Res 2003;15:5607-15.
24.	Sager JA, Benvenuti S, Bardelli A: PRL-3: a phosphatase for metastasis? Cancer Biol Ther 2004;3:952-3.
25.	Bénard J, Douc-Rasy S, Ahomadegbe JC. TP53 family members and human cancers. Hum Mutat 2003;21:182-91.
26.	Sun XF. p73 overexpression is a prognostic factor in patients with colorectal adenocarcinoma. Clin Cancer Res. 2002;8:165-70.
27.	Malhotra K, Luehrsen KR, Costello LL, Raich TJ, Sim K, Foltz L, et al. Identification of differentially expressed mRNAs in human fetal liver across gestation. Nucleic Acids Res 1999;27:839-47.
28.	Murphy M, Pykett MJ, Harnish P, Zang KD, George DL. Identification and characterization of genes differentially expressed in meningiomas. Cell Growth Differ 1993;4:715-22.
29.	Moparthi S, Arbman G, Wallin Â, Kayed H, Kleeff J, Zentgraf H, et al. Expression of MAC30 protein is related to survival and biological variables in primary and metastatic colorectal cancers. int J Oncol 2007;30:91-5.
30.	Kayed H, Kleeff J, Ding J, Hammer J, Giese T, Zentgraf H, et al. Expression analysis of MAC30 in human pancreatic cancer and tumors of the gastrointestinal tract. Histol Histopathol 2004;19:1021-31.
Efficacy of first-line systemic treatment in correlation with BRAF V600E and different KRAS mutations in metastatic colorectal cancer - a single institution retrospective analysis
Martina Rebersek1, Marko Boc1, Petra Cerkovnik2, Jernej Benedik1, Zvezdana Hlebanja1, Neva Volk1, Srdjan Novakovic2, Janja Ocvirk1
1 Department of Medical Oncology, 2 Department of Molecular Diagnostics, Institute of Oncology Ljubljana, Ljubljana, Slovenia
Received 4 August 2011 Accepted 20 September 2011
Correspondence to: Martina Reberšek, Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška 2, 1000 Ljubljana Slovenia. Phone: +386 1 5879 220; Fax: +3861 5879 400; E-mail: mrebersek@onko-i.si
Disclosure: No potential conflicts of interest were disclosed.
Background. KRAS mutation status in codons 12 and 13 is recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Despite having a wild type KRAS (wt-KRAS), not all patients with wt-KRAS respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway. Consequently, other molecular markers in colorectal cancer are needed to be evaluated to predict the response to therapy.
Patients and methods. In this retrospective study, objective responses (OR), time to progression (TTP), overall survival (OS) were analyzed in 1 76 metastatic colorectal cancer (mCRC) patients treated with first-line chemotherapy in combination with monoclonal antibodies in respect of KRAS status in codons 12 and 13 and BRAF mutational status. Results. The KRAS mutations were found in 63 patients (35.8 %), the KRAS mutation in codon 12 in 53 patients (30.1%) and the KRAS mutation in codon 13 in 10 patients (5.7%). The BRAF V600E mutation was detected in 13 of 176 patients (7.4%). In the subgroup of mCRC patients having wt-KRAS and wild type BRAF (wt-BRAF), the objective response rates were higher (OR 54.0% ,CR 14.7%, PR 39.3%) than in the patients with wt-KRAS and mt-BRAF (OR 38.5%,CR 15.4%, PR 23.1%), the difference was not statistically significant (p= 0.378). Median OS in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 107.4 months and 45 months, respectively. The difference was statistically significant (p= 0.042). TTP in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 16 months and 12 months, respectively. The difference was not statistically significant (p= 0.558).
Conclusions. Patients with BRAF V600E mutation have statistically significantly worse prognosis than the patients with wt-BRAF and progress earlier during treatment. The definitive role of the BRAF V600E mutation as a prognostic and predictive factor for the response to anti-EGFR monoclonal antibodies needs to be analyzed in large prospective clinical studies.
Key words: metastatic colorectal cancer; KRAS; BRAF; prognostic factors
Introduction
Colorectal cancer (CRC) is the fourth most common cancer and one of the leading causes of cancer death in the world. It is the most common cancer in Slovenia and, according to the Cancer Registry of Slovenia, 1279 new patients were diagnosed with CRC in 2007.1 The majority of patients need
combined modality treatment and carful post-treatment surveillance is necessary to offer patient an optimal treatment approach.23 Metastatic disease is still incurable, with 5% five-year survival without treatment. With the introduction of new chemotherapy, using oxaliplatin and irinotecan in the current management of metastatic disease, in combination with biologicals, targeting epidermal
growth factor- mediated growth regulatory pathway and the vascular endothelial growth factor-mediated angiogenesis pathway, we can prolong the progression-free survival (PFS) and overall surviva! (OS) of these patients.4-8 In selected patients with appropriate combination of therapy and surgery we can achieve approximately a 50% five-year survival.
The development of CRC is a multistep process which accumulates different gene mutations, chromosomal abnormalities and epigenetic chang-es.910 The mutations within KRAS proto-oncogen, predominately within codons 12 and 13, activate RAS/RAF signalling and are thought to occur early in carcinogenesis of CRC. The KRAS status is the first molecular marker to predict the response to anti-EGFR monoclonal antibodies cetuximab and panitumumab in metastatic CRC (mCRC) patients, and it needs to be determined before deciding in favor of treatment with anti-EGFR antibodies. As the KRAS mutations occur early in CRC formation, there is a high concordance between the KRAS mutations of primary tumour and metastases, which was confirmed in previous studies.11-13 In a recent retrospective study, de Roock with his colleagues raised the possibility that the patients with the KRAS mutation in codon 13 might have benefited from anti- EGFR antibodies treatment.14 The mutations in KRAS gene are found in approximately 30 to 40% of mCRC patients, reported in previous literature, but only 40 to 60% of these patients with wt-KRAS will respond to anti-EGFR antibodies treatment.1516 Therefore, other molecular markers downstream of EGFR in the RAS/RAF/MAPK pathway and other effector pathways are found to be involved to predict the response to specific systemic therapy.
The BRAF gene encodes a serine/threonine protein kinase of the RAS/RAF/MEK/ERK kinase pathway and it is also involved in CRC carcinogenesis.910 The most common mutation of the BRAF gene is V600E which is found in approximately 5 to 9% of mCRC.1718 The same was reported in our previous study carried on Slovenian patients with CRC where the BRAF V600E mutation was found in 5.1% of patients.19 Previous retrospective studies suggested that mt-BRAF was a marker of resistance to anti-EGFR therapy and that the patients with mt-BRAF had significantly shorter PFS and OS than the patients with wt-BRAF tumours.20 The mutations in the KRAS and BRAF genes have been reported to be mutually exclusive.2122 In the retrospective analysis by Farina- Sarasqueta et al., it was also shown that the BRAF V600E mutation was
an independent prognostic factor for the survival of patients with colon cancer in stages II and III, while the KRAS mutations did not have any effect on the overall survival of these patients. They concluded that the prognostic role of the KRAS mutations in an adjuvant setting has to be determined.23 In recent clinical studies, it was published that the BRAF V600E mutation in metastatic colorectal cancer is conferred to a poor prognosis regardless of treatment, but these patients may have some benefit from the treatment with cetuximab in combination with chemotherapy as the first-line therapy, but not when used in the patients in whom the disease has progressed after the first-line therapy.17
The aim of this retrospective study was to analyze objective responses, time to progression and overall survival of the patients with metastatic colorectal cancer treated with first-line systemic therapy in respect of KRAS and BRAF status.
Patients and methods
Patients
In the study, 176 patients with histologically confirmed metastatic colorectal cancer (mCRC), primarily metastatic or progressed during or after adjuvant therapy were retrospectively analyzed. They were treated according to the national and NCCN guidelines, including performance status of patients and comorbidity. They were treated with chemotherapy, including fluoropirimidins, capecitabine or 5-fluorouracil (5- FU), oxaliplatin or irinotecan in combination with biologicals, be-vacizumab or cetuximab in respect of previously determined KRAS status. The treatment was continued according to the RECIST criteria, until the planned operation or until the progression of disease or toxicity occurred.
Methods and assessment of response
All relevant data from medical files were collected and entered into the data base. Baseline data was analyzed with regard to age, sex, primary site (colon and rectum), number and location of metastases. Efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumours (RECIST, version 1.1) by using computed tomografy (CT) scans, magnetic resonance scans, abdominal ultrasound, chest X-ray, bone scans, clinical examination and laboratory tests.24 The study was conducted in the conformance with the principles of the Declaration of Helsinki.
Molecular analysis of KRAS and BRAF mutations
DNA for molecular analysis was extracted from formalin-fixed, paraffin-embedded tumour tissue of primary tumours or metastases with at least 70% of tumour cells. TheraScreen KRAS Mutation Kit® (Roche Applied Science, Mannheim, D) was used to determine seven most common mutations in codons 12 and 13 of the KRAS gene. The V600E mutation in BRAF was detected by end-point gen-otyping using the TaqMan MGB probes (Applied Biosystems, Warrington, UK) as described previously.19 The mutation V600E in BRAF in positive tumour samples was confirmed by direct sequencing after amplification of the exon 15 of the BRAF gene.19
Statistical analysis
The primary end-points of the analysis were overall response rate (ORR), based on RECIST criteria, overall survival (OS) and time to progression (TTP) according to the KRAS and BRAF status.
The x2-test was used to compare ORR, OS and TTP between groups, with 95% confidence intervals (CI) calculated for the medians. OS and TTP were estimated by using Kaplan-Meier Estimates and compared using the log-rank test. TTP was measured in all patients from the beginning of the first-line systemic chemotherapy to the first evidence of progression. The duration of survival was calculated from the beginning of systemic treatment until the date of death. p value < 0.05 was considered statistically significant. Statistical data were obtained using the SPSS software package PASW statistics 18.0.
Results
Patients' characteristics
In total, 176 patients with mCRC who received first-line therapy between May 2005 and October 2010 were included in the retrospective analysis. The cut-off date for the present analysis was April 2011. All patients were treated at the Institute of Oncology Ljubljana, all were Caucasian. The median age was 62 years (range 27-86 years) and the majority of the patients were males (61.4%). Most of the patients had metastatic colon cancer (71.4%). One hundred and four patients had primary metastatic disease (59.1%). The most common sites of metastases were liver and lung. The most com-
TABLE 1. Baseline and disease characteristic of patients
Caracteristics	Patients , n= 174 (%)
Gender	
Male	108 (61.4)
Female	68 (38.6)
Age(years)	
Median	62
Range	(27- 86)
WHO PS*	
0	126 (71.6)
1	50 (28.4)
Primary tumour localization,	
Colon	125 (71)
Rectum	51 (28)
Metastatic site	
Liver	68 (38.6)
Lung	11 (6.3)
Liver and lung	12 (6.8)
Other	85 (49.3)
KRAS status	
KRASw	113 (64.2)
KRASm 12	53 (84.0)
KRASm 13	10 (16.0)
BRAF status	
BRAFw	163 (92.6)
BRAFm	13 (7.4)
*WHO PS- World Health Organization performance status
mon therapies the patients received were irinote-can, capecitabine with bevacizumab (29.5%) and oxaliplatin, capecitabine with cetuximab (22.1%). Twenty-four patients (13.6%) were treated only with chemotherapy, capecitabine in monotherapy, or with fluoropirimidines in combination with oxaliplatin or irinotecan. Patients' baseline and disease characteristics are shown in Table 1.
KRAS mutations were found in 63 patients (35.8%), to be more precise, the KRAS mutation in codon 12 in 53 patients (84.0%) and the KRAS mutation in codon 13 in 10 patients (16.0%). The BRAF V600E mutation was detected in 13 of 176 patients (7.4%).
The mutations of the KRAS or BRAF gene were detected in total in 76 patients (43.4%) (Table1).
Efficacy
The response rates according to RECIST criteria with regard to the KRAS and BRAF status are shown in Table 2. The overall response rates in pa-
' (mondii}
figure 1. Time to progression in patients with wt-KRAS/wt-BRAF and wt-KRAS/mt-BRAF.
T kit* Itti&rvttlt)
figure 2. Overall survival in patients wt-KRAS/wt-BRAF and wt-KRAS/mt-BRAF.
tients with wt-KRAS and wt-BRAF and in patients with wt- KRAS and mt- BRAF were CR 14.7 % + PR 39.3 % + SD 35.5 % and CR 15.4 % + PR 23.1 % + SD 46.1 % respectively. The objective response rates in the group of patients with wt-KRAS and wt-BRAF tumours were 54.0% (CR 14.7%, PR 39.3%), while in the group of patients with wt-KRAS and mt-BRAF were 38.5% (CR 15.4%, PR 23.1%). The difference was not statistically significant (p= 0.378). The median OS in the group of patients with wt-KRAS and wt-BRAF tumours was 107.4 months (95% CI: 82- 132.9 months) and in the group of patients with wt-KRAS and mt-BRAF tumours 44.9
months (95% CI: 28.4- 61.5 months) (Figure 1). The difference in median OS between those two groups was statistically significant (p= 0.042). TTP in the group of patients with wt-KRAS and wt-BRAF tumours and in the group of patients with wt-KRAS and mt-BRAF tumours was 16 months (95% CI: 10.7- 21.2 months) and 12 months (95% CI: 4.0- 15.0 months), respectively (Figure 2). It was not statistically significant (p= 0.558).
In the KRAS mutation subgroups, the objective response rate of 53 patients with the mutation in codon 12 was 47% (CR 20.7%, PR 26.4%) and, in 10 patients with the mutation in codon 13, the objective response was 33% (CR 11.1%, PR 22.2%). The difference was not statistically significant (p= 0.08). TTP in the patients with the mutation in codon 12 and the patients with the mutation in codon 13 was 13.5 months (95% CI: 9- 18 months) and 9.3 months (95% CI: 5.1- 13.5 months), respectively. The difference was not statistically significant (p= 0.106).
Surgical resection of liver metastases was performed in 47/176 patients (26.7%); more specifically, in 31 patients with wt-KRAS tumours and in 16 patients with mt-KRAS tumours. R0 resection was achieved in 38/176 patients (21.6 %), of whom 37 patients had wt-BRAF and only one had mt-BRAF tumour.
Discussion
In our study population, the KRAS mutations in codons 12 and 13 were found in 35.8% of patients, in most of them in codon 12; while the mutation V600E in BRAF gene was detected in 13 patients (7.4%). The results of testing are comparable with those previously reported, where the KRAS mutations were found in 30 to 40% and the BRAF V600E mutation in 5 to 9% of the patients.11-1317'19
The presented data demonstrate that the patients with the BRAF V600E mutation have worse prognosis than the patients with wt-BRAF tumour and progress early during treatment. The patients with wt-BRAF tumours have higher response rates than the patients harbouring the BRAF V600E mutation, but the difference was not statistically significant. One third of the patients with wt-KRAS or mt-BRAF tumour still respond to the treatment alluding that the BRAF status is not predictive for the response to anti-EGFR antibody therapy. This was also reported in previously published analyses and, in recently published retrospective meta-analysis of the CRYSTAL and OPUS studies, it was also concluded, that the patients with BRAF muta-
TABLE 2. Response rates in KRAS wild type patients according to BRAF status in first-line therapy
wKRAS
wKRAS/wBRAF
wKRAS/mBRAF
Overall response rate (CR+ PR), n (%)
Disease control rate (CR+PR+SD), n (%)
CR
PR
SD
PD
Median OS, months estimate
Median TTP, months estimate
93 (52.8)
157 (89.2) 26 (14.8) 67 (38.0) 64 (36.4) 19 (10.8)
129.4 (95% CI: 52.4- 206.4) 15.9 (95% CI: 10.8- 21.0)
88 (54.0)
146 (89.5) 24 (14.7) 64 (39.3) 58 (35.5) 17 (10.5)
107.4 (95% CI: 82- 132.9)* 16.0 (95% CI: 10.7-21.2)**
5	(38.5)
11 (84.6)
2	(15.4)
3	(23.1)
6	(46.1) 2 (15.4)
44.9 (95% CI: 28.4- 61.5) * 12.0 (95% CI: 4.0-15.0) **
' p= 0.042
' p= 0.558
tion might have also benefited from the treatment with anti-EGFR antibodies.171821-2325 At this point it should be highlighted that not all patients in our retrospective analysis with wt-KRAS received cetuximab-based first-line systemic therapy; the therapy was selected in accordance to the patients' baseline characteristics, the purpose of treatment or planned operation for metastases.
The difference in TTP between the patients with wt-KRAS and wt-BRAF tumours and the patients with wt-KRAS and mt-BRAF tumours was 4 months. The difference was not statistically significant, probably due to our small group of patients and, consequently, small proportion of the patients with BRAF mutation. The comparison of median OS of those two groups showed a statistically significant difference which was also accompanied with a better prognosis of patients with wt-KRAS and wt-BRAF tumour. These results are comparable with those reported earlier.172021 The results of retrospective pooled analysis from randomized CRYSTAL and OPUS trials showed that cetuximab as the first-line chemotherapy based on irinotecan or oxaliplatin significantly improved OS, ORR and PFS the in patients with wt-KRAS tumours. According to the results of the same meta-analysis, the patients with BRAF mutations also appeared to have benefited from cetuximab as the first-line systemic treatment.2526
In our retrospective study, the KRAS mutations were most frequently detected in codon 12. This is in accordance with the results of our previous study.19 Comparing the patients having KRAS mutations in codon 12 with the ones having the muta-
tion in codon 13 after the treatment with chemotherapy and bevacizumab, the response rates were higher in the patients with the mutations in codon 12. Nevertheless, the differences in response rates, OS and TTP between these two groups were not statistically significant; we assume that the groups of patients were too small. In the contrast, in their retrospective study, De Roock et al. showed that the patients with the mutation in codon 13 KRAS who were treated with cetuximab had better overall and progression-free survival than the patients with other KRAS mutations and might have benefited from the treatment with cetuximab.14 In an abstract recently published in the 2011 ASCO Annual Meeting Proceedings, Tejpar et al. retrospectively analyzed the influence of KRAS G13D mutations on the efficacy of treatment with cetuximab as the first-line systemic therapy and compared it with the pooled results of randomized studies CRYSTAL and OPUS. The patients with the KRAS mutation in codon 13 had a much lower treatment effect compared to the patients with wt-KRAS tumours and might have nevertheless benefited from treatment with cetuximab.27
Although not studied in our retrospective analysis, other KRAS mutations were also reported to predict the response to anti- EGFR monoclonal antibodies. The results of a small study of 74 patients, conducted by Loupakis with his colleagues, suggested that rare KRAS mutations in codon 61 and in codon 146 might also be responsible for in the treatment resistance to anti-EGFR monoclonal an-tibodies.2829 In contrast, in their large retrospective analysis, De Roock et al. concluded that the codon
146 mutations did not affect the response to cetuxi-mab and that the patients with codon 61 mutant tumours had lower response rate.20 According to the analysis of other mutations, they proposed testing of KRAS status, if not mutated, then of BRAF and NRAS status, and PIK3CA exon 20 mutation in order to improve the objective response up to 40% in selected patients.
In our retrospective study, 26.7% of patients, all with KRAS wild-type tumours, who had previously unresectable liver-only metastases, underwent surgical resection after systemic therapy, with R0 resection achieved in 38 patients (21.6%); one of those was patient with the BRAF V600E mutation. Although it is difficult to make any comparison, because our patients were not selected according to specific systemic therapy, these results are comparable with those reported in previous studies claiming that 19 to 23% patients treated with beva-cizumab- and irinotecan-based chemotherapy and with previously unresectable liver-only metastases underwent resection.30-32 In a recently published clinical study BOXER, where the patients with un-resectable liver-only metastases were treated with oxaliplatin, capecitabine and bevacizumab, R0 resection was achieved in 40% of patients.33 The proportion of patients with resected liver metastases in our retrospective study was higher than that reported in earlier studies including the patients with previously unresectable liver-only metastases and treated with cetuximab in combination with irinotecan- or oxaliplatin-based chemotherapy; resection was achieved in 4 to 10%.3435 In the randomized phase II CELIM study, in which the patients with liver-only metastases were treated with irinotecan- or oxaliplatin-based chemotherapy with cetuximab as the first-line systemic therapy, the proportion of R0 resection was higher; it was achieved in 34% of patients.36 In another phase II POCHER trial, the proportion of R0 resection was even higher; it was achieved in 60% of patients who were treated with chronomodulated chemotherapy with irinotecan, oxaliplatin, 5- fluorouracil and leucovorin.37
In conclusion, the results of our retrospective study showed that the patients with BRAF V600E mutation had worse prognosis than those with wt-BRAF, with lower response rates and progressed early during systemic treatment, consequently, with less possibilities to achieve resectability of metastatic disease. The definitive role of the BRAF V600E mutation as a prognostic and predictive factor to response to the anti-EGFR monoclonal antibodies needs to be analyzed in large prospec-
tive clinical studies. Different KRAS mutations in codon 12 and 13 and other molecular markers, predictive or prognostic, downstream of EGFR in the RAS/RAF/MAPK pathway, and other effector pathways, are needed to be defined to select the patients, who will benefit from specific systemic therapy in a way of individualized treatment.
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7.	Kohne CH. How to integrate molecular targeted agents in the continuum of care. Ann Oncol 2010; 21: vii134-9.
8.	Ocvirk J. Advances in the treatment of metastatic colorectal carcinoma. Radiol Oncol 2009; 43: 1-8.
9.	Cohen SJ, Cohen RB, Meropol NJ. Targeting signal transducting pathways in colorectal cancer- More than skin deep. J Clin Oncol 2005; 23: 5374-85.
10.	Markowitz SD, Bertagnolli MM. Molecular basis of colorectal cancer. N Engl J Med 2009; 361: 2449-60.
11.	Artale S, Sartore-Bianchi A, Veronese SM, Gambi V, Sarnataro CS, Gambacorta M, et al. Mutations of KRAS and BRAF in primary and matched metastatic sites of colorectal cancer. J Clin Oncol 2008; 26: 4217-9.
12.	Etienne- Grimaldi M- C, Formento J- L, Francoual M, François E, Formento P, Renée N, et al. K- Ras mutations and treatment outcome in colorectal cancer patients receiving exclusive fluoropyrimidine therapy. Clin Cancer Res 2008; 14: 4830-5.
13.	Ličar A, Cerkovnik P, Ocvirk J, Novaković S. KRAS mutations in Slovene patients with colorectal cancer: frequency, distribution and correlation with the response to treatment. int J Oncol 2010, 36: 1137-44.
14.	De Roock W, Jonker DJ, Di Nicolantonio F, Sartore-Bianchi A, Tu D, Siena S, et al. Association of KRAS p.G13D mutation with outcome in patients with outcome with chemotherapy- refractory metastatic colorectal cancer treated with cetuximab. JAMA 2010; 304: 1812-20.
15.	Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, et al. Wild- type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26: 1626-34.
16.	Roth AD, Tejpar S, Dolorenzi M, Yan P, Fiocca R, Klingbiel D, et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: Results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010; 28:466-74.
17.	Van Cutsem E, Lang I, D'haens G, Moiseyenko V, Zaluski J, Kohne C, et al. The crystal study: Assessment of the predictive value of KRAS status on clinical outcome in patients with mCRC receiving first-line treatment with cetuximab or cetuximab plus folfiri. [Abstract]. Ann Oncol 2008; 19(Suppl 6): vi17-8.
18.	Tol J, Nagtegaal ID, Punt CJA. BRAF mutation in metastatic colorectal cancer. N Engl J Med 2009; 361: 98-99.
19.	Ličar A, Cerkovnik P, Novaković S. Distribution of some activating KRAS and BRAF mutations in Slovene patients with colorectal cancer. Med Oncol 2010 Jul 20. [Epub ahead of print]
20.	De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Fountzilas G, et al. Effect of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010; 11: 753-62.
21.	Tej par S, De Roock W. PIK13CA, BRAF and KRAS mutations and outcome prediction in chemorefractory metastatic colorectal cancer (mCRC) patients treated With EGFR targeting monoclonal antibodies (MoAbs): results of a European Consortium. Eur J Cancer 2009; 7: 322.
22.	Lambrechts D, De Roock W, Prenen H, De Schutter J, Jacobs B, Biesmans B, et al. The role of KRAS, BRAF, NRAS and PIK3CA mutations as a marker of resistence of cetuximab in chemorefractory metastatic colorectal cancer. [abstract] J Clin Oncol 2009; 27 (Suppl): 172.
23.	Farina-Sarasqueta A, van Lijnschoten G, Moerland E, Creemers GJ, Lemmens VE, Rutten HJ, et al. The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol 2010; 21: 2396-2402.
24.	Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45: 228-247.
25.	Kohne CH, Rougier P, Stroh C, Schlichting M, Bokemeyer C, Van Cutsem E, et al. Cetuximab with chemotherapy as 1st- line treatment for metastatic colorectal cancer: A meta- analysis of the CRYSTAL and OPUS studies according to KRAS and BRAF mutation status. ASCO Gl 2010 [abstract 406].
26.	Bookemeyer C, Kohne CH, Rougier P, Stroh C, Schlichting M, Van Cutsem E, et al. Cetuximab with chemotherapy as 1st- line treatment for metastatic colorectal cancer: Analysis of the CRYSTAL and OPUS studies according to KRAS and BRAF mutation status [abstract]. J Clin Oncol 2010; 28 (Suppl): 3506.
27.	Tejpar S, Bukemeyer C, Celik I, Schlichting M, Sartorius U, Van Cutsem E, et al. Influence of KRAS G13D mutations on outcome in patients with meta-static colorectal cancer (mCRC) treated with first- line chemotherapy with or without cetuximab. ASCO 2011 [abstract 3511].
28.	Loupakis F, Ruzzo A, Cremolini C, Vincenzi B, Salvatore L, Santini D, et al. KRAS codon 61, 146 and BRAF mutations predict resistence to cetuximab + irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer 2009; 101: 715-21.
29.	Smith G, Bounds R, Wolf H, Steele RJ, Carey FA, Wolf CR, et al. Activating K-ras mutations outwith 'hotspot' codons in sporadic colorectal tumours-implications for personalised cancer medicine. Br J Cancer 2010; 102: 1-11.
30.	Okines A, Del Puerto O, Cunningham D, Chau I, Van Cutsem E, Saltz L, et al. Surgery with curative-intent in patients treated with first-line chemotherapy plus bevacizumab for metastatic colorectal cancer First BEAT and the randomised phase-III NO16966 trial. Br J Cancer 2009; 101: 1033-8.
31.	Terrebonne E, Smith D, Becouarn Y, Michel P, Guimbaud M, Fourrier- Reglat A, et al. Resection of colorectal cancer (CRC) metastases after bevacizumab (BV) treatment for first-line therapy: results of the ETNA cohort study (abstract 3594). J Clin Oncol 2010; 28: (Suppl.):15s.
32.	Ocvirk J, Rebersek M, Boc M. Bevacizumab in first- line therapy of meta-static colorectal cancer: A retrospective comparison of FOLFIRI and XELIRI. Anticancer Res 2011; 31: 1777-82.
33.	Wong R, Cunningham D, Barbachano Y, et al. A multicentric study of capecit-abine, oxaliplatin plus bevacizumab as perioperative treatment of patients with poor- risk colorectal liver- only metastases not selected for upfront resection. Ann Oncol 2011; 22: 2042-8.
34.	Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 2009; 360: 1408-17.
35.	Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de Braud F, et al. Fluorouracil, leucovorin, and oxaliplatin with or without cetuximab in the first- line treatment of metastatic colorectal cancer. J Clin Oncol 2009; 27: 663-71.
36.	Folprecht G, Gruenberger T, Bechstein WO, Raab HR, Lordick F, Hartmann JT, et al. Tumour response and secondary resectability of colorectal liver metastases following neoadjuvant chemotherapy with cetuximab: the CELIM randomised phase 2 trial. Lancet Oncol 2010; 1: 38-47.
37. Garufi C, Torsello A, Tumolo S, Ettorre GM, Zeuli M, Campanella C, et al. Cetuximab plus chronomodulated irinotecan, 5- fluorouracil, leucovorin and oxaliplatin as neoadjuvant chemotherapy in colorectal liver metastases: POCHER trial. Br J Cancer 2010; 103: 1-6.
Hepatocellular carcinoma with subcutaneous metastasis of the scalp
Yilmaz Tezcan and Mehmet Koc
Selcuk University, Meram Faculty of Medicine, Department of Radiation Oncology, Konya, Turkey
Received 6 April 2011 Accepted 23 May 2011
Correspondence to: Yilmaz Tezcan, M.D. Department of Radiation Oncology, Meram Faculty of Medicine, Selcuk University, 42090-Konya, Turkey. Phone: +332 223 6942; Fax; +0332 223 6182; E-mail: yilmaztezcan@yahoo.com
Disclosure: No potential conflicts of interest were disclosed.
Background. The majority of subcutaneous metastases from hepatocellular carcinoma (HCC) originate from needle tracks or surgical wound contamination. Non-iatrogenic subcutaneous metastasis from hepatocellular carcinoma was rarely reported.
Case report. A 70-year-old man presented with a mass in his left occipital region of the scalp. The surgical complete resection was performed. The histopathology report of the scalp mass showed a characteristic metastatic HCC. Computed tomography (CT) of the abdomen showed no primary or metastatic lesion in the abdomen; that's why the adjuvant treatment was not given after the surgery. Five months later, magnetic resonance imaging (MRI) of the brain revealed a 6 x 5.5 cm mass at the left posterior parietal region of the scalp. Second surgery was performed and histopathology of the specimen excised was again metastatic HCC. The external beam radiation therapy (XRT) was administered after the surgery. A follow-up MRI of the brain showed no recurrent disease after 9 months from XRT. Conclusions. HCCs should be considered in the differential diagnosis of carcinomas metastatic to the skin, even in the absence of liver symptoms.
Key words: hepatocellular carcinoma; radiation therapy; cutaneous metastases
Introduction
Hepatocellular carcinoma (HCC) is the most common primary tumour of the liver. Lungs, abdominal lymph nodes, and bones are the most common extrahepatic metastatic sites of HCC.
Cutaneous metastases from HCC are very rare.1-2 We report a case who has a subcutaneous mass on his scalp which was the first clue for the diagnosis of the HCC. Aggressive recurrence was occurred three months after surgery that was well controlled with radiation therapy.
Case report
In November 2009, a 70-year-old man presented with a mass in his left occipital region of the scalp. His ECOG status was 0. He has a history of hepati-tis-C virus (HCV) positivity for 30 years. The magnetic resonance imaging (MRI) revealed a 6.5 x 6.0
cm mass invading bone in the left occipital region of the scalp which has extra and intracranial components (Figure 1). Fine needle aspiration biopsy showed a malign tumour.
The mass was completely resected. The macroscopic size of mass was measured 6.0 x 5.5 x 2.0 cm. Pathology of the mass showed a characteristic met-astatic hepatocellular carcinoma (HCC) invading the occipital bone. All surgical margins were free of tumour. Immunohistochemical staining showed Pan-CK, CK8, CEA(p) and CD10 positivity.
Complete blood count, liver function tests, and a-fetoprotein (AFP) level were normal. Abdomino-pelvic computed tomography (CT) showed no abnormalities; that's why no adjuvant treatment was given after the surgery. Five months later (on April 2010) he noticed a mass at the same region. MRI revealed a 5.2 x 2.3 cm (Figure 2) mass invading bone in his left posterior parietal portion of the scalp. For the second time the surgery was performed and once again the metastasis of HCC was
FIGURE 1. Brain MRI with a subcutaneous metastasis from hepa-tocellular carcinoma.
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FIGURE 3. Brain MRI after second surgery which is contrast enhanced residual lesion.
confirmed on the histopathological examination of the resected tissue specimen. Postoperative MRI of the brain revealed a contrast enhancing mass on the left parietal region (Figure 3). CT of the abdomen showed a hypodens lesion in the right lobe of the liver (Figure 4). Due to the bone invasion and
FIGURE 2. Brain MRI with a recurrent subcutaneous metastasis from hepatocellular carcinoma.
FIGURE 4. CT scan of hepatocellular carcinoma in right lobe of the liver.
residual disease, the palliative external beam radiation therapy (XRT) was applied after the surgery. Three-dimensional treatment planning was used and the radiation dose to the scalp was 300 cGy per day for 5 days a week; the total dose was 3000 cGy (Figure 5). Radiofrequency ablation (RFA) was administered to the metastatic mass of the right liver and the systemic therapy with a targeting agent (sorafenib) treatment was started. A follow-up MRI of the brain showed no recurrent disease 9 months
FIGuRE 5. The typical dose distribution from 2 posterior oblique field using 6 MV photon beams from conformal radiotherapy plan.
from XRT (Figure 6). After 17-month follow-up from the diagnosis, the patient could perform his daily activities although he developed hypo albu-minemia and fatigue.
Discussion
The majority of subcutaneous metastasis from HCC originates from needle tracks or the surgical wound contamination.3-5 The non-iatrogenic subcutaneous metastasis from hepatocellular carcinoma was rarely reported. Since these patients are usually considered at their terminal period, they are usually observed without any treatment. However, the surgical resection of the metastatic lesion has been performed in a few cases.56 In one study, skin metastases were detected in only 2.7 % of cirrhotic HCCs, and none in noncirrhotic HCC.7 figure 6. mri of the brain 9-month later of the xrt.
Although the subcutaneous metastasis of HCC is unusual, it could be presented as the sole and initial sign of the HCC.8
Huang et al.9 reported that the radiation therapy was found XRT an efficient treatment modality when subcutaneous metastases of HCC are in question. They observed at least a partial response in 20 of 24 lesions (83.3%), with radiation doses ranging from 8 to 64 Gy. No severe squeals were recorded. The overall 6-month survival was 43.4%, and the overall 1-year survival was 22.8%. At these patients, the treatment response was good, and the side-effect profile was acceptable. Due to our palliative aim, we applied 30 Gy in 10 fraction XRT over 2 weeks and achieved a good local control during last 9-month.
HCCs should be considered in the differential diagnosis of carcinomas metastatic to the skin, even in the absence of liver symptoms or absence of imaging finding with ultrasonography or CT that usually reveal the primary lesion.10 Surgery is the primary treatment choice, like in some other cases of metastases11, in particular, when superficial (skin) metastases are to be resected. Radiotherapy seems to be a reasonable alternative in patients with advanced disease and poor performance status and in other clinical scenarios when surgery could not be implemented.
10.	Botros M, Quevedo JF, Miller RC. Angiosarcoma of the liver after multimo-dality therapy for gallbladder carcinoma. Radiol Oncol 2009; 43: 126-31.
11.	Strojan P. Role of radiotherapy in melanoma management. Radiol Oncol 2010; 44: 1-12.
References
1.	Okuda K, Konda Y. Primary carcinoma of the liver. In: Haubrich WS, Schaffner F, Berk JE, editors. Bockus gastroenterology. 5th edition. Philadelphia, PA: WB Saunders; 1995. p. 2444-89.
2.	MacDonald RA. Primary carcinoma of the liver: a clinicopathological study of 108 cases. Arch Intern Med 1967; 99: 266-79.
3.	Casella G, Cacopardo E, Rovere G, Buda CA, Cascinu S, Baldini V. Cutaneous seeding after ultrasound-guided percutaneous ethanol injection for treatment of hepatocellular carcinoma. J Clin Ultrasound 2001; 29: 354-8.
4.	Navarro F, Taourel P, Michel J, Perney P, Fabre JM, Blanc F, et al. Diaphragmatic and subcutaneous seeding of hepatocellular carcinoma following fine-needle aspiration biopsy. Liver 1998; 8: 251-4.
5.	Koffi E, Moutardier V, Sauvanet A, Noun R, Flejou JF, Belghiti J. Wound recurrence after resection of hepatocellular carcinoma. Liver Tanspl Surg 1996; 2: 301-3.
6.	Koffi E, Moutardier V, Sauvanet A, Noun R, Flejou JF, Belghiti J. Wound recurrence after resection of hepatocellular carcinoma. Liver Tanspl Surg 1996; 2: 301-3.
7.	Peters RL. Metastatic patterns of HCC. In: Okuda K, Peters RL, editors. Hepatocellular Carcinoma. New York: John Wiley & Sons, Inc; 1976. p. 156-64.
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9.	Huang YJ, Tung WC, Hsu HC, Wang CY, Huang EY, Fang FM. Radiation therapy
to non-iatrogenic subcutaneous metastasis in hepatocellular carcinoma:
results of a case series. Br J Radiol 2008; 81: 143-50.
Genotyping of BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes in a male patient with secondary breast cancer
Ana Lina Vodusek1, Srdjan Novakovic2, Vida Stegel2, Berta Jereb1
1	Department of Radiation Oncology, Institute of Oncology, Ljubljana, Slovenia
2	Department of Molecular Diagnostics, Institute of Oncology, Ljubljana, Slovenia
Received 12 May 2011 Accepted 23 June 2011
Correspondence to: Ana Lina Vodušek, MD, Institute of Oncology, Zaloška 2, 1000 Ljubljana, Slovenia. Phone:+386 1 5879 629; Fax: +386 1 5879 304; E-mail: avodusek@onko-i.si
Disclosure: No potential conflicts of interest were disclosed.
Background. Some tumour suppressor genes (BRCA2) and mismatch repair genes (MSH2, MLH1) are correlated with an increased risk for male breast cancer.
Case report. Our patient developed secondary breast cancer after the treatment for Hodgkin's disease in childhood. DNA was isolated from the patients' blood and screened for mutations, polymorphisms and variants in BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes. We found no mutations but common polymorphisms, and three variants in mismatch repair genes.
Conclusions. Nucleotide variants c.2006-6T>C and p.G322D in MSH2 might be correlated with male breast cancer. Key words: gene screening; breast cancer; male; secondary neoplasm
Introduction
Secondary neoplasms (SN) are the most serious late effects of the treatment of childhood cancers. Their incidence is increasing with time of observation to 25% at 25 years from diagnosis of the primary tumor.12 Patients treated with radiotherapy for Hodgkin's disease (HD) are at highest risk for SN.3 Male breast cancer (MBC) is rare, accounting for less than 1% of all breast cancers.45 Risk factors for primary MBC include testicular disease, benign breast conditions, age, family history, the Klinefelter syndrome, gynecomasty and non-therapeutic radiation exposure.56 The highest risk for primary MBC is among the carriers of mutations in the BRCA2 gene. Besides mutations in the BRCA2 gene, additional germ line mutations in MBC have been reported also in the androgen receptor gene and PTEN.7 There is likely to be a number of genes more commonly mutated correlated with a modest increase in primary MBC, such as mismatch repair (MMR) genes - MSH2, MLH1, PMS1, PMS2.7,8
In literature we found two case reports on secondary MBC, one after the treatment for HD and
one after the treatment for acute lymphoblastic leukaemia including total body irradiation and bone marrow transplantation.39 There are no data on genetic factors predisposing the development of secondary MBC after the treatment for HD.
The aim of this case report was to elucidate the genetic conditions in a patient with secondary MBC, screening the genes already known to be correlated with primary MBC - BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2.
Case report
Patient
A 37 year old man developed breast cancer 24 years after the treatment for HD. In 1983, when 13 years old, he was treated with chemotherapy (6 cycles of MOPP/ABVD) and irradiation of the neck and upper thorax, retroperitoneal and inguinal nodes (25 Gy) for stage IIIBS HD. Afterwards he was followed, at the Department of Paediatrics and after 1995 at the Late Effect Clinic at the Institute of Oncology in Ljubljana.
TABLE 1. Nucleotide variations detected in male breast cancer patient
Gene BIC*	HGVS nomenclature**	Genotype	Clinical significance
MLH1	c.453+79A>G	heterozygote AG	polymorphism
	c.1668-19A>G	homozygote AA	polymorphism
	c.655A>G (p.I219V)	heterozygote AG	unclassified variant
MSH2	c.211+9G>C	homozygote GG	polymorphism
	c.1511-9A>T	heterozygote AT	polymorphism
	c.1661 + 11G>A	heterozygote GA	polymorphism
	c.2006-6T>C	heterozygote TC	unclassified variant
	c.965G>A (p.G322D)	heterozygote AG	unclassified variant
BRCA1 2201C>T	c.2082C>T (p.S694S )	heterozygote CT	polymorphism
2430T>C	c.2311T>C (p.L771L )	heterozygote TC	polymorphism
2731C>T	c.2612C>T (p.P871L )	heterozygote CT	polymorphism
3232A>G	c.3113A>G (p.E1038G )	heterozygote AG	polymorphism
3667A>G	c.3548A>G (p.K1183R )	heterozygote AG	polymorphism
4427T>C	c.4308T>C (p.F1436S )	heterozygote TC	polymorphism
4956A>G	c.4837A>G (p.S1613G )	heterozygote AG	polymorphism
BRCA2			
203G>A	c.1-25G>A	homozygote GG	polymorphism
1342C>A	c.1114C>A (p.H372N )	homozygote CA	polymorphism
3624A>G	c.3396A>G (p.L1132L )	heterozygote AG	polymorphism
4035T>C	c.3807T>C (p.V1269V)	heterozygote TC	polymorphism
7470A>G	c.7242A>G (p.S2414S )	heterozygote AG	polymorphism
IVS16-14C>T	c.7806-14C>T	heterozygote TC	polymorphism
P53	c.96 + 41 56 del CCCCAGCCCTCCAGGT c.215 C>T (p. Pro72Arg)	homozygote heterozygote CT	polymorphism polymorphism
	c.782 + 72 A>C	heterozygote AG	polymorphism
	c.782 + 92 A>G	heterozygote AC	polymorphism
CDKN2A			
	c.1-191A>G	homozygote GG	polymorphism
	c.471+69C>T	heterozygote CT	polymorphism
*Nucleotide variations described as in BIC (Breast Cancer Information Core) database. DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1, or U43746 for mRNA of BRCA2. First nucleotide of mRNA is numerated as 1.
**Description of nucleotide variations is in accordance with HGVS (Human Genome Variation Society) nomenclature. DNA variants are numerated according to NCBI reference sequence NM_000249 for MLH1, NM_000251 for MSH2, NM_007294.2 for BRCA1, NM_000059.3 for BRCA2, NM_000546 for p53 and NM_000077.3 for CDKN2A. First nucleotide of start codon ATG is numerated as 1.
Eleven years after the treatment we observed primary hypogonadism with low levels of testosterone, bilateral gynecomasty and azoospermia and at the age of 37 a palpable tumour 6 cm in diameter in the central part of the left breast, infiltrating the skin, with an inverted nipple. Ultrasounds of both axillae, of the abdomen, chest X-ray films and Technetium bone scan were normal.
Histology showed an invasive ductal carcinoma, grade III, with negative progesterone and estrogen receptors and positive Her2 status. His family history was negative. He was treated with neoadju-vant chemotherapy, left mastectomy with axillary node dissection, adjuvant treatment with trastuzu-mab and postoperative irradiation of the left mammary region.
Methods of genetic investigations
Patient's DNA was isolated from peripheral blood using the DNA blood isolation kit Quiagen (Hilden, Germany). It was screened for variants in tumour suppressor genes (BRCA1, BRCA2, p53, CDKN2A) and MMR genes (MLH1 and MSH2).
Four methods were used: DGGE (denaturizing gradient gel electrophoresis) or HRM (high resolution melting), direct sequencing and MLPA (multiplex ligation-dependent probe amplification). The screening of BRCA1/2 genes was performed for all exons by the DGGE while MLH1 and MSH2 genes were screened using the HRM. Positive fragments were subsequently sequenced to determine the nucleotide change. Genes BRCA1, BRCA2, MLH1 and MSH2 were also screened for large deletions or insertions using the MLPA method. All coding regions of p53 and CDKN2A were sequenced.
HRM was used for discrimination between two DNA molecules with different sequences for the detection of SNPs (single nucleotide polymorphism) and small deletions and insertions. PCR (polymer-ase chain reaction) was performed on LC480 instrument using the LC 480 High-resolution Melting Master Kit (Roche, Manheim, Germany) according to manufacturer's instructions.
DGGE was used for detection of SNPs and small deletions or insertions. The PCR amplification of DNA samples was performed using a set of GC-clamped primers (Ingeny International BV, Goes, Netherlands) according to the cycling conditions provided by the primer-manufacturer. Different denaturing and running conditions were used for specific primer sets. When electrophore-sis was complete, gels were stained with EtBr and documented using the GelDoc system.
Direct sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) and products analyzed on the ABI Prism® 310 Genetic Analyzer (Applied Biosystems). Data were collected with ABI Prism 310 software (Applied Biosystems), and the results analyzed with the ABI Prism DNA sequencing analysis software (Applied Biosystems). Sequence data were analyzed utilizing the Gene Runner software tool.
The MLPA was applied for the detection of large deletions and insertions. The probe-mixes labelled as P003 for MLH1 and MSH2, P045-B1 for BRCA2, and P002 for BRCA1 from MRC Holland (MRC Holland, Amsterdam, Netherlands) were used, according to manufacturer's instructions.
Results of genetic investigations
BRCA1 in BRCA2
With MLPA, DGGE and direct sequencing we did not find any changes in the sample. We detected some polymorphisms at the frequently polymorphic loci but no mutations.
However, we found a rare polymorphism in BRCA2 gene p.V1269V which did not code a different amino acid; therefore, it did not affect protein function (Table 1). The sample was also tested for the presence of mutation 1100delC in CHEK2 gene with MLPA kit P045-B1. In the samples mutation 1100delC was not detected.
P53
Tumour suppressor gene p53 was sequenced in total. No mutations were found, but some common polymorphisms were (Table 1).
CDKN2A
By direct sequencing of exons of CDKN2A gene (p16 and p14ARF) we detected two polymorphisms: c.1-191A>G in 5'-UTR (untranslated region) and c.471+69C>T in 3'-UTR (Table 1).
MLH1 in MSH2
We detected no changes by means of the MLPA. The screening of the patients' DNA was therefore continued through HRM and sequencing. With these two methods no mutations were discovered, but we found some polymorphisms which were frequently detected in a general population. We also detected three variants of DNA which were described as variants with unknown influence on the protein function (Table 1).
Discussion
Secondary breast cancer is the most common SN among women who have received high dose radi-otherapy.10 However, secondary breast cancer is a rare disease in men and very little is known about its aetiology. It has been suggested that the carcinogenic effect of ionizing radiation may be similar in the male and prepubertal female breast.11
Beside the radiation exposure, some of the risk factors for primary MBC6 were also found in our
patient with secondary MBC - i.e. hormonal imbalances and gynecomasty.
Genetic factors associated with an increased risk of primary MBC include BRCA2 mutations that account for 4% to 14% of all primary MBC.612 We found no such mutations; only a rare V1269V polymorphism that does not affect protein function.
Since p53 is often mutated in female breast cancers, it could be mutated also in MBC. Even though the mutations in tumour suppressor gene p53 are correlated with numerous malignancies1314, no mutations were found in our patient while screening this gene. Also in MMR genes (MLH1 and MSH2) and in CDKN2A no deleterious mutations have been detected (Table1).
We found polymorphisms in MMR genes -MLH1 and MSH2, frequently detected in a general population, but also three variants (two in MSH2 and one in MLH1) which are described as variants with unknown influence on the protein function. This finding could be of interest since recent reports allude that two of these variants in MSH2 (c.2006-6T>C and p.G322D) might influence the process of cancer development. Nucleotide variant c.2006-6T>C may be associated with an increased risk for of Non-Hodgkin's Lymphomas.15 Additionally, the risk of some haematological malignancies in individuals carrying c.2006-6T>C variant is increased after the treatment with alkylating agents such as procarbazine, dacarbazine, cyclophospahmide.16
According to some recent reports the variant p.G322D is associated with changes in MSH2 protein function. Individuals with this variant have slightly reduced release efficiency of mismatched targeted DNA compared to the wild type.17
Conclusions
To our knowledge there are no reports of genetic screening in secondary MBC. We found three unclassified variants that could be correlated with an increased risk of secondary MBC but further studies should be performed.
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8.	Murata H, Khattar NH, Gu L, Li GM. Roles of mismatch repair proteins hMSH2 and hMLH1 in the development of sporadic breast cancer. Cancer Lett 2005; 223: 143-50.
9.	Latz D, Alfrink M, Nassar N, Beyerle C. Breast cancer in a male patient after treatment of acute lymphoblastic leukemia including total body irradiation and bone marrow transplantation. Onkologie 2004; 27: 477-9.
10.	Zebic-SinkovecM, Kadivec M, PodobnikG, Skof E, Snoj M. Mammographycally occult high grade ductal carcinoma in situ (DCIS) as second primary breast cancer, detected with MRI: a case report. Radiol Oncol 2010; 44: 228-31.
11.	Thomas DB, Rosenblatt K, Jimenez LM, McTiernan A, Stalsberg H, Stemhagen A, et al. Ionizing radiation and breast cancer in men (United States). Cancer Causes and Control 1994; 5: 9-14.
12.	Eeles RA. Future possibilities in the prevention of breast cancer: intervention strategies in BRCA1 and BRCA2 mutation carriers. Breast Cancer Res 2000; 2: 283-90 .
13.	Malkin D. The role of p53 in human cancer. J Neurooncol 2001; 51: 231-43.
14.	Zager V, Cemazar M, Hreljac I, Lah TT, Sersa G, Filipic M. Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression. Radiol Oncol 2010; 44: 42-51.
15.	Cesar Paz-y-Mino, Perez CJ, Fiallo BF, Leone PE. A polymorphism in the hMSH2 gene (gIVS12-6T>C) associated with non-Hodgkin lymphomas. Cancer Genet Cytogenet 2002; 133: 29-33.
16.	Worrillow LJ, Travis LB, Smith AG, Rollinson S, Smith AJ, Wild CP, et al. An intron splice acceptor polymorphism in hMSH2 and risk of leukemia after treatment with chemotherapeutic alkylating agents. Clin Cancer Res 2003; 9: 3012-20.
17.	Ollila S, Delmadi Bebek D, Jiricny J, Nystrom M. Mechanisms of pathogenic-ity in human MSH2 missense mutants. Human Mutation 2008: 29: 1355-63.
Periampullary localized pancreatic intraepithelial neoplasia-3 (PanIN-3): evaluation with contrast-enhanced MR cholangiography (MRCP)
Oktay Algin1, Evrim Ozmen1, Pamir Eren Ersoy2, Mustafa Karaoglanoglu1
1	Department of Radiology, Atatürk Training and Research Hospital, Bilkent, Ankara, Turkey
2	Department of General Surgery, Atatürk Training and Research Hospital, Bilkent, Ankara, Turkey
Received 19 January 2011 Accepted 19 July 2011
Correspondence to: Oktay Algin, Department of Radiology, Atatürk Training and Research Hospital, Bilkent-Ankara, Turkey. Phone: 0090 312 2912525; Fax: 0090 312 2912707; E-mail: droktayalgin@gmail.com
Disclosure: No potential conflicts of interest were disclosed.
Background. The early determination of premalignant lesions of pancreas can prevent unnecessary excessive surgical procedures and can reduce morbidity and mortality. Pancreatic intraepithelial neoplasia-3 (PanIN-3) is a preinvasive form of adenocarcinoma (carcinoma in situ). PanINs have not taken place in the literature of radiology yet, it should be considered in differential diagnosis of pancreatic cystic lesions.
Case report. A patient with preliminary diagnosis of chronic cholecystitis who had choledocolithiasis and periampullary pancreatic cyst detected by noncontrast-enhanced (NCE) and contrast-enhanced (CE) magnetic resonance cholangiography (MRCP) is presented. Pathological examination results of gallbladder and pancreatic cyst were reported as gallbladder adenocarcinoma and PanIN-3, respectively.
Conclusions. Pancreatic cystic lesions with thin septa which enhances slightly with the administration of contrast material may represent PanIN-3. In patients with cystic pancreatic lesion localized at periampullary region, using CE-MRCP together with NCE-MRCP could be useful in the evaluation of pancreatic cystic masses as well as other abdominal pathologies.
Key words: multidetector computed tomography; pancreatic cysts; magnetic resonance cholangiography; carcinoma; pancreatic intraepithelial neoplasia; magnetic resonance imaging
Introduction
Pancreatic cystic lesions are often detected with imaging techniques incidentally and can be differentiated from other lesions with some characteristic imaging findings. These findings could be useful for optimal classification, accurate clinical approach, and early diagnosis and correct therapy planning. An early determination of premalignant lesions can prevent unnecessary excessive surgical procedures and can reduce morbidity and mortality. It may increase the survival of patients by providing simple surgical procedures as well.1
Most of pancreatic cystic lesions are pseudo-cyts.2 Serous micro-cystic adenomas, mucinous cystic neoplasms, intraductal papillary mucinous
neoplasms and solid pseudopapillary tumours consist about 90% of all pancreatic tumors.1 The remaining 10% is made by metastases, cystic endocrine tumours, teratomas, lymphangiomas, primary pancreatic adenocarcinomas and acinar cell cystadenomas-carcinomas, etc.1-3 Although there are some reports related to the diagnostic features of the diseases mentioned above, as far we are aware, there is no paper about pancreatic in-traepithelial neoplasia-3 (PanIN-3) which is a premalignant lesion (carcinoma in situ) in radiology literature. In this paper, we present a patient with preliminary diagnosis of chronic cholecystitis who had choledocolithiasis and periampullary pancreatic cystic lesion detected by noncontrast-enhanced (NCE) and contrast-enhanced (CE) magnetic reso-
FIGuRE 1. Sequential T2 weighted (noncontrast-enhanced) MR cholangiography images of the patient. The images show stone (yellow arrow) in ductus choledochus (orange arrow) and cystic lesion (red arrow) in the pancreas.
nance cholangiography (MRCP) examinations. Pathological examination results of gallbladder and pancreatic cyst were reported as gallbladder adenocarcinoma and PanIN-3, respectively. In this case report we aimed to discuss the role of NCE-MRCP and CE-MRCP in cystic tumours of periam-pullary region, to evaluate radiologic features of PanINs and review the literature.
Case report
A 58-year-old man was admitted to the emergency department with right upper quadrant pain and jaundice. Though gallbladder could not be demonstrated optimally, the ultrasonographic examination revealed an increase in gallbladder wall thickness but it was contracted. Dilatation of intra and extrahepatic bile duct was detected. As a result, 64-detector multidetector computed tomography (MDCT) was performed since we could not evaluate gallbladder and choledochus optimally with ultrasound. In MDCT, gallbladder was contracted and could not be demonstrated clearly. The enlargement of choledochus and multiple choledochus stones were determined. For a better evaluation of the biliary stones, T2 weighted (T2W) MRCP (NCE-MRCP) was performed. T2W images showed multiple stones in ductus choledochus and intrahepatic bile ducts. A 15x20 mm cystic lesion including thin septa was demonstrated at the head of pancreas at periampullary region (Figure 1).
There was no relationship between cystic lesion and pancreatic duct. Gadoxetic-acid enhanced MRCP (CE-MRCP) was performed at the same session to examine the bile duct obstruction and to evaluate the pancreatic cystic lesion (Figure 2). A slight enhancement was detected at the wall and septi of the cystic lesion in CE-MRCP. There was no relationship between cyst and choledochus or pancreatic duct. There was no obstruction in the biliary tract as well. The transition of contrast material to the lumen of gallbladder was not seen at either early or late phase images. The patient was planned to undergo the surgical treatment according to these findings.
At surgical exploration, the size of gallbladder was significantly reduced. There were fibrotic adhesions between gallbladder, liver and transverse colon. Cholecystectomy was done; the pancreatic cystic lesion was aspirated and excised, as well. The histopathologic examination revealed a moderately differentiated adenocarcinoma of gallbladder with positive surgical margins and the pancreatic lesion was reported as PanIN-3. The patient was referred to the medical oncology department. The patient was in good condition the 6th month after the operation.
Discussion
The most important features of pancreatic adeno-carcinomas are their progressive course and their
FIGURE 2. Axial contrast enhanced multidetector computed tomography (upper left), noncontrast-enhanced T1 weighted (upper medium), arterial phase contrast-enhanced T1 weighted (upper right), portal phase contrast-enhanced T1 weighted (left below), delayed phase contrast-enhanced T1 weighted with and without fat saturation (middle below and right below, respectively) images of the patient. Pancreatic cystic lesion with contrast-enhanced thin septi-wall (red arrows) can be differentiated from ductus choledochus (yellow arrows) by gadoxetic-acid enhanced T1 weighted images. Also, delayed phase T1 weighted images show stone in the ductus choledochus.
high mortality rate. The majority of patients have locally advanced or distant metastatic disease (inoperable stage) during the diagnosis. Therefore, a detection of premalignant and malignant pancreatic lesions at early stage is necessary for curative surgery and improving the survival rates like in other oncological diseases.4-6
PanIN is the most common and histologically well-defined precursor that leads to pancreatic ductal carcinoma.7 Histologically PanINs could be divided into 3 subtypes. PanIN-1 and PanIN-2 are low-grade lesions, whereas PanIN-3 (carcinoma in situ) is a preinvasive form of adenocarcinoma.7 In conclusion PanIN-3 is a premalignant lesion similar to intraductal papillary mucinous neoplasm and mucinous cystic neoplasm and these lesions should be diagnosed early before they improve to invasive carcinoma. Although PanINs have not taken place in the literature of radiology yet, it should be considered in differential diagnosis of pancreatic cystic lesions.3 The existence of genetic, biochemical, and histological relation between PanINs and hepatobiliary/pancreatic carcinomas was reported in recent pathology literature.478 Gallbladder adenocarcinoma of our patient is a good example for this situation.
To evaluate the relation between the lesion and pancreatic duct at the heavily T2W sequences (NCE-MRCP) may differentiate PanIN-3 from intraductal papillary mucinous neoplasm.2 Although NCE-MRCP cannot always show the relationship, it often contributes to the diagnosis.3 Endoscopic ultrasound and endoscopic ultrasound-guided fine needle aspiration might be useful when NCE-MRCP is inadequate.3 Mucinous cystic neoplasms are often seen among women during the 4-6th decades (also called mother lesions).1 They consist of one or multiple cystic lesions and 10-25 % of the lesions may include peripheral curvilinear calcification. They are well demarcated and generally larger than 2 cm.1 Wall or septa of cysts could be enhanced after the contrast material administration similar to PanIN-3.2
Serous cystadenomas are generally seen among women after the 6th decade (also called grandmother lesions).1 They include fibrous central scars with or without a characteristic stellate pattern of calcification and these fibrous central scars are enhanced with contrast material.1 Demonstration of fibrous central scars with or without other characteristic features could provide differentiation serous cystadenomas from other cystic lesions ra-
diologically. Pseudocysts should be included to the differential diagnosis of pancreatic cystic lesions. Also, percutaneous or endoscopic ultrasound-guided fine needle aspiration of cystic fluid may be helpful in many cases with pancreatic cystic mass; since levels of amylase increase in the cystic fluid of pseudocysts, CEA levels are elevated in many mucinous cystic neoplasms, keratinous and amorphous debris can be seen in lymphoepithelial cysts, and mucin-rich fluid and columnar mucinous cells within this fluid can be observed in intraductal papillary mucinous neoplasm.1-3 Solid pseudopap-illary tumour and acinar cell cystadenoma/carcino-ma are the other rare cystic neoplasms that should be considered in differential diagnosis as well.129
MDCT, abdominal magnetic resonance imaging (MRI) and MRCP were reported as most useful techniques to evaluate pancreatic cystic lesions.310 Nevertheless, periampullary cystic lesions cannot be evaluated optimally by the tomographic examination in patients with dilated distal choledochus and choledocholithiasis as in our case.11 In such cases MRI and NCE-MRCP are supposed to be better in determination of cyst morphology and classifica-tion.11 However, there is not a published report related to the role of CE-MRCP in the evaluation of pancreatic cysts. CE-MRCP could be useful in determination of the relationship between the cystic lesions adjacent to choledochus and pancreatic duct or choledochus itself. It may also detect possible causes of the pancreatic duct obstruction at the periampul-lary region and it can provide functional data. CE-MRCP could evaluate the entire abdomen (especially in hepatobiliary system, duodenum, and pancreatic evaluation) at a single session with the addition of T2W images (NCE-MRCP).12 Moreover, CE-MRCP could supply an optimal assessment in case with suspicious malignant cyst by the detection of the contrast enhancement of cystic lesion with dynamic sequences, without a risk for radiation exposure or nefrotoxic contrast-material application.
MRCP together with NCE-MRCP could be useful in the evaluation of pancreatic cystic masses as well as other abdominal pathologies.
References
1.	Acar M, Tatli S. Cystic tumors of the pancreas: a radiological perspective. Diagn Interv Radiol 2011; 17: 143-9.
2.	Gumus M, Ugras S, Algin O, Gundogdu H. Acinar cell cystadenoma (acinar cystic transformation) of the pancreas: the radiologic-pathologic features. Korean J Radiol 2011; 12: 129-34.
3.	Dumitrescu D, Saftoiu A, Stoica M, Ciurea T, Popescu C. Natural evolution of an intraductal papillary mucinous neoplasm of the pancreas: A case report. J Gastrointestin Liver Dis 2007; 16: 105-8.
4.	Feldmann G, Beaty R, Hruban RH, Maitra A. Molecular genetics of pancreatic intraepithelial neoplasia. J Hepatobiliary Pancreat Surg 2007; 14: 224-32.
5.	Botros M, Quevedo JF, Miller RC. Angiosarcoma of the liver after multimo-dality therapy for gallbladder carcinoma. Radiol Oncol 2009; 43: 126-31.
6.	Debevec L, Jeric T, Kovac V, Bitenc M; Sok M. Is there any progress in routine management of lung cancer patients? A comparative analysis of an institution in 1996 and 2006. Radiol Oncol 2009; 43: 47-53.
7.	Recavarren C, Labow DM, Liang J, Zhang L, Wong M, Zhu H, et al. Histologic characteristics of pancreatic intraepithelial neoplasia associated with different pancreatic lesions. Human Pathol 2011; 42: 18-24.
8.	Sanada Y, Yoshida K, Ohara M, Tsutani Y. Expression of orotate phosphori-bosyltransferase (OPRT) in hepatobiliary and pancreatic carcinoma. Pathol Oncol Res 2007; 13: 105-13.
9.	Chung WJ, Byun JH, Lee SS, Lee MG. Imaging findings in a case of mixed aci-nar-endocrine carcinoma of the pancreas. Korean J Radiol 2010; 11: 378-81.
10.	Sahani DV, Saokar A, Hahn PF, Brugge WR, Fernandez-Del Castillo C. Pancreatic cysts 3 cm or smaller: how aggressive should treatment be? Radiology 2006; 238: 912-9.
11.	Sainani NI, Saokar A, Deshpande V, Fernandez-del Castillo C, Hahn P, Sahani DV. Comparative performance of MDCT and MRI with MR cholangiopancreatography in characterizing small pancreatic cysts. AIR Am J Roentgenol 2009; 193: 722-31.
12.	Algin O, Ozlem N, Kilic E, Karaoglanoglu M, Arslan H. Gd-BOPTA enhanced MR cholangiography findings in gall bladder perforation. Emerg Radiol 2010; 17: 487
Conclusions
Pancreatic cystic lesions with thin septa which enhances slightly with the administration of contrast material may represent PanIN-3. Early diagnosis and treatment of these lesions increase the duration and quality of lifetime by preventing excessive surgical procedures. Therefore, radiologists should be aware of the premalignant pancreatic cystic lesions. In patients with cystic pancreatic tumour localized at the periampullary region, using CE-
Brain meningioma invading and destructing the skull bone: replacement of the missing bone in vivo
Tomaz Velnar1, Rado Pregelj1, Clara Limbaeck-Stokin2
1	University Medical Centre Ljubljana, Department of Neurosurgery, Ljubljana, Slovenia
2	Institute of Pathology, Medical Faculty, Ljubljana, Slovenia
Received 12 May 2011 Accepted 18 June 2011
Correspondence to: Tomaž Velnar, MD, MSc, Department of Neurosurgery, University Medical Centre Ljubljana, Ljubljana, Slovenia. Phone: +368 1 522 32 62; Fax: +386 1 522 22 18; E-mail: tvelnar@hotmail.com or t.velnar.s06@cranfield.ac.uk
Disclosure: No potential conflicts of interest were disclosed.
Background. Meningiomas are frequently encountered tumours. In those invading locally into the adjacent tissue, reconstructions may pose a problem.
Case report. We report a case of a benign convexity brain meningioma with invasion into the skull bone and subcutaneous tissue. The tumour was removed completely, together with the infiltrated tissue and the defects were successfully closed with in vivo bone reconstruction.
Conclusions. The reconstruction of the skull bone is sometimes needed after the benign meningioma excision. Artificial bone may be a suitable material, allowing fast intraoperative reconstruction with excellent brain protection and cosmetic effect during the one-stage procedure.
Key words: meningioma; brain; invasion; bone reconstruction
Introduction
In neurosurgical practice, meningiomas are frequently encountered tumours and they represent about 20% to 25% of central nervous system neo-plasms.1-3 Arising from arachnoidal cells lining the brain and spinal cord, they may be found intrac-ranially and intraspinally. In addition, meningi-omas may originate as extracranial or extraspinal masses. These types of tumours are referred to as ectopic and have been described in various locations, the most frequent being the head, neck and soft tissue alongside the vertebral column.124
Meningiomas usually affect middle aged and older adults and contrary to oligodenroglial tumours are twice as frequent in women as in men.45 Progesterone receptors have been found in menin-geal tumour cells and possibly this hormone positively influences tumour development and progression. Genetic mutations in the neurofibroma-tosis 2 gene (NF2), immunological factors and
exposure to both high and low dose of ionizing radiation have been recognised among risk factors.2 Ninety percent of meningiomas are slow growing and benign tumours, the remaining ones are invasive or truly malignant.1 Most meningiomas have good long-term prognosis after the treatment, some display an aggressive clinical behaviour. The vicinity and compression of the eloquent brain zones, venous sinuses, skull base location and adjacent bone destruction may often lead to serious and potentially lethal consequences.14 Clinically, meningiomas are revealed by various symptoms including neurologic deficits and epileptic sei-zures.1 Surgery still remains the principal form of the treatment and must be preceded by appropriate preoperative diagnostics.6-8
Although the majority of meningiomas behave as expansive lesions, compressing the brain tissue, they may cause erosion on the neighbouring structures, especially bone. It is the location and particularly invasion of tumour into adjacent tissue that
may hamper radical resections and reconstructions by simple surgical means.1910 In such cases, the reconstruction of the skull bone is problematic due to tissue deficit. Many alternatives exist, from autografts, allografts or artificial replacement material.1112 We report an unusual case of a men-ingioma of the brain, located in the premotor and motor cortex in the frontoparietal region, invading and destroying the skull bone and subcutaneous tissue. The tumour was removed completely, together with the infiltrated tissue and the defects after the operation were successfully reconstructed with in vivo bone reconstruction.
Case report
A 66-year old gentleman in otherwise good general health was admitted to the neurosurgical department due to a skull deformation in the left frontoparietal region, which was growing progressively. He first noticed it approximately seven months ago and complained of dull headaches, located in the left half of the head that were noticed a few times weekly. No other complaints in connection with his health status were reported at the admission.
The neurological status during the clinical examination was normal. Locally, a skull tumour of 5 cm in diameter was felt. It was immobile and insensitive on palpation and the skin covering it was normal.
The computer tomography (CT) and magnetic resonance (MR) imaging revealed an intracranial expansive lesion of 8 cm in diameter, compressing the cortex and invading the skull bone and subcutaneous tissue (Figures 1A and 1B). The CT angiog-raphy did not show any signs about sinus invasion. Surgery was indicated.
The operation was performed via midline incision. In the subcutis, the tumour mass growing through the bone was seen, infiltrating the periosteum and galea (Figure 2A). A round section of the skull bone was performed, encircling the tumour first. Then, the bone in the very vicinity of the tumour was drilled in such a way that two circular bone flaps were formed around the tumour as it was not possible to elevate the first bone flap due to the tumour adhesion to the bone without damaging the bridging veins and the dura. The tumour was then microsurgically removed, carefully dissected and elevated off the brain substance (Figure 2B). The tumour origin was in the dural convexity over the left motor and premotor cortex. The cortex was
FIGURES 1A, 1B. The axial view of the CT scan showing homogenous lesion in the mo-tor/premotor area, radiologically classified as meningioma, with hyperostotic bone (arrow) (A). The coronal view of the tumour, growing intra- and extracranially (B).


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relatively spared, though severely compressed and the superior sagittal sinus was also compressed but otherwise intact. However, the bone was po-rotic and invaded by the tumour, which spread through the periosteum into the galea. The tumour was completely excised together with all infiltrated extracranial tissue.
Extensive defects of the dura mater and bone were well reconstructed with dura replacement material (lyophilised dura) and water tightly sealed with fibrin glue in order to avoid liquorrhea. Artificial bone was modelled in vivo from two component polymethylmetacrylate material, which was moulded and modelled according to the shape of the removed bone just before closure (Figures 2C and 2D). The original bone flap was used as a template. The fit was very good, giving an excellent cosmetic result as well as brain protection. The new artificial bone flap was fixed to the skull bone with titanium plates. Finally, the wound was closed in layers. After the operation, the patient was neuro-logically intact. The control CT scan showed a good position of the implant with no fluid collection underneath (Figure 3). The rest of the postoperative course was uneventful.
Histology showed that the tumour was a conventional fibrous meningioma, WHO grade 1. It indeed originated from the dural convexity and spread through the bone into the subcutis (Figure 4). No additional treatment was recommended.
Discussion
Our patient was operated on for a benign menin-gioma, which was especially interesting because of the invasion into the skull bone, its destruction and invasion into the subcutaneous tissue as well as the postoperative question about tissue reconstruction. The majority of meningiomas are benign tumours
FIGURES 2A, 2B, 2C, 2D. Intraoperative view of the meningioma growing through the skull bone into the periosteum and the subcutis. The scalp has been retracted laterally (A). Removal of the intracranial part of the meningioma: the tumour was dissected from the brain tissue, gently lifted off the brain via suspension and removed together with the infiltrated dura. Special care was taken not to damage the vessels (B). The artificial bone modelling and its template. Green material is polymethylmetacrylate. During the operation, the outer ring of the bone visible on the right was removed first, only then the tumour dissection and removal of the inner bony ring started (C). The artificial bone in place, fixed with titanium clamps at the edge (D).
that behave as expansive lesions.1310 Symptoms usually arise due to compression of the brain and erosion of the neighbouring tissue.
Some of meningiomas are invasive and about 5% of meningiomas are malignant, more likely causing direct invasion.1313'15 Besides invasive and malignant meningiomas, benign meningiomas may also invade bone. In all cases, the reconstruction of the removed bone is necessary.
Patients with meningiomas are often elderly people with associate diseases that preclude radical resections and complicate a postoperative course. Because of tissue deficit and extensive operation, the reconstruction of the missing tissue, especially the skull bone and soft tissue, is problematic.16-18 There are many alternatives to repair the missing tissue, nowadays three main techniques are used: autografts, allografts and artificial replacement material.91019 The selection of the material and operative technique depends on surgeons' experience and preferences in addition to size, location, shape and depth of the bone defect.
About 50% of cases show hyperostosis of the bone overlaying the tumour, with meningothelial tumour cells infiltrating the bone itself.910 A lot of technical difficulties may arise during the operation and because of that, many skull base tumours, principally those of the anterior or middle cranial fossa and those extending into the orbit were not excised completely. Recently, improved techniques of craniofacial surgery have been developed, allowing a wide range reconstruction and leading to more successful clinical result.19
In order to accomplish a complete resection, a combined intra- and extracranial resection is required, involving the removal of the hypertroph-ic bone. It was suggested that strict adherence to oncological principles should be applied also in the case of benign neoplasms in order to prevent contamination of wounds with tumour cells and potential recurrence.20 Often, a radical resection may be attained with low morbidity in operated patients, providing a significantly better long-term clinical outcome.10 In such extensive resections, the aesthetic reconstruction of large bone defects may pose a significant issue during the operation. Viable tissue in the form of autografts and allografts is one attractive option, another one is artificial replacement material.91112
In this particular case, we decided for in vivo reconstruction of the missing bone with artificial replacement material for several reasons. The autografts, which are available in the form of free tissue transfer, rotational flaps and combined as
figure 3. The axial view of the postoperative CT scan with the artificial bone (arrows) covering slightly oedematous brain tissue.
well, allow coverings of large volume tissue defects, especially those of soft tissue.1921 Vascularised tissue is less prone to infection and will survive easily than grafts without direct vascular supply. Autografts are safe in terms of disease transmission and exhibit no immune response reactions. Bone grafts show low infection and desorption rate that leads to relatively short term of graft incorpo-ration.21 Moreover, vascularised tissue is relatively resistant to postoperative irradiation, which may also become necessary after the resection of grade III and sometimes of grade II meningiomas.11192122 However, to protect healthy tissue, specially brain and vessels, we use limited postoperative irradiated fields in contrast with prophylactic cranial irradiation.23
On the other hand, the use of viable tissue has many drawbacks, especially morbidity at the donor site after the operation, higher infection rate due to more extensive operative process, longer intraoperative time and a need of plastic surgeon. When allografts are considered, the infection risk is
FIGURE 4. The histological image of the extracranial part of the fibrous meningioma, showing tumour cell invasion into the muscle tissue of the galea.
higher and immunological reactions are possible, complicating the recovery and slowing down the healing process.21
Further possibilities are cranioplasty implants. Some are manufactured in advance, according to the shape of the bone defect, other may be modelled intraoperatively from titanium meshes or various composite materials. In our case, we decided for the artificial bone reconstruction. Custom made cranipolasty implants are made in advance according to the shape of the bone defect, therefore, the fit is not always appropriate, increasing the possibilities that the implant may shift. Furthermore, a second operation for the implantation is needed. Our goal was to complete the tumour resection and the reconstruction in one leg, posing less operation-associated risks and enabling faster recovery. We reconstructed the defect from polymethylmethacrylate, which is an efficient and relatively straightforward procedure. It can be performed immediately after the tumour resection, it is fast and yields excellent cosmetic results. After mixing the two components of polymethylmeth-acrylate, the material is soft and it may be modelled in the shape of the removed bone, exactly filling the bone defect. It hardens in about ten minutes, allowing subtle additional adjustments in shape and fit during modelling. The curvature of the implant may be adjusted according to the curvature of the skull and the implant may be easily fixed with titanium plates and screws, titanium clamps
or absorbable clamps, providing good stability. The implant, when in place and air-dried, yields a solid construct that perfectly matches the patient's natural head shape and has good strength in both compression and tension. Our operative procedure with bone modelling did not pose any particular technical problem and was time saving with good result of the reconstruction.
Among the artificial materials, artificial bone is an efficient alternative to titanium plate or mesh for intraoperative bone reconstruction.9 According to our experience, this technique is useful in appropriate conditions, as it is time-saving, straightforward and enables brain protection with good cosmetic result after the extensive cranial vault defect at the time of tumour resection.
Conclusions
This case illustrates how reconstruction of the skull bone is sometimes needed after the benign menin-gioma excision. Artificial bone may be a suitable material, allowing fast intraoperative reconstruction with excellent brain protection and cosmetic effect during the one-stage procedure.
References
1.	Kalamarides M, Goutagny S. Meningiomas. Rev Prat 2006; 56: 1792-8.
2.	Perry A, Gutmann DH, Reifenberger G. Molecular pathogenesis of meningiomas. J Neurooncol 2004; 70: 183-202.
3.	Vranic A. Antigen expression on recurrent meningioma cells. Radiol Oncol 2010; 44: 107-12.
4.	Wiemels J, Wrensch M, Claus EB. Epidemiology and etiology of meningioma. J Neurooncol 2010; 99: 307-14.
5.	Velnar T, Smrdel U, Popovic M, Bunc G. Genetic markers in oligodendroglial tumours. Radiol Oncol 2010; 44: 13-8.
6.	Wen PY, Quant E, Drappatz J, Beroukhim R, Norden AD. Medical therapies for meningiomas. J Neurooncol 2010; 99: 365-78.
7.	Norden AD, Drappatz J, Wen PY. Advances in meningioma therapy. Curr Neurol Neurosci Rep 2009; 9: 231-40.
8.	Prabhu SS, Demonte F. Treatment of skull base tumors. Curr Opin Oncol 2003; 15: 209-12.
9.	Bloch O, McDermott MW. In situ cranioplasty for hyperostosing meningiomas of the cranial vault. Can J Neurol Sci 2011; 38: 59-64.
10.	Shrivastava RK, Sen C, Costantino PD, Della Rocca R. Sphenoorbital meningiomas: surgical limitations and lessons learned in their long-term management. J Neurosurg 2005; 103: 491-7.
11.	Moyer JS, Chepeha DB, Teknos TN. Contemporary skull base reconstruction. Curr Opin Otolaryngol Head Neck Surg 2004; 12: 294-9.
12.	Salyer KE, Bruce DA, Hardin CE, Hopkins KS, Gendler E. Craniofacial neuro-surgical approach for extensive hyperostotic meningioma. J Craniofac Surg 1993l; 4: 128-34.
13.	Singh RVP, Yeh JS, Campbell DA. Implantation meningioma in temporalis muscle: case report. Br J Neurosurg 1994; 8: 93-5.
14.	Akai T, Shiraga S, Iizuka H, Kishibe M, Kawakami S, Ueda Y. Recurrent meningioma with metastasis to the skin incision. Neurol Med Chir (Tokyo) 2004; 44: 600-2.
15.	Menal P, Garcia-Tirado FJ, Embun R, Rivas JJ. Is a benign meningioma always an indolent tumor? Interact Cardiovasc ThoracSurg 2011; 13: 94-5.
16.	Sughrue ME, Rutkowski MJ, Shangari G, Chang HQ, Parsa AT, Berger MS, et al. Risk factors for the development of serious medical complications after resection of meningiomas. J Neurosurg 2011; 114: 697-704.
17.	Black P, Morokoff A, Zauberman J, Claus E, Carroll R. Meningiomas: science and surgery. Clin Neurosurg 2007; 54: 91-9.
18.	Li PL, Mao Y, Zhu W, Zhao NQ, Zhao Y, Chen L. Surgical strategies for petro-clival meningioma in 57 patients. Chin Med J 2010; 123: 2865-73.
19.	Cansiz H, Cambaz B, Papila I, Tahami R, Güne§ M. Use of free composite graft for a large defect in the anterior skull base. J Craniofac Surg 1998; 9: 76-8.
20.	Velnar T, Bunc G. Iatrogenic metastasis of a benign meningioma to the periosteum at the site of previous craniotomy: a case report. Wien Klin Wochenschr 2008; 120: 766-9.
21.	Bauer TW, Muschler GF. Bone graft materials. An overview of the basic science. Clin Orthop Relat Res 2000; 371: 10-27.
22.	Angelos PC, Downs BW. Options for the management of forehead and scalp defects. Facial Plast Surg Clin North Am 2009; 17: 379-93.
23.	Stanic K, Kovac V. Prophylactic cranial irradiation in patients with small-cell lung cancer: the experience at the Institute of Oncology Ljubljana. Radiol Oncol 2010; 44: 180-6.
Dosimetric verification of compensated beams using radiographic film
Slaven Jurkovic1, Gordana Zauhar2, Dario Faj3, Deni Smilovic Radojcic1, Manda Svabic1, Mladen Kasabasic3, Ana Diklic1
1	University Hospital Rijeka, Radiotherapy Department, Physics Division, Rijeka, Croatia
2	School of Medicine, Department of Physics, Rijeka, Croatia
3	University Hospital Osijek, Department of Radiotherapy and Oncology, Osijek, Croatia
Received 19 January 2011 Accepted 25 April 2011
Correspondence to: Slaven Jurković, UH Rijeka, Radiotherapy Department, Physics Division, Krešimirova 42, 51000 Rijeka, Croatia. Phone: + 385 51 658 398; Fax:+385 51 658 391; E-mail: slaven.jurkovic@ri.t-com.hr
Disclosure: No potential conflicts of interest were disclosed.
Introduction. External photon beam modulation using compensators in order to achieve a desired dose distribution when brachytherapy treatment is followed by external beam radiation is a well-established technique. A compensator modulates the central part of the beam, and the dose beneath the thickest part of the compensator is delivered mostly by scattered, low energy photons. A two-dimensional detector with a good spatial resolution is needed for the verification of those beams. In this work, the influence of different types of detectors on the measured modulated dose distributions was examined.
Materials and methods. Dosimetric verification was performed using X-Omat V, Eastman Kodak radiographic films at different depths in a solid water phantom. The film measurements were compared with those made by ionization chambers. Photon beams were also modelled using EGSnrc Monte Carlo algorithm to explain the measured results. Results. Monte Carlo calculated over-response of the film under the thickest part of the compensator was over 15%, which was confirmed by measurements. The magnitude of over-response could be associated with changes in the spectra of photon energy in the beam.
Conclusions. The radiographic film can be used for the dosimetry of compensated high energy photon beams, with limitations in volumes where photon spectra are hardly degraded.
Key words: radiation therapy; dosimetry; compensators
Introduction
Intracavitary application of brachytherapy sources followed by external beam radiation is a common practice in radiotherapy of carcinoma of the cervix. Since the application of brachytherapy sources results in characteristic dose distributions, modulated external photon beams should be added in a way to achieve the desired cumulative dose distribution over the target volume. Several techniques used in practice have been described.1-4
On the other hand, the dosimetry of modulated linear accelerator's photon beams is rather complex, mainly due to dose distribution in homogeneity within the radiation field with large dose gradients. Therefore, dosimetric verification needs
a high spatial resolution and this demand makes the radiographic film a dosimeter of choice.5-8 Nevertheless, it has been shown that the absorbed dose could be related to the energy absorption coefficient.9 Furthermore, energy absorption coefficients for film emulsions and water differ significantly in the low energy region bellow 400 keV, as the data calculated according to Seltzer have shown.10 Since there is an enhanced contribution of scattered radiation to the total dose in modulated photon beams, a disadvantage of the film which shows over-response to low energy photons may become important.11-13
In this work, we compared the measured dose distributions of high energy photon beams acquired by different detectors. In order to discuss ex-
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perimental results, Monte Carlo simulation of particle transport for the measured beams was done.
Materials and methods
Dosimetric verification of open and modulated 6 MV photon beams from Siemens Oncor Impression linear accelerator was performed using X-Omat V (Eastman Kodak) radiographic films at different depths in solid water (PTW Solid Water Phantom). We used fixed source-to-surface (SSD)
geometry with SSD=100 cm on the phantom surface. Film dosimetry was performed using Vidar DosimetryPro Advantage scanner with Coherence Physicist (Siemens Medical Solutions) and PIPSPro (Standard Imaging) software packages for film dosimetry. The dose profiles measured by the film were compared with those made by ioniza-tion chambers (IBA Dosimetry, compact chambers CC13 and CC 01) in the water phantom (IBA Dosimetry, Blue Phantom). Regarding a better spatial resolution of a small volume ionisation chamber (CC01), data measured with those chambers in
FIGURE 2. Compensator's shape calculated to conform the dose distribution given by an external beam according to the dose distribution around brachytherapy sources. The insert shows thickness of the compensator in a form of level curves in mm.
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FIGURE 4. Mean energy distributions for open and modulated beams in air and on 10 cm depth in water. Calculations were done with SSD=100 cm and 20*20 cm2 field size for open and modulated beams. Regarding the symmetry of the energy distributions only half-profiles are shown.
the high gradient region of the beams were superimposed on measurements with CC13 ionisation chamber which had a better signal-to-noise ratio.
Photon spectra for Siemens Oncor Impression linear accelerator photon beams were calculated at the measuring planes using Monte Carlo simulation of particle transport (EGSnrc). The simulation for 6 MV photons with a field size of 20x20cm2 (defined at SSD=100 cm) was performed using OMEGA/BEAM code, developed by the National Research Council of Canada (NRCC). This is an EGSnrc user code capable of complex linear accelerator geometric coding.14 The detailed geometry and composition of each individual device in the Siemens Oncor Impression linear accelerator were obtained from the manufacturer. Open and modulated beams were modelled using BEAMnrc software. The modelled geometry of compensated beam is shown in Figure 1. Calculated shape of the compensator is shown in Figure 2.
The compensator shape was calculated using the dose distribution around the brachytherapy sources as a pattern according to which the open photon beam was modulated in order to achieve the desired total dose distribution.4
Therefore, the shape of the dose distribution is rather characteristic and, from the dose profiles point of view, three different areas can be distinguished: the area under the compensator and the open beam area where the measurements can be performed with high reproducibility and the area near the edge of the compensator which is characterized by high dose gradients and a lower level of measurement reproducibility. In the last area, Monte Carlo calculation is especially used as a guideline for the interpretation of the measured dose distributions. On the other hand, changes in the energy spectrum were expected in the area under the thickest part of the compensator.
The absorbed dose in a material depends on energy absorption coefficients9 and there is a large difference in those coefficients for film and water in low energy area.10 Therefore, an over-response of the film under the thickest part of the compensator was expected at larger depths because the Compton scattered low-energy photons dominate there.8 Dose calculations in a material were performed according to:9
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P J E P	P
Tite BEAM code was implemented using variance reduction techniques: photon forcing, bremsstrahlung splitting and range rejection to speed up
the simulation. The lower charged particle cutoff energy, AE, was 0.7 MeV, and the lower photon cutoff energy, AP, was 0.01 MeV. The energy loss per transport step of the electron, ESTEPE, was controlled by PRESTA.15 Scored plane was set at Z=100cm to collect the particles after transportation from the accelerator, and to form the phase space file. Information concerning particles in the phase space file included the position (X, Y, Z), direction (U, V, W), energy, charge, weighting, and origin (LATCH). Five to ten million particles were collected in the scored plane. The phase space file served as the source for the following water phantom simulation using DOSXYZ, an EGSnrc user code for 3D absorbed dose calculation in Cartesian coordinates.16 In DOSXYZ, the water phantom size was 40x40x40cm3 and the phase space source position was on the water surface (Z=0). The origin was at the centre of the radiation field. Voxels with size of 0.5x0.5x0.5 cm3 (Xx YxZ) were set at the depth of the maximum dose for dose profile simulation. 50 voxels from water surface (Z=0) with size 2.0x2.0x0.2 cm3 and 20 voxels with size 2.0x2.0x0.4 cm3 were set along the central axis for central percent depth dose (PDD) simulation. The particles in the phase space file were redistributed and reused to obtain better accuracy in dose calculation.16 Physical parameters of original electron beam that may influence the dose profile and central-axis PDD curve are the beam energy, the beam spot size and the distance from the point source.1718 These parameters were adjusted to allow dose profiles and percentage depth dose curve to match measured data. Since we calculated changes in beam energies, for the purpose of our work, the accuracy of the beam profiles was not essential. We decided that 3% discrepancy from measurements is acceptable in the high dose region and 20% in the low dose region. Recommended values are 2% and 20% respective-ly.19-21
Results
From the analysis of measured beam profiles, we observed significant discrepancies between measurements with the radiographic film and ioniza-tion chambers when measuring beam profiles of modulated beams on larger depths in water. The discrepancies were pronounced under the thickest part of the compensator (Figure 3).
Calculated mean energy distributions in open and modulated beams are shown in Figure 4.
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FIGuRE 5A. Photon spectral distributions for open and modulated 6MV photon beams on central axis in air. Calculations were done with SSD=100 cm and 20*20 cm2 field size.
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FIGuRE 5B. Photon spectral distributions in water for open and modulated 6MV photon beams, on 10 cm depth in water. Calculations were done with SSD=100 cm and 20*20 cm2 field size at central axis for open beam and also at central axis and under steep part of the compensator (6.5 cm off axis) for modulated beam.
From the 'in air' simulation analyses, we can see that the compensator removed low energy photons from the beam, so the mean energy of the modulated beam is higher than the one of the open beam (Figure 4). This can also be seen in Figure 5A. On the other hand, at larger depths in water, the Compton scattering low-energy photons dominate, especially under the thickest part of the compensator, so the Figure 4 shows the decrease of the mean energy of the modulated beam there.
Calculated photon spectral distributions for open and modulated beams are shown in Figures 5A and 5B, respectively.
Taking into account the dependence of mass absorption coefficients on photon energy for used
dosimeters and calculated energy distribution of photons in small volumes, we can estimate changes in the film response. Regarding data shown in Figures 4, 5A and 5B, it follows that the largest differences could be expected under the thickest part of the compensator at larger depths in water because of the largest energy degradation. Dose calculations for the film and water were done according to Equation [1]. Calculated over-response of the film in this region was over 15% which was confirmed by measurements (Figure 3).
Discussion
In this paper, we showed that magnitude of over-response of the radiographic film of modulated high energy photon beams could be associated with the changes in the spectra of photon energy in the beam. Since the largest spectral change was under the thickest part of the compensator, there was the largest difference between film and ionisation chamber measurements.
Regarding a high dose gradient beneath the steep part of the compensator, it was not possible to measure doses in this area accurately. Nevertheless, spectra in this area resemble open rather than modulated beam beneath the thickest part of the compensator. In this way, the over-response of the film under the steep part of the compensator would be small.
The radiographic film is often used for verifications of modulated photon beams.57822 Despite of described limitations, it can be used either on build up depth for the evaluation of compensators shape or for measuring dose distributions of modulated high energy photon beams in phantoms. Special attention should be paid to the interpretation of measured values in volumes where photon spectra are hardly degraded.
5.	Bucciolini M, Banci F, Casati M. Verification of IMRT fields by film dosimetry. Med Phys 2004; 31: 161-8.
6.	Bailey DW, Kumaraswamy L, Podgorsak MB. A fully electronic intensity-modulated radiation therapy quality assurance (IMRT QA) process implemented in a network comprised of independent treatment planning, record and verify, and delivery systems. Radiol Oncol 2010; 44: 124-30.
7.	Salz H, Wiezorek T, Scheithauer M, Schwedas M, Beck J, Wendt TG. IMRT with Compensators for Head-and-Neck Cancers. Strahlenter Onkol 2005; 181: 665-72.
8.	Srivastava RP, Wagter De C. The value of EDR2 film dosimetry in compensator-based intensity modulated radiation therapy. Phys Med Biol 2007; 52: 449-57.
9.	Brahme A, Andreo P. Dosimetry and quality specification of high energy photon beams. Acta Rad Onc 1986; 25: 213-23.
10.	Seltzer SM. Calculation of photon mass energy-transfer and mass energy-absorption coefficients. Radiat Res 1993; 136: 147-70.
11.	Yeo IJ, Wang CC, Burch SE. A filtration method for improving film dosimetry in photon radiation therapy. Med Phys 1997; 24: 1943-53.
12.	Wang X, Spirou S, Lo Sasso T, Stein J, Chui C, Mohan R. Dosimetric verification of intensity-modulated fields. Med Phys1996; 23: 317-27.
13.	Ju SG, Ahn YC, Huh SJ, Yeo IJ. Film dosimetry for intensity modulated radiation therapy:Dosimetric evaluation. Med Phys 2002; 29: 351-55.
14.	Rogers DWO, Faddegon BA, Ding GX, Ma CM, We J, Mackie TR. BEAM: a Monte Carlo code to simulate radiotherapy treatment units. Med Phys 1995; 22: 503-24.
15.	Bielayew AF, Rogers DWO. PRESTA: the parameter reduced electron-step transport algorithm for electron Monte Carlo transport. Nucl Instrum Methods B 1987; 18: 165-81.
16.	Ma CM, Reckwerdt P, Holmes M, Rogers DWO, Geiser B. DOSXYZ users manual. National Research, Council of Canada Report PIRS-0509B; 1995.
17.	Lin SY, Chu TC, Lin JP. Monte Carlo simulation of a clinical linear accelerator. App Rad Isotopes 2001; 55: 759-65.
18.	Faj D, Vrtar M, Krajina Z, Jurković S, Margaretić D. Model of total skin electron treatment using the 'six-dual-field' technique. Coll Antropol 2003; 27: 713-21.
19.	Verhaegen F, Seuntjens J. Monte Carlo modeling of external radiotherapy photon beams. Phys Med Biol 2003; 48: R107-64.
20.	Rivest DRC, Riauka TA, Murtha AD, Fallone BG. Dosimetric implications of two registration based patient positioning methods in prostate image guided radiation therapy (IGRT). Radiol Oncol 2009; 43: 203-12.
21.	Mesbahia A, Reillyb AJ, Thwaites DI. Development and commissioning of a Monte Carlo photon beam model for Varian Clinac 2100EX linear accelerator. Applied Radiation 2006; 64: 656-62.
22.	Ting JY, Davis L. Dose verification for patients undergoing IMRT. Med Dosim 2001; 26: 205-13.
References
1.	Han IH, Malviya V, Chuba P, Orton CG, Devi S, Deppe G, et al. Multifractioned HDR brachytherapy with concomitant daily teletherapy for cervical cancer Gynecol Oncol 1996; 63: 71-7.
2.	Popple RA, Kim RY, Pareek P, Duan J, Shen S, Brezovich IA. Custom step wedge blocking using dynamic multileaf collimation for parametrial pelvic boost irradiation following brachytherapy carcinoma of the cervix. Med
Phys 2003; 30: 2699-702.
3.	Jurković S, Žauhar G, Bistrović M, Faj D, Kaliman Z, Smilović Radojčić D. An alternative approach to compensators design for photon beams used in radiotherapy. Nucl Instrum Methods A 2007; 580: 530-3.
4.	Jurković S, Žauhar G, Faj D, Smilović Radojčić Đ, Švabić M, Radiation therapy photon beams dose conformation according to dose distribution around intracavitary applied brachytherapy sources. Med Dos 2010; 35: 49-52.
Radiol Oncol 2011; 45(4):227-247.
doi:10.2478/v10019-011-0037-0
Titanijev dioksid v vsakdanji uporabi. Je varen?
Skočaj M, Filipič M, Petković J, Novak S
Izhodišča. Na splošno ocenjujejo, da je titanijev dioksid (TiO2) inerten in varen, zato ga že desetletja uporabljajo v različne namene. Z razvojem nanotehnologij pa se je povečala proizvodnja in uporaba TiO2 v nanoobliki. Ta ima številne nove koristne lastnosti, vendar lahko pričakujemo povečano izpostavljenost ljudi in okolja. Zato je pomembno boljše poznavanje toksikoloških lastnosti nanodelcev TiO2. Mehanistične toksikološke raziskave kažejo, da nanodelci TiO2 povzročajo toksične učinke predvsem prek oksidativnega stresa. Ta povzroča poškodbe celic, genotoksičnost, vnetja, imunski odziv itd. Obseg in vrste poškodb sta v veliki meri odvisna od fizikalnih in kemijskih lastnosti delcev TiO2. Te lastnosti določajo biološko dostopnost in reaktivnost TiO2. Na osnovi dokazov poskusov inhalacijske izpostavljenosti živali je Mednarodna agencija za raziskavo raka (IARC) delce TiO2 opredelila kot »možno karcinogene za ljudi«, Nacionalni inštitut za varnost in zdravje v poklicih (NIOSH) pa je nanodelce TiO2 opredelil kot karcinogene pri poklicni izpostavljenosti. Raziskave kožne izpostavljenosti, ki je pri ljudeh zelo velika zaradi uporabe v zaščitnih kremah za sončenje, na splošno kažejo, da je prehod prek kože zanemarljiv. Vendar pa ni podatkov o dolgotrajni izpostavljenosti in podatkov o morebitnih škodljivih učinkih fotooksidacijskih produktov. Čeprav je TiO2 dovoljen kot aditiv v hrani (E1 71 ) in v farmacevtskih proizvodih, ni zanesljivih podatkov o njegovi absorpciji, porazdelitvi, izločanju in toksičnosti pri oralni izpostavljenosti. TiO2 vstopa tudi v okolje, za vodne organizme ni akutno toksičen, pri dolgotrajni izpostavljenosti pa povzroča vrsto subletalnih učinkov.
Zaključki. Dokler ne bodo na voljo relevantni toksikološki podatki in podatki o izpostavljenosti ljudi, ki bodo omogočili zanesljivo oceno tveganj, je pri uporabi nanodelcev TiO2 potrebna velika previdnost.
Radiol Oncol 2011; 45(4): 248-258.
doi:10.2478/v10019-011-0038-z
Ocena ledvične funkcije pri otrocih s hidronefrozo -dodatna možnost magnetnoresonančne tomografije
Hadjidekov G, Hadjidekova S, Tonchev Z, Bakalova R, Aoki I
Izhodišča. Magnetnoresonančna urografija (MRU) je ena izmed najbolj primernih slikovnih metod v pediatrični urologiji. Z eno samo preiskavo dobimo največ diagnostičnih podatkov. Namen raziskave je bil opredeliti diagnostično vrednost MRU pri otrocih z različnimi prirojenimi razvojnimi nepravilnostmi sečil in rodil, predvsem nepravilnostmi ledvičnega meha in sečevodov, ter naknadno oceniti ledvično funkcijo z različnima računalniškima programoma. Bolniki in metode. V raziskavo smo zajeli 96 otrok (starost 7 dni do 18 let). Pri 54 smo statično T2 MRU dopolnili z ekskretorno T1 MRU po aplikaciji Gadolinijevega kontrastnega sredstva. Oceno ledvične funkcije smo opravili z dvema računalniškima programoma, s "CHOP-fMRU" in "ImageJ".
Rezultati. Z MRU smo lahko pri vseh otrocih zelo kvalitetno prikazali razvojne nepravilnosti ledvic in celotnega urotrak-ta. Ob primerih obstrukcije zaradi pielouretralne stenoze smo z MRU potrdili že z ostalimi slikovnimi metodami ugotovljeno diagnozo. Ob primerih razvojnih nepravilnosti končnega dela sečevoda pa je bila MRU bolj natančna metoda. Ugotovili smo veliko skladnost med diagnozo postavljeno z MRU in dokončno kirurško diagnozo. Z računalniškima programoma "CHOP-fMRU" in "ImageJ" smo izračunali čas ledvičnega izločanja, volumen ledvic in volumetrično ledvično funkcijo, ki so bili zelo skladni s scintigrafijo 99mTc-DTPA in tudi med seboj.
Zaključki. Zaradi svojih prednosti postaja MRU najpomembnejša slikovna metoda v pediatrični uroradiologiji. Omogoča sintezo anatomskega prikaza in oceno ledvične funkcije. Na ta način omogoča optimalno izbiro primernih kandidatov za kirurško zdravljenje. Z uporabljenima programoma smo lahko odkrili tudi majhne nepravilnosti v delovanju ledvic.
Radiol Oncol 2011; 45(4): 259-266.
doi: 10.2478/v10019-011-0034-3
Katepsin H posredno regulira kostni morfogenetski protein 4 (BMP-4) v človeških celičnih linijah
Rojnik M, Jevnikar Z, Mirkovic B, Janes D, Zidar N, Kikelj D, Kos J
Izhodišča. Katepsin H je cisteinska proteaza, ki ima pomembno vlogo pri napredovanju raka, vendar pa je njegova natančna funkcija v tem procesu nejasna. Pred kratkim se je uveljavila domneva, da naj bi bil katepsin H udeležen pri izoblikovanju kostnega morfogenetskega proteina 4 (BMP-4) pri miših. Da bi pojasnili, ali je katepsin H udeležen pri regulaciji BMP-4 tudi pri ljudeh, smo raziskovali njegov vpliv na izražanje, izoblikovanje in razgradnjo BMP-4 v človeških celičnih linijah raka prostate (PC-3), osteosarkoma (HOS) in pro-monocitov (U937).
Materiali in metode. Z uporabo tehnologije mikromrež polimerazne verižne reakcije (PCR) smo ugotovili, da katepsin H regulira izražanje BMP-4, kar smo potrdili s PCR v realnem času. S pomočjo prenosa Western ter konfokalne mikroskopije smo raziskovali vlogo katepsina H pri izoblikovanju BMP-4.
Rezultati. Izražanje BMP-4 je v celicah HOS v odvisnosti od katepsina H povečano, vendar pa nasprotno katepsin H značilno zniža izražanje BMP-4 v celicah U937 in PC-3. Drugačno regulacijo BMP-4 bi lahko povezali z odsotnostjo zrele 28 kDa oblike katepsina H v celicah HOS, saj smo tu opazili le vmesno 30 kDa obliko katepsina H. BMP-4 v človeških celičnih linijah ni v neposredni odvisnosti s katepsinom H, poleg tega pa specifični inhibitor katepsina H ne vpliva na večstopenjsko izoblikovanje BMP-4. Ugotovili smo tudi, da izoliran katepsin H ne razgrajuje BMP-4 tako z aminopepti-dazno kot tudi ne z endopeptidazno aktivnostjo.
Zaključki. Naši rezultati izključujejo možnost neposredne regulacije kostnega morfogenega proteina 4 (BMP-4) s katepsinom H, vendar pa podpirajo hipotezo, da katepsin H posredno regulira izražanje BMP-4.
Radiol Oncol 2011; 45(4): 267-272.
doi:10.2478/v10019-011-0033-4
Sinonazalni invertni papilom združen s ploščatoceličnim karcinomom
But-Hadžić J, Jenko K, Poljak M, Kocjan BJ, Gale N, Strojan P
Izhodišča. Namen raziskave je bil ovrednotiti naše izkušnje s sinonazalnim invertnim papilomom združenim s plošča-toceličnim karcinomom (IP/PCK), analizirati prisotnost humanega virusa papiloma (HPV) in oceniti vlogo radioterapije. Bolniki in metode. V prospektivnih podatkovnih bazah (1995-2005) smo ugotovili pet bolnikov z IP/PCK. Prisotnost HPV smo določili v vseh petih tumorjih.
Rezultati. Štirje izmed petih bolnikov so imeli tumorje stadijev T3-T4; vsi bolniki so bili brez področnih zasevkov. Štiri bolnike smo zdravili z namenom ozdravitve: z operacijo, ki smo jo pri treh bolnikih dopolnili z radioterapijo. Pri bolniku z neoperabilnim tumorjem smo naredili zmanjševalno operacijo in nato bolnika radikalno obsevali. Lokalno kontrolo tumorja smo dosegli pri treh bolnikih 8, 46 in 58 mesecev po operaciji. Bolezen se je ponovila lokalno pri dveh bolnikih: po endoskopski odstranitvi T1 tumorja (recidivni tumor je bil uspešno zdravljen z dodatno operacijo) in pri bolniku z neoperabilnim tumorjem. Področne ali sistemske ponovitve bolezni nismo videli. HPV status je bil določen pri vseh petih tumorjih in trije izmed njih so bili pozitivni na HPV tip 11.
Zaključki. Pri operabilnih IP/PCK priporočamo zdravljenje z operacijo in pooperativno radioterapijo, omejeno na področje ležišča tumorja ter z uporabo doz, primerljivih s tistimi, ki jih uporabljamo pri invazivnem PCK. V primerih neo-perabilnih tumorjev priporočamo radikalno radioterapijo z dozami med 66 in 70 Gy.
Radiol Oncol 2011; 45(4): 273-278.
doi:10.2478/v10019-011-0029-0
Primerjava preživetja bolnikov z laparoskopsko in odprto radikalno resekcijo raka debelega črevesa stadija II
Fan CZ, Chu YP, Wei P, Dai H, Chen W
Izhodišča. Namen raziskave je bil primerjati preživetje bolnikov z laparoskopsko in odprto radikalno resekcijo raka debelega črevesa stadija II.
Bolniki in metode. V raziskavo je bilo vključenih 220 bolnikov z rakom debelega črevesa stadija II, ki so bili obravnavani v Chaoyang Hospital of Capital Medical University v Pekingu od januarja 2000 do decembra 2009. Laparoskopska radikalna resekcija je bila narejena pri 61 bolnikih, odprta radikalna resekcija pa pri 159 bolnikih. Primerjava preživetja bolnikov v obeh skupinah je bila narejena s pomočjo Kaplan Meierjevih krivulj preživetja in statističnega testa log rank. Rezultati. Med skupinama ni bilo statistično značilnih razlik v 3 letnem (88,5% proti 80,5%; X2=1,98, p=0,159) in 5 letnem preživetju (81,9% proti 69,2%; X2=1,98, p=0,159). Statistično značilno razliko smo ugotovili v srednjem preživetju, ki je bilo v skupini z laparoskopsko resekcijo 102,6 (95 % CI: 76,8-122,7) mesecev, v skupini z odprto radikalno resekcijo pa 90,0 (95% CI: 70,4-109,6) mesecev (X2=4.183, p=0.041). Pri bolnikih s pooperativno kemoterapijo je bilo preživetje 96,0 (95% CI: 68,6-111,4) mesecev in pri bolnikih brez kemoterapije 92,6 (95% CI: 56,8-107,2) mesecev (X2=6.389, p=0.011). Pri bolnikih starejših od 75 let je bilo preživetje v skupini z laparoskopsko resekcijo 90,0 (95% CI: 25,3-105,0) mesecev in v skupini z odprto radikalno resekcijo 83,4 (95% CI: 13,1-96,9) mesecev (X2=6.191, p=0.013).
Zaključki. Srednje preživetje bolnikov s karcinomom debelega črevesa stadija II, pri katerih je bila narejena laparoskopska radikalna resekcija, je bilo boljše kot pri tistih, pri katerih je bila narejena odprta resekcija. To zlasti velja za bolnike starejše od 75 let.
Radiol Oncol 2011; 45(4): 279-284
doi:10.2478/v10019-011-0030-7
Izražanje NF-kB p65, fosfori li ranega na serinu-563 pri raku danke brez ali z preoperativnim obsevanjem
Lewander A, Gao J, Adell G, Zhang H, Sun XF
Izhodišča. Namen študije je bil preučiti izražanje NF-kB p65, fosforiliranega na serinu-536 (fosfor-Ser-p65) pri raku danke. Zanimalo nas je njegovo izražanja pri bolnikih z ali brez preoparativnega obsevanja (RT) ter povezava s klinič-nopatološkimi značilnostmi bolnikov in z njihovimi biološkimi dejavniki.
Bolniki in metode. Fosfor-Ser-p65 smo določali pri 141 tumorjih danke, pri 149 normalnih mukozah in pri 48 metastazah v limfnih vozlih bolnikov z rakom danke, ki so bili vključeni v švedsko klnično študijo.
Rezultati. Izražanje fosfor-Ser-p65 je bilo statistično značilno povečano v tumorjih (p<0,0001 za obe skupini, ki sta ali nista prejeli RT) v primerjavi z nomalno mukozo. Izražanje pa ni bilo še bolj povečano v metastazah, v primerjavi s primarnimi tumorji (p>0,05). Izražanje fosfor-Ser-p65 je v precejšnji meri koreliralo z izražanjem označevalca za tumorki endotelij 1 (TEM1, p=0,02), FXYD-3 (p=0,001), fosfatazo za regeneracijo jeter (PRL, p=0,02), p73 (p=0,048) in s proteinom povezanim z meningeomom (MAC30, p=0,05) v skupini, ki je bila obsevana, vendar ne pri skupini, ki ni bila obsevana (p>0,05).
Zaključki. Povečano izražanje fosfor-Ser-p65 je lahko udeleženo pri razvoju raka danke. Po radioterapiji tumorjev izražanje fosfor-Ser-p65 korelira z biološkimi dejavniki, ki so povezani z bolj malignimi značilnostmi tumorjev. Vendar izražanje fosfor-Ser-p65 ni bilo direktno povezano z odgovorom na radioterapijo glede na ponovitev bolezni in preživetje.
Radiol Oncol 2011; 45(4): 285-291.
doi:10.2478/v10019-011-0039-y
učinkovitost sistemskega zdravljenja prvega reda pri bolnikih z razsejanim rakom debelega črevesa in danke v povezavi z BRAF in različnimi KRAS mutacijami
Reberšek M, Boc M, Cerkovnik P, Benedik J, Hlebanja Z, Volk N, Novaković S, Ocvirk J
Izhodišča. Mutacije v kodonu 12 in 13 gena KRAS so napovedni dejavniki za odgovor na zdravljenje z anti-EGFR monoklonalnimi protitelesi pri bolnikih z razsejanim rakom debelega črevesa in danke. Vsi bolniki z nemutiranim tipom gena KRAS žal ne odgovorijo na zdravljenje s temi tarčnimi zdravili. Vzrok so še drugi mehanizmi odpornosti na zdravljenje. Ti nastanejo z aktivacijo mutacij v predelu drugih EGFR signalnih poti in napovedujejo odgovor na specifično sistemsko zdravljenje.
Bolniki in metode. V retrospektivni analizi smo ovrednotili objektivni odgovor na zdravljenje, čas do napredovanja bolezni in celokupno preživetje glede na status KRAS v kodonih 12 in 13 ter status BRAF. Analizirali smo podatke pri 1 76 bolnikih z razsejanim rakom debelega črevesa in danke, ki smo jih zdravili s prvim redom sistemske kemoterapije v kombinaciji z monoklonalnimi protitelesi.
Rezultati. KRAS mutacije smo ugotovili pri 63 bolnikih (35,8%), pri 53 bolnikih v kodonu 12 (30,1%), pri 10 bolnikih pa v kodonu 13 (5,7%). BRAF V600E mutacijo je imelo 13 od 176 bolnikov (7,4%). V podskupini bolnikov z nemutiranim tipom KRAS so tudi bolniki z nemutiranim tipom BRAF odgovorili na zdravljenje v 54,0%, bolniki z mutiranim BRAF pa v 38,5% (p = 0,378). Srednje preživetje bolnikov z nemutiranim tipom BRAF je bilo 107,4 mesecev, bolnikov z mutiranim BRAF pa 45 mesecev (p = 0,042). Čas do napredovanja bolezni pri bolnikov z nemutiranim tipom BRAF je bil daljši, 16 mesecev, v primerjavi z 12 mesecev pri bolnikih z mutiranim BRAF (p = 0,558).
Zaključki. Bolniki z BRAF V600E mutacijo imajo značilno krajše preživetje kot bolniki z nemutiranim tipom BRAF gena, njihova bolezen hitreje napreduje med sistemskim zdravljenjem. Dokončno vlogo BRAF V600E mutacije kot progno-stični in napovedni dejavnik za odgovor na sistemsko zdravljenje s kemoterapijo in monoklonalnimi protitelesi bodo opredelili rezultati prospektivnih randomiziranih kliničnih raziskav.
Radiol Oncol 2011; 45(4): 292-295.
doi:10.2478/v10019-011-0022-7
Hepatocelularni rak s podkožnim zasevkom v predelu skalpa
Tezcan Y, Koc M
Izhodišča. Največ podkožnih zasevkov hepatocelularnega raka (HCC) nastane zaradi prenosa rakastih celic ob igelni punkciji ali ob operaciji. O nejatrogenih podkožnih zasevkih hepatocelularnega raka redko poročajo. Prikaz primera. Opisujemo primer 70-letnega bolnika s tumorsko maso v podkožju levega zatilja. Bolnika smo radikalno operirali, v kirurških robovih ni bilo rakastih celic. Histopatološki pregled pa je pokazal zasevke HCC. Pregled z računalniško tomografijo ni pokazal znakov primarnega tumorja ali zasevkov v trebuhu, zato se nismo odločili za dodatno zdravljenje. Pet mesecev kasneje smo z magnetno resonančno preiskavo (MRI) ugotovili ponovitev bolezni. Tumorska masa je bila velika 6,0 x 5,5 cm in je ležala v podkožju levega posteriornega parietalnega dela. Bolnika smo ponovno operirali in histopatološki pregled je ponovno pokazal zasevke HCC. Operacija ni bila radikalna in bolnika smo pooperativno obsevali. Po 9 mesecih MRI ni pokazal morebitne ponovitve bolezni v predelu glave. Zaključki. Ob ugotovljenih podkožnih zasevkih moramo v diferencialni diagnozi pomisliti tudi na HCC, čeprav bolniki nima simptomov jetrnega obolenja.
Radiol Oncol 2011; 45(?): 296-299.
doi:10.2478/v10019-011-0031-6
ugotavljanje genotipov BRCA1, BRCA2, p53, CDKN2A, MLH1 in MSH2 pri moškem s sekundarnim rakom dojke
Vodušek AL, Novaković S, Stegel V, Jereb B
Izhodišča. Nekatere tumor supresorske gene (BRCA2) in gene za popravljanje neujemanja (MSH2, MLH1) povezujejo s povišanim tveganjem za nastanek raka dojke pri moških.
Prikaz primera. Bolnik je zbolel za sekundarnim rakom dojke po zdravljenju Hodgkinove bolezni v otroštvu. Iz bolnikove krvi smo izolirali DNA ter v genih BRCA1, BRCA2, p53, CDN2A, MLH1 in MSH2 iskali mutacije, polimorfizme in variante. Našli smo tri variante v genih za popravljenje neujemanja ter navadne polimorfizme.
Zaključki. Nukleotidne variante c.2006-6T>C in p.G322D v genu MSH2 bi lahko povezovali s povišanim tveganjem za nastanek raka dojke pri moških.
Radiol Oncol 2011; 45(4): 300-303.
doi: 10.2478/v10019-011-0035-2
ugotavljanje periampularne lokalizirane pankreatične intraepitelijske neoplazije-3 (PanIN-3) z MR holangiografijo, ojačeno s kontrastom
Algin O, Ozmen E, Ersoy P E, Karaoglanoglu M
Izhodišča. Zgodnja določitev premaligne lezije pankreasa prepreči obsežne kirurške posege in tako zmanjša obolelost in smrtnost. Pankreatična intraepitelijska neoplazija-3 (PanIN-3) je preinvazivna oblika adenokarcinoma (karcinom in situ). Pankreatične intraepitelijske neoplazije še nimajo mesta v radiološki literaturi, vendar pa je potrebno v diferencialni diagnostiki pankreatičnih lezij upoštevati tudi te premaligne lezije.
Prikaz primera. Predstavljamo bolnika, ki smo mu postavili začasno diagnozo kroničnega holecistitisa z žolčnimi kamni in periampularno pankreatično cisto. Preiskavi smo naredili z nekontrastno MR holangiografijo in z MR holangiografijo, ojačano s kontrastom. Kasnejši patološki pregled žolčnika in pankreatične ciste je pokazal adenokarcinom žolčnika in PanIN-3.
Zaključki. Cistične lezije pankreasa s tankimi septi, ki so ojačena z dodajanjem kontrastnega sredstva, lahko predstavljajo PanIN-3. Pri bolnikih s cističnimi lezijami pankreasa v periampularni regiji je MR holangiografija, ojačena s kontrastom, skupaj z nekontrastno metodo lahko koristna pri odkrivanju cističnih lezij pankreasa kot tud druge abdo-minalne patologije.
Radiol Oncol 2011; 45(4): 304-309.
doi:10.2478/v10019-011-0036-1
In vivo nadomestitev kosti pri meningeomu, ki je vraščal in uničeval lobanjsko kost
Velnar T, Pregelj R, Limbaeck-Stokin C
Izhodišča. Meningeomi so pogosti možganski tumorji. Nekateri z lokalnim vraščanjem uničujejo okoliško kostno tkivo, ki ga je med operacijo skupaj s tumorjem potrebno odstraniti. Rekonstrukcija po operaciji je lahko zato znatno otež-kočena.
Prikaz primera. Opisujemo bolnika z benignim meningeomom konveksitete, ki je vraščal skozi lobanjsko kost v podkožje. Tumor smo skupaj z infiltriranim tkivom v celoti odstranili. Vrzeli okoliškega tkiva, ki so nastale po odstranitvi tumorja, smo že med operacijo oskrbeli z rekonstrukcijo kosti iz umetnih materialov.
Zaključki. Po operaciji benignega meningeoma moramo včasih narediti rekonstrukcijo lobanjske kosti. Pri tem lahko uporabimo kost iz umetnih materialov, ki nam omogoča sorazmerno hitro medoperativno rekonstrukcijo z odlično zaščito možganov in dobrim kozmetskim učinkom.
Radiol Oncol 2011; 45(4): 310-314.
doi:10.2478/v10019-011-0020-9
Dozimetrično preverjanje kompenziranih žarkov z radiografskim filmom
Jurković S, Žauhar G, Faj D, Smilović Radojčić Đ, Švabić M, Kasabašić M, Diklić A
Izhodišča. Modulacija zunanjih fotonskih žarkov, kadar teleterapiji sledi brahiterapija, je uveljavljena tehnika. Pomaga nam, da bolnika obsevamo z želeno dozo. Kompenzator modulira centralni del žarka; pod najdebelejšim delom kompenzatorja pa je doza odvisna predvsem od sipanih nizko energijskih fotonov. Za preverjanje teh žarkov potrebujemo dvo-dimenzionalni detektor z dobro prostorsko ločljivostjo. Proučili smo vpliv različnih tipov detektorjev na merjeno modulirano dozo.
Materiali in metode. Dozimetrično preverjanje smo opravili z X-Omat V, Eastman Kodak radiografskim filmom na različnih globinah v fantomu trde vode. Filmske meritve smo primerjali z meritvami opravljenimi z ionizacijsko celico. Da bi razložili merjene rezultate, smo fotonske žarke modelirali z algoritmom EGSnrc Monte Carlo.
Rezultati. Z algoritmom Monte Carlo izračunan odziv filma pod najdebelejšim delom kompenzatorja je bil več kot 15% prevelik, kar smo potrdili z drugimi meritvami. Velikost prevelikega odziva filma lahko povezujemo s spremembami v spektru fotonskih energij žarka.
Zaključki. Čeprav radiografski film ni vedno najprimernejši dozimeter, ga lahko uporabimo za dozimetrijo kompenziranih visoko energijskih fotonskih žarkov, pri tem pa moramo biti pozorni na volumne, kjer je fotonski spekter močno energijsko oslabljen.
Authors Index 2011
Adell G: 4/279-284 Adim S: 2/116-122 Algin O: 4/300-303 Anderluh F: 3/209-212 Aoki I: 4/248-258 Ardebili S: 2/102-115 Ari F: 2/116-122 Aurer I: 1/27-30
Bakalova R: 4/248-258 Bayol U: 1/53-58 Benedik J: 4/285-291 Bešenski N: 2/147-158 Boc M: 4/285-291 Borštnar S: 1/46-52 Bregant P: 1/64-67 But-Hadžić J: 4/267-272
Campos B: 2/102-115 Cebulski W: 1/59-63 Cerkovnik P: 4/285-291 Chen Weicai: 2/123-128; Chen Wenming: 4/273-278 Chryssou EG: 1/22-26 Chu YP: 4/273-278 Cör A: 1/30-39 Coskun A: 2/129-131 Cui MH: 3/196-203
Čemazar M: 1/30-39 Čirić E: 3/209-212
Dai H: 4/273-278
de Bree E: 1/22-26
de Denaro M: 1/64-67; 2/143-146
Diklić A: 4/310-314
Ding W: 2/82-90
Dobrenić M: 3/189-195
Dogan M: 1/53-58 Drmota S: 2/102-115
Erkan N: 1/53-58; 2/129-131 Evrensel T: 2/116-122
Faj D: 4/310-314 Fan CZ: 4/273-278 Filipič M: 4/227-247 Fornasier MR: 2/143-146 Fu N: 2/82-90
Gale N: 4/267-272 Gao J: 4/279-284 Gao LF: 3/196-203; Ghavamnasiri MR: 3/184-188 Giannikaki E: 1/22-26 Glavina K: 2/147-158 Gole B: 2/102-115 Gornicka B: 1/59-63 Grazio Frkovič S: 1/46-52 Grošev D: 3/189-195 Guan H: 2/123-128 Gundogdu H: 4/300-303
Hadjidekov G: 4/248-258
Hadjidekova S: 4/248-258
He J: 2/123-128
Herold - Mende C: 2/102-115
Hlebanja Z: 4/285-291
Hodolič M: 1/17-21
Horvat M: 2/75-81
Hrabak Paar M: 1/27-30
Hrovatič Podvršnik N: 3/209-212
Huić D: 3/189-195
Janeš D: 4/259-266 Jangjoo A: 3/184-188
Jenko K: 4/267-272 Jereb B: 4/296-299 Jevnikar Z: 4/259-266 Jurković S: 4/310-314
Kakhki VRD: 3/184-188 Kandušer M: 3/204-208 Karaagac E: 2/116-122 Karantanas A: 1/22-26 Karaoglanoglu M: 4/300-303 Kasabašić M: 4/310-314 Kikelj D: 4/259-266 Kiraly R: 1/68-74 Klarić - Čustović R: 2/147-158 Koc M: 3/213-219; 4/292-295 Kocjančič B: 4/267-272 Kos J: 4/259-266 Krasnodebski IW: 1/59-63 Kristić S: 3/174-179
Lah Turnšek T: 2/102-115 Lavrenčak J: 1/40-45 Letonja M: 3/180-183 Lewander A: 4/279-284 Liang H: 2/91-96 Limbaeck-Stokin C: 4/304-309 Lincender L: 3/174-179 Lu G: 2/82-90
Malhotra H: 2/132-142 Matos E: 1/46-52 Meng Q: 2/82-90 Miklavčič D: 3/204-208 Mirković B: 4/259-266 Momennezhad M: 3/184-188 Mowlavi A: 2/143-146 Možina B: 1/46-52; 3/209-212
Novak S: 4/227-247	Skočaj M: 4/227-247	Weisz C: 1/68-74
Novaković S: 4/285-291;	Slodkowski M: 1/59-63	Wronski M: 1/59-63
4/296-299	Smilović Radojčić Đ: 4/310-314	Wu H: 2/123-128
	Stegel V: 4/296-299	Wu V: 2/132-142
Oblak I: 3/209-212	Steinman J: 2/132-142	
Ocvirk J: 3/209-212; 4/285-291	Strojan P: 1/40-45; 4/267-272	Xu L: 2/91-96
Oral A: 2/116-122	Strojan Fležar M: 1/40-45	Xu M: 2/91-96
Ovčariček T: 1/46-52	Sun XF: 4/279-284	Yadav P: 3/220-226
Ozmen E: 4/300-303		Yang B: 2/82-90
	Šantl Letonja M: 3/180-183	Yilmaz C: 1/53-58
Paliwal B: 3/220-226	Šimunić S: 2/147-158	
Pavlin D: 1/30-39	Štabuc B: 2/75-81	Zajc I: 2/102-115
Pavliša Goran: 2/97-101	Štern-Padovan R: 1/27-30	Zakavi SR: 3/184-188
Pavliša Gordana: 2/97-101	Švabić M: 4/310-314	Zarand P: 1/68-74
Perdikakis E: 1/22-26		Zhang H: 4/279-284
Pesznyak C: 1/68-74	Tezcan Y: 3/213-219; 4/292-295	Zhao F: 3/196-203;
Petković J: 4/227-247	Tokullugil A: 2/116-122	Zhao SH: 3/196-203
Petrović R: 3/189-195	Tonchev Z: 4/248-258	Zhao XJ: 3/196-203
Podgorsak M: 2/132-142	Tozon N: 1/30-39	Zheng JY: 3/196-203
Pogačnik A: 1/30-39	Tran T: 2/132-142	Zhou R: 2/123-128
Polgar I: 1/68-74		Ziarkiewicz-Wroblewska B
Poljak M: 4/267-272	Ulukaya E: 2/116-122	1/59-63
Prasad D: 2/132-142		Zidar N: 4/259-266
Pregel R: 4/304-309	Valatsou C: 1/22-26	Zuvić M: 3/189-195
Prijič S: 1/1-16	Vardar E: 1/53-58	Žauhar G: 4/310-314
	Vegar-Zubović S: 3/174-179	Žganec M: 1/40-45
Qiu S: 2/123-128	Velenik V: 3/209-212	
	Velnar T: 4/304-309	
Radoš M: 2/97-101	Vitral GSF: 3/166-173	
Ramasubramanian V: 3/220-226	Vodušek A: 4/296-299	
Raposo NRB: 3/166-173	Volk N: 4/285-291	
Reberšek Matej: 3/204-208	Vranič A: 3/159-165	
Reberšek Martina: 4/285-291		
Rojnik M: 4/259-266	Wang M: 2/123-128	
	Wang Xianming: 2/123-128	
Sadeghi R: 3/184-188	Wang Xuhui: 2/91-96	
Samardzić T: 3/189-195	Wang Z: 2/123-128	
Serša G: 1/1-16; 1/30-39	Wei P: 4/273-278	
Subject Index 2011
131I radionuclide: 2/143-146
18F-choline PET/CT: 1/17-21
adaptive planning: 3/220-226
adenoma: 2/129-131
anaemia: 2/129-131
apoptosis: 2/116-122; 3/196-203
apparent diffusion coefficient: 2/97-101
applications, safety: 4/227-247
backward wave: 2/82-90
biopsy: 1/22-26
body contour: 3/184-188
body outlining: 3/184-188
bone morphogenetic protein 4: 4/259-266
bone reconstruction: 4/304-309
BRAF: 4/285-291
brain: 4/304-309
breast cancer: 2/116-122; 3/166-173; 3/184-188; 4/296-299
Brunner's gland: 2/129-131
cancer: 4/259-266
cancer therapy: 1/1-16
carcinoma: 4/300-303
cathepsin H: 4/259-266
CD133/prominin1: 2/102-115
central venous catheterization: 1/27-30
cerebrospinal leak: 2/91-96
chemotherapy: 2/75-81; 2/116122; 4/273-278
children: 4/248-258
colorectal cancer: 2/75-81
compensators: 4/310-314
computed tomography: 3/180-183
computed tomography dose index: 1/64-67
cone-beam computerized tomography, kV CBCT: 3/220-226
conformal radiotherapy: 3/213-219
Croatian Medical Association, Croatian Society of Radiology: 2/147-158
CT: 2/91-96
CT to density table: 3/220-226
CT, MR imaging, diffusion weighted imaging: 1/22-26
cutaneous metastases: 4/292-295
cysteine cathepsins: 2/102-115
delivery systems: 1/1-16
desmoid tumour: 1/59-63
diagnosis: 1/17-21; 2/91-96
differential diagnosis: 1/59-63
differentiated thyroid cancer: 3/189-195
dose profile: 1/64-67
dosimetry: 2/132-142; 4/310-314
echocardiography: 3/180-183
effective circulating blood volume: 2/82-90
elastic recoil: 2/82-90
electrodes: 3/204-208
electrogene therapy: 1/30-39
electron density phantom: 3/220-226
electronic portal imaging device: 1/68-74
electroporation: 1/30-39
electrotransfection: 1/30-39
ependymoma: 2/97-101
fluorescence-guided resection: 3/159-165
follow-up: 1/17-21
functional analysis: 4/248-258
Gafchromic film: 1/64-67
gamma knife: 2/132-142
gastrointestinal stromal tumour: 1/59-63
gene electrotransfer: 3/204-208
gene screening: 4/296-299
GIST: 1/59-63
glioblastoma: 2/102-115
glioblastoma multiforme: 3/213-219
glioma stem cells: 2/102-115
head and neck cancer: 1/40-45
healthy blood donors: 3/209-212
helical computed tomography: 1/27-30
hemangiopericytoma: 1/22-26
hepatocellular carcinoma: 4/292-295
history: 2/147-158
human cell lines: 4/259-266
human papillomavirus infection: 4/267-272
IL-12: 1/30-39
image cytometry: 1/40-45
image-guided surgery: 3/159-165
immunohistochemistry: 4/279-284
INF-y: 1/30-39
intradermal injection: 3/184-188 invasion: 2/102-115; 4/304-309 inverted papilloma: 4/267-272
KRAS: 4/285-291
laparoscopy: 4/273-278
left ventricular
pseudoaneurysm: 3/180-183
low iodine diet: 3/189-195
lymphoma, non-Hodgkin: 1/27-30
lymphoscintigraphy: 3/184-188 M30: 2/116-122
magnetic nanoparticles: 1/1-16
magnetic resonance
cholangiography: 4/300-303
magnetic resonance imaging: 4/300-303
magnetic targeting: 1/1-16
magnetofection: 1/1-16
male: 4/296-299
malignnt glioma: 3/159-165
mast cell tumors: 1/30-39
MCNPX code: 2/143-146
meningioma: 4/304-309
mesenteric fibromatosis: 1/59-63
metastatic colorectal cancer: 4/285-291
metastatic tumours: 1/53-58
microsatellite instability: 2/75-81
MR urography: 3/174-179; 4/248-258
MRI: 2/91-96
multidetector computed tomography: 4/300-303
multiple basilar skull fracture: 2/91-96
multislice CT: 1/64-67
nanoparticles: 4/227-247 nanotechnology: 1/1-16 neural stem cells: 2/102-115 neuronavigation: 3/159-165 NF-kB, serine-536: 4/279-284 nuclear features: 1/40-45
oncology: 1/1-16 outcome: 4/267-272 ovarian cancer: 3/196-203
paediatrics: 3/174-179
pancreatic cysts: 4/300-303
pancreatic intraepithelial neoplasia: 4/300-303
pelvis: 1/22-26
pencil chamber: 1/64-67
perfexion: 2/132-142
pilocytic astrocytoma: 2/97-101
pipette tip, CHO cell line: 3/204-208
portal film: 1/68-74
principal wave: 2/82-90
prognosis: 1/40-45; 2/123-128; 4/273-278; 4/279-284
prognostic factors: 1/46-52; 3/213-219; 4/285-291
prostate carcinoma: 1/17-21
proteolytic enzymes: 4/259-266
quality control: 1/68-74
radiation therapy: 4/310-314; 4/292-295
radioiodine: 3/189-195
radiological contrast: 3/166-173
radiology: 2/147-158
radiotherapy: 1/22-26; 4/267-272; 4/279-284
rectal cancer: 3/209-212
rectal cancer: 4/279-284
recurrence: 4/279-284
resistance: 2/82-90
response to chemotherapy: 2/116-122
RNA interference: 3/196-203
ROLL technique: 3/166-173
scattered photons: 3/184-188 secondary malignancies: 1/53-58 secondary neoplasm: 4/296-299 shutter effect: 2/132-142
SKOV3 cells: 3/196-203
squamous cell carcinoma: 4/267-272
stage II colon cancer: 4/273-278
staging: 1/17-21
stat3: 3/196-203
stereotactic radiosurgery: 2/132-142
surgery: 3/159-165; 3/166-173
targeted chemotherapy: 2/123-128
temozolomide: 3/213-219
thyroid gland: 2/143-146
thyroid malignancy: 1/53-58
TIMP-1: 3/209-212
titanium dioxide: 4/227-247
total energy deposition: 2/143-146
toxicity: 4/227-247
transcranial magnetic stimulation: 3/159-165
treatment outcome: 1/46-52
triple negative breast cancer: 1/46-52
triple-negtive breast cancer: 2/123-128
urinary tract: 3/174-179; 4/248-258
urine iodine concentration: 3/189-195
velocity tracing: 2/82-90
FUNDACIJA DR. JXHQLEWA
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.JXHOLEWA
Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education - a report for the second half of 2011
The Dr. J. Cholewa Foundation for Cancer Research and Education is a non-profit, non-government and non-political association of individuals, institutions and organisations, its main aim is to support novel initiatives and forward thinking in cancer education, research and prevention. One of its most important aims is to facilitate the flow of relevant information and knowledge from major oncology centres in the world to medical professionals, other experts and general public in Slovenia.
The Foundation thus distributes different types of grants and support to applicants from Slovenia wishing to extend existing or gain new knowledge in oncology. It helps professional and other associations in Slovenia to organise scientific and other meetings of specific interest in different fields of advanced cancer research and education. One of its most important activities is to support the publication of various cancer information and cancer awareness brochures and booklets for the general public. The Dr. J. Cholewa Foundation for Cancer Research and Education is especially proud to continue its support for the publication of "Radiology and Oncology", an important international medical scientific journal that is edited, published and printed in Ljubljana, Slovenia. As its name suggests, "Radiology and Oncology" is a journal that publishes scientific articles, reviews, case reports, short reports and letters that deal with problems in radiology, radiophysics, experimental and clinical oncology, supportive therapy, prevention and early diagnostics of different types of cancer. It is an open access journal, available free of charge on its website, with an important Science Citation Index impact factor. The Dr. J. Cholewa Foundation for Cancer Research and Education plans to add a number of new activities to the now well established projects in course in the near future, as the need for changes is becoming ever more necessary. The need for up to date prevention and early detection measures for certain types of cancers has grown substantially in the last few years in Slovenia. An increase in a number of incidence and prevalence rates of various types of cancer has been observed in the last two or three decades in Slovenia and these changes warrant the identification of new priorities and new goals in the national setting. Hopefully, the Foundation may be able to respond and to support at least some of the proposals by the experts active in these new and challenging aspects of oncology in Slovenia. The Foundation has continued with its activities throughout 2011 with the aim to spread the latest knowledge about cancer and related problems to specialists and other professionals in Slovenia, with important part of its activities being the education and information of the lay public. These activities may already in the near future lead to greater practical application of the latest methods and protocols in the treatment of cancer in Slovenia.
Borut Štabuc, MD, PhD
Andrej Plesničar, MD
Tomaž Benulič, MD

THE
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ndividualne ženske
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CETUKSI MAB
Erbitux 5 mg/ml raztopina za infundiranje (Skrajšan povzetek glavnih značilnosti zdravila)
Sestava: En ml raztopine za infundiranje vsebuje 5 mg cetuximaba in pomožne snovi. Cetuksimab je himerno monoklonsko IgG1 protitelo. Terapevtske indikacije: Zdravilo Erbitux je indicirano za zdravljenje bolnikov z metastatskim kolorektalnim rakom z ekspresijo receptorjev EGFRin 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 indicirano za zdravljenje bolnikov z rakom skvamoznih celic glave in vratu v kombinaciji z radioterapijo za lokalno napredovalo bolezen in v kombinaciji s kemoterapijo na osnovi platine za ponavljajočo se in/ali metastatsko bolezen. Odmerjanje in način uporabe: Zdravilo Erbitux pri vseh indikacijah infundirajte enkrat na teden. Pred prvo infuzijo mora bolnik prejeti premedikacijo z antihistaminikom in kortikosteroidom. 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 NCI-CTC), morate prekiniti terapijo s cetuksimabom. Z zdravljenjem smete nadaljevati le, če se je reakcija izboljšala do 2. stopnje. Zaradi možnosti pojava znižanja nivoja magnezija v serumu se pred in periodično med zdravljenjem priporoča določanje koncentracije elektrolitov. Če se pojavi sum na nevtropenijo, je potrebno bolnika skrbno nadzorovati. Potrebno je upoštevati kardiovaskularno stanje bolnika in sočasno dajanje kardiotoksičnih učinkovin kot so fluoropirimidini. Interakcije: farmakokinetične značilnosti cetuksimaba ostanejo nespremenjene po sočasni uporabi enkratnega odmerka irinotekana, tudi farmakokinetika irinotekana je nespremenjena pri sočasni uporabi cetuksimaba. Pri kombinaciji s fluoropirimidini se je povečala pogostnost srčne ishemije, vključno z miokardnim infarktom in kongestivno srčno odpovedjo ter pogostnost sindroma dlani in stopal. V kombinaciji s kemoterapijo na osnovi platine se lahko poveča pogostnost hude levkopenije ali hude nevtropenije. Neželeni učinki: Zelo pogosti (> 1/10): hipomagneziemija, povečanje ravni jetrnih encimov, kožne reakcije, blage ali zmerne reakcije povezane z infundiranjem, blag do zmeren mukozitis. Pogosti (> 1/100, < 1/10): dehidracija, hipokalciemija, anoreksija, glavobol, konjunktivitis, driska, navzeja, bruhanje, hude reakcije povezane z infundiranjem, utrujenost. Posebna navodila za shranjevanje: Shranjujte v hladilniku (2 °C - 8 °C). Pakiranje: 1 viala z 20 ml ali 100 ml raztopine. Način in režim izdaje: H. Imetnik dovoljenja za promet: Merck KGaA, 64271 Darmstadt, Nemčija. Datum zadnje revizije besedila: november 2010.
Pred predpisovanjem zdravila natančno preberite celoten Povzetek glavnih značilnosti zdravila. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila (EMEA) http://www.emea.europa.eu. Dodatne informacije so na voljo pri: Merck d.o.o., Dunajska cesta 119, 1000 Ljubljana, tel.: 01 560 3810, faks: 01 560 3831, el. pošta: info@merck.si
ta www.merckserono.net
Ki
J www.Erbitux-international.com
iMerckSerono
Merck Serono is a division of Merck.
.MERCK
I
POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA
Ime zdravila: Temodal 20 mg, 100 mg, 140mg, 180 mg, 250 mg, Temodal 2,5 mg/ml prašek za raztopino za infundiranje Kakovostna in količinska sestava: Vsaka kapsula zdravila Temodal vsebuje 20 mg, 100 mg, 140 mg, 180 mg ali 250 mg temozolomida. Ena vlala vsebuje 100 mg temozolomida Po rekonstitucijl 1 ml raztopine za infundiranje vsebuje 2,5 mg temozolomida. Pomožna snov: Ena vlala vsebuje 2,4 mmol natrija. Terapevtske indikacije: Zdravilo Temodal 2,5 mg/ml je Indlclrano za zdravljenje: odraslih bolnikov z novo diagnosticlranim multiformnim glioblastomom, sočasno z radioterapijo (RT) in pozneje kot monoterapija in otrok starih 3 leta In več, mladostnikov in odraslih bolnikov z malignimi glloml, npr. multlformnimi glloblastoml ali anaplastlčnlmi astrocrtomi ki se po standardnem zdravljenju ponovijo ali napredujejo. Odmerjanje in način uporabe: Zdravilo Temodal 2,5 mg/ml smejo predpisati le zdravniki, ki Imajo izkušnje z zdravljenjem možganskih tumoijev. Odrasli bolniki z novo diagnosticiianim multiformnim glioblastomom Zdravilo Temodal 2,5 mg/ml se uporablja v kombinaciji z žariščno radioterapijo (faza sočasne terapije), temu pa sledi do 6 ciklov monoterapije (monoterapijska faza) z temozolomidom (TMZ). Faza sočasne templjeTUl naj bolnik jemlje v odmerku 75 mg/m2 na dan 42 dni, sočasno z žariščno radioterapijo (60 Gy, danih v 30 delnih odmerkih). Zmanjševanje odmerka ni priporočeno, vendar se boste vsak teden odločili o morebitni odložitvi jemanja TMZ ali njegovi ukinitvi na podlagi kriterijev hematološke in nehematološke toksičnosti. TMZ lahko bolnik jemlje ves čas 42-dnevnega obdobja sočasne terapije (do 49 dni), če so izpolnjeni vsi od naslednjih pogojev:
•	absolutno število nevtrofilcev (ANC - Absolute Neutrophil Count) s 1,5 x 109/I;
•	število trombocitov 2100 x 109/I;
•	skupna merila toksičnosti (SMT) za nehematološko toksičnost s 1. stopnje (z izjemo alopecije, navzee in bruhanja). Med zdravljenjem morate pri bolniku enkrat na teden pregledati celotno krvno sliko.
Faza monoterapije Štiri tedne po zaključku faze sočasnega zdravljenja s TMZ in RT naj bolnik jemlje TMZ do 6 ciklov monoterapije. V1. ciklu (monoterapije) 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 SMT za nehematološko toksičnost za 1. cikel stopnje s 2 (z izjemo alopecije, slabosti in bruhanja), absolutio število nevtrofilcev (ANC) :> 1,5 x 109/I in število trombocitov s 100 x 109/I. Če odmerka niste povečali v 2. ciklu, ga v naslednjih ciklih ne smete povečevati. Ko pa odmerek enkrat povečate, naj ostane na ravni 200 mg/m2 na dan v prvih 5 dneh vsakega naslednjega cikla, razen če nastopi toksičnost. Zmanjšanje odmerka in ukinitev zdravila med fazo monoterapije opravite, kot je opisano v preglednicah 2 in 3. Med zdravljenjem morate 22. dan pregledati celotno krvno sliko (21 dni po prvem odmerku TMZ). Odrasli in pediatrični bolniki, stari 3 leta ali več, s ponavljajočim se ali napredujočim malignim gliomom: Posamezen cikel zdravljenja traja 28 dni. Bolniki, ki še niso bili zdravljeni s kemoterapijo, naj jemljejo TMZ 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 ciklu pa se poveča na 200 mg/m2 enkrat na dan 5 dni, če ni bilo hematoloških toksičnih učinkov. Kontraindikacije: Preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. Preobčutljivost za dakarbazin PTIC). Posebna opozorila in previdnostni ukrepi: Pljučnica, ki jo povzroča Pneumocystis carinii Pilotno preskušanje podaljšane 42-dnevne sheme zdravljenja je pokazalo, da pri bolnikih, ki so sočasno prejemali TMZ in RT, obstaja še posebej veliko tveganje za nastanek pljučnice zaradi okužbe s Pneumocystis carinii (PCP). Malignosti Zelo redko so poročali tudi o primerih mielodisplastičnega sindroma in sekundarnih malignostih, vključno z mieloidno levkemijo. Antiemetično zdravljenje Navzea in bruhanje sta pogosto povezana z zdravljenjem s TMZ. Antiemetično zdravljenje se lahko da pred uporabo TMZ ali po njej. Odrasli bolniki z novo diagnosticiranim multiformnim glioblastomom Antiemetična profilaksaje priporočljiva pred začetnim odmerkom sočasne faze in je močno priporočljiva med fazo monoterapije. Ponavljajoči se ali napredujoči maligni gliom Pri bolnikih, ki so močno bruhali (stopnja 3 ali 4) v prejšnjih ciklih zdravljenja, je potrebno antiemetično zdravljenje. Laboratorijske vrednosti Pred jemanjem zdravila morata biti izpolnjena naslednja pogoja za laboratorijske izvide: ANC 21,5 x 109/1 in število trombocitov :>100 x 109/I. 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 > 1,5 x 109/I in število trombocitov > 100 x 109/I. Če med katerimkoli ciklom ANC pade na < 1,0 x 109/I ali število trombocitov na < 50 x 109/I, morate odmerek zdravila v naslednjem ciklu zmanjšati za eno stopnjo (glejte poglavje 4.2). Stopnje odmerka so 100 mg/m2,150 mg/m2 in 200 mg/m2. Najmanjši priporočeni odmerek je 100 mg/m2. Pediatrična uporaba Kliničnih izkušenj z uporabo TMZ pri otrocih, mlajših od 3 let, ni. Izkušnje z uporabo tega zdravila pri starejših otrocih in mladostnikih so zelo omejene. Starejši bolniki (stari > 70 lei) Videti je, daje pri starejših bolnikih tveganje za nevtropenijo ali trombocitopenijo večje, kot pri mlajših. Zato je pri uporabi zdravila TMZ pri starejših bolnikih potrebna posebna previdnost.
Moški bolniki Moškim, ki se zdravijo s TMZ je treba svetovati, naj ne zaplodijo otroka še šest mesecev po prejetem zadnjem odmerku in naj se pred zdravljenjem posvetujejo o možnostih za shranitev zmiznjene sperme. NatrijJo zdravilo vsebuje 2,4 mmol natrija na vialo. To je treba upoštevati pri bolnikih na nadzorovani dieti z malo natrija. Medsebojno delovanje z dragimi zdravili in druge oblike interakcij: Študije medsebojnega delovanja so izvedli le pri odraslih. V ločeni študiji 1. faze, sočasna uporaba TMZ in ranitidina ni povzročila spremembe obsega absorpcije temozolomida ali izpostavljenosti njegovem aktivnem presnovku monometiltriazenoimidazol karboksamidu (MTIK). Analiza populacijske farmakokinetike v preskušanjih 2. faze je pokazala, da sočasna uporaba deksametazona, proklorperazina, fenitoina, karbamazepina, ondanse-trona, antagonist» receptoijev H2 ali fenobarbitala ne spremeni očistka TMZ. Sočasno jemanje z valprojsko kislino je bilo povezano z majhnim, a statistično pomembnim zmanjšanjem očistka TMZ. Študij za določitev učinka TMZ na presnovo ali izločanje drugih zdravil niso izvedli. Ker pa se TMZ ne presnavlja v jetrih in se na beljakovine veže le v majhni meri, je malo verjetno, da bi vplival na farmakokinetiko drugih zdravil.
Uporaba TMZ v kombinaciji z drugimi mielosupresivnimi učinkovinami lahko poveča verjetnost mielosupresije. Neželeni učinki: Pri bolnikih, ki se zdravijo s TMZ v kombinaciji z RT ali monoterapijo po RT zaradi novo diagnosticiranega multiformnega glioblastoma ali z monoterapijo pri bolnikih s ponavljajočim se ali napredujočim gliomom, so bili zelo pogosti neželeni učinki podobni; slabost, bruhanje, zaprtje, neješčnost, glavobol in utrujenost. Pri bolnikih z novo diagnosticiranim glioblastomom multiforme na monoterapiji so zelo pogosto poročali o konvulzijah, medtem ko je bil izpuščaj opisan zelo pogosto pri bolnikih z novo diagnosticiranim multiformnim glioblastomom, ki so prejemali TMZ sočasno z RT, ter pri tistih, ki so zdravilo prejemali v obliki monoterapije, pogosto pa pri tistih s ponavljajočim se gliomom. Pri obeh indikacijah so o večini hematoloških neželenih reakcij poročali pogosto ali zelo pogosto. Imetnik dovoljenja za promet Schering-Plough Europe, Rue de Stalle 73, Bruselj Belgija Način in režim izdaje zdravila: Zdravilo Temodal 20 mg, 100 mg, 140mg, 180 mg, 250 mg se izdaja na recept (Rp/Spec), Temodal 2,5 mg/ml prašek za raztopino za infundiranje pa je namenjeno uporabi samo v bolnišnicah (H). Datum priprave informacije: februar 2010
1.	Stiipp Ft, et.al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised III study: 5-year analysis of tfie EORTC-NCIC trial
2.	Povzetek temeljnih značilnosti zdravila Temodal
5 jakosti v 5 barvah za lažje in
natančnejše dnevno odmerjanje2
Schering-Plough CE AG Dunajska cesta 22,1000 Ljubljana tel: 01 30010 70 fax: 01 30010 80
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Resnični napredek
Pomembno izboljšanje preživetja potrjeno tudi ob daljšem spremljanju bolnikov1
liULWm
n-iiin-i^
SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA
Samo za strokovno javnost.
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 in-dicirano za prvo linijo zdravljenja bolnikov z lokalno napredovalim ali me-tastatskim nedrobnoceličnim rakom pljuč z EGFR-aktivirajočimi mutacijami. Zdravilo Tarceva je indicirano tudi za samostojno vzdrževalno zdravljenje bolnikov z lokalno napredovalim ali metastatskim nedrobnoceličnim rakom pljuč s stabilno boleznijo po 4 ciklih standardne kemoterapije na osnovi platine v prvi liniji zdravljenja. Zdravilo Tarceva je indicirano tudi 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-negati-vnimi tumorji (glede na rezultat imunohistokemije). 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. Pri bolnikih z lokalno napredovalim ali metastatskim nedrobnoceličnim rakom pljuč, ki še niso prejeli kemoterapije, je treba testiranje za določanje mutacij EGFR opraviti pred začetkom zdravljenja z zdravilom Tarceva. Zdravilo Tarceva vzamemo najmanj eno uro pred zaužitjem hrane ali dve uri po tem. Kadar je potrebno odmerek prilagoditi, ga je treba zmanjševati 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 150 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: Preobčutljivost za erlotinib ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Pri določanju bolnikovega statusa mutacij EGFR je pomembno izbrati dobro validirano in robustno metodologijo, da se izognemo lažno negativnim ali lažno pozitivnim rezultatom. 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 treba zdravljenje z zdravilom Tarceva prekiniti, dokler ni znana diagnoza. Bolnike, ki se sočasno zdravijo z erlotinibom in gemcitabinom, je treba skrbno spremljati zaradi možnosti pojava toksičnosti, podobni intersticijski bolezni pljuč. Če je ugotovljena intersticijska bolezen pljuč, zdravilo Tarceva ukinemo in uvedemo ustrezno zdravljenje. Pri približno polovici bolnikov, ki so se zdravili z zdravilom Tarceva, se je pojavila driska (vključno z zelo redkimi primeri, ki so se končali s smrtnim izidom). 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 treba zdravljenje z zdravilom Tarceva 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. Bolniki, ki prejemajo zdravilo Tarceva, imajo večje tveganje za razvoj perforacij v prebavilih, ki so jih opazili občasno (vključno z nekaterimi primeri, ki so se končali s smrtnim izidom). Pri bolnikih, ki sočasno prejemajo zdravila, ki zavirajo angiogene-zo, kortikosteroide, nesteroidna protivnetna zdravila (NSAID) in/ali kemoterapijo na osnovi taksanov, ali so v preteklosti imeli peptični ulkus ali diver-tikularno bolezen, je tveganje večje. Če pride do tega, je treba zdravljenje z zdravilom Tarceva dokončno ukiniti. Poročali so o primerih kožnih bolezni z mehurji in luščenjem kože, vključno z zelo redkimi primeri, ki so nakazovali na Stevens-Johnsonov sindrom/toksično epidermalno nekrolizo in so bili v nekaterih primerih smrtni. Zdravljenje z zdravilom Tarceva je treba prekiniti ali ukiniti, če se pri bolniku pojavijo hude oblike mehurjev ali luščenja kože. Bolniki, pri katerih se pojavijo znaki in simptomi, ki nakazujejo na keratitis
in so lahko akutni ali se poslabšujejo: vnetje očesa, solzenje, občutljivost na svetlobo, zamegljen vid, bolečine v očesu in/ali rdeče oči, se morajo takoj obrniti na specialista oftalmogije. V primeru, da je diagnoza ulcerativnega ke-ratitisa potrjena, je treba zdravljenje z zdravilom Tarceva prekiniti ali ukiniti. V primeru, da se postavi diagnoza keratitisa, je treba skrbno razmisliti o koristih in tveganjih nadaljnega zdravljenja. Zdravilo Tarceva je pri bolnikih, ki so v preteklosti imeli keratitis, ulcerativni keratitis ali zelo suhe oči, uporabljati previdno. Uporaba kontaktnih leč je prav tako dejavnik tveganja za keratitits in ulceracijo. Med uporabo zdravila Tarceva so zelo redko poročali o primerih perforacije ali ulceracije roženice. 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. flu-voksamina) z erlotinibom je potrebna previdnost. V primeru pojava neželenih učinkov, povezanih z erlotinibom, lahko odmerek erlotiniba zmanjšamo. Predhodno ali sočasno zdravljenje z zdravilom Tarceva ni spremenilo očistka prototipov substratov CYP3A4, midazolama in eritromicina. Inhibicija gluko-ronidacije 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 šentja-nževka. 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 kumarinske antikoagulante, je treba redno kontrolirati protrombinski čas ali INR. Sočasno zdravljenje z zdravilom Tarceva in statinom lahko poveča tveganje za miopatijo, povzročeno s statini, vključno z rabdomiolizo; to so opazili redko. Sočasna uporaba zaviralcev P-glikoproteina, kot sta ciklospo-rin 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 anta-cidov 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 paklitaksela na farmakokinetiko erlotiniba ni bilo. Kapecitabin lahko poveča koncentracijo erlotiniba. Pomembnih učinkov erlotiniba na farmakokinetiko kapecitabina ni bilo.
Neželeni učinki: Zelo pogosti neželeni učinki so kožni izpuščaj in driska, kot tudi utrujenost, anoreksija, dispneja, kašelj, okužba, navzea, bruhanje, stomatitis, bolečina v trebuhu, pruritus, suha koža, suhi keratokonjunktivi-tis, konjunktivitis, zmanjšanje telesne mase, depresija, glavobol, nevropatija, dispepsija, flatulenca, alopecija, okorelost, pireksija, nenormalnosti testov jetrne funkcije. Pogosti neželeni učinki so krvavitve v prebavilih, epistaksa, keratitis, paronihija, fisure na koži. Občasno so poročali o perforacijah v prebavilih, hirzutizmu, spremembah obrvi, krhkih nohtih, odstopanju nohtov od kože, blagih reakcijah na koži (npr. hiperpigmentacija), spremembah trepalnic, hudi intersticijski bolezni pljuč (vključno s smrtnimi primeri). Redko pa so poročali o jetrni odpovedi. Zelo redko so poročali o Stevens-Johnsonovem sindromu/toksični epidermalni nekrolizi ter o ulceracijah in perforacijah ro- < ženice.	't
Režim izdaje zdravila: H/Rp. Imetnik dovoljenja za promet: Roche Re- — gistration Limited, 6 Falcon Way, Shire Park, Welwyn Garden City, AL7 1TW, ^ Velika Britanija. Verzija: 1.0/11. Informacija pripravljena: Oktober 2011. o
DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o. Vodovodna cesta 109, 1000 Ljubljana. Povzetek glavnih značilnosti zdravila je dosegljiv na www.roche.si ali www.onkologija.si.
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DOKAZANO PODALJŠA PREŽIVETJE PRI BOLNIKIH:
•	z lokalno napredovalim ali metastatskim nedrobnoceličnim rakom pljuč1
•	z metastatskim rakom trebušne slinavke1
1 Povzetek glavnih značilnosti zdravila TARCEVA, www.ema.europa.eu

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Ta vključuje zdravila kot so Glivec® (imatinib), Tasigna® (nilotinib), Afinitor® (everolimus), Zometa® (zoledronska kislina), Femara® (letrozol), Sandostatin® LAR® (oktreotid/i.m. injekcije) in Exjade® (deferasiroks).
Novartis Oncology ima tudi obširen razvojni program, ki izkorišča najnovejša spoznanja molekularne genomike, razumskega načrtovanja in tehnologij za odkrivanje novih učinkovin.
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SKRAJSAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA: MEGACE 40 mg/ml peroralna suspenzija
Sestava: 1 ml peroralne suspenzije vsebuje 40 mg megestrolacetata. TERAPEVTSKE INDIKACIJE: Zdravljenje anoreksije-kaheksije ali nepojasnjene, pomembne izgube telesne mase pri bolnikih z AIDS-om. Zdravljenje anorektično-kahektičnega sindroma pri napredovalem raku. ODMERJANJE IN NAČIN UPORABE: Pri aidsu je priporočeni začetni odmerek Megace za odrasle 800 mg (20 ml peroralne suspenzije) enkrat na dan eno uro pred jedjo ali dve uri po jedi in se lahko med zdravljenjem prilagodi glede na bolnikov odziv. V raziskavah bolnikov z aidsom so bili klinično učinkoviti dnevni odmerki od 400 do 800 mg/dan (10 do 20 ml), uporabljani štiri mesece. Pri anorektično-kahektičnem sindromu zaradi napredovalega raka je priporočljiv začetni odmerek 200 mg (5 ml) na dan; glede na bolnikov odziv ga je mogoče povečati do 800 mg na dan (20 ml). Običajni odmerek je med 400 in 800 mg na dan (10-20 ml). V raziskavah bolnikov z napredovalim rakom so bili klinično učinkoviti dnevni odmerki od 200 do 800 mg/dan (5 do 20 ml), uporabljani najmanj osem tednov. Pred uporabo je potrebno plastenko s suspenzijo dobro pretresti. Uporaba pri otrocih: Varnosti in učinkovitosti pri otrocih niso dokazali. Uporaba pri starostnikih: Zaradi pogostejših okvar jeter, ledvic in srčne funkcije, pogostejših sočasnih obolenj ali sočasnega zdravljenja z drugimi zdravili je odmerek za starejšega bolnika treba določiti previdno in običajno začeti z najnižjim odmerkom znotraj odmernega intervala. KONTRAINDIKACIJE: Preobčutljivost za megestrolacetat ali katerokoli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Uporaba gestagenov med prvimi štirimi meseci nosečnosti ni priporočljiva. Pri bolnikih s tromboflebitisom v anamnezi je treba zdravilo Megace uporabljati previdno. Zdravljenje z zdravilom Megace se lahko začne šele, ko so bili vzroki hujšanja, ki jih je mogoče zdraviti, ugotovljeni in obravnavani. Megestrolacetat ni namenjen za profilaktično uporabo za preprečitev hujšanja. Učinki na razmnoževanje virusa HIV niso ugotovljeni. Med zdravljenjem z megestrolacetatom in po prekinitvi kroničnega zdravljenja je treba upoštevati možnost pojava zavore nadledvične žleze. Morda bo potrebno nadomestno zdravljenje s stresnimi odmerki glukokortikoidov. Megestrolacetat se v veliki meri izloči prek ledvic. Ker je verjetnost zmanjšanega delovanja ledvic pri starostnikih večja, je pri določitvi odmerka potrebna previdnost, prav tako je koristno spremljanje ledvične funkcije. Peroralna suspenzija vsebuje saharozo. Bolniki z redko dedno intoleranco za fruktozo, malabsorpcijo glukoze/galaktoze ali pomanjkanjem saharoza-izomaltaze ne smejo jemati tega zdravila. Peroralna suspenzija vsebuje tudi majhne količine etanola (alkohola), in sicer manj kot 100 mg na odmerek. INTERAKCIJE: Aminoglutetimid: poročali so o zmanjšanju koncentracije progestogena v plazmi z možno izgubo terapevtskega delovanja zaradi inducirane presnove. Sočasno jemanje megestrolacetata (v obliki peroralne suspenzije) in zidovudina ali rifabutina ne povzroča sprememb farmakokinetičnih parametrov. NEŽELENI UČINKI: Pogosti (> 1/100, < 1/10): navzea, bruhanje, driska, flatulenca, izpuščaj, metroragija, impotenca, astenija, bolečina, edem. Neznana pogostnost (pogostnosti ni mogoče oceniti iz razpoložljivih podatkov): poslabšanje osnovne bolezni (širjenje tumorja), adrenalna insuficienca, kušingoidni izgled, Cushingov sindrom, diabetes mellitus, motena toleranca za glukozo, hiperglikemija, spremembe razpoloženja, sindrom karpalnega kanala, letargija, srčno popuščanje, tromboflebitis, pljučna embolija (v nekaterih primerih usodna), hipertenzija, navali vročine, dispneja, zaprtje, alopecija, pogosto uriniranje. Vrsta ovojnine in vsebina: Plastenka z 240 ml suspenzije. Režim izdaje: Rp/Spec. Imetnik dovoljenja za promet: Bristol-Myers Squibb spol. s r.o., Olivova 4, Praga 1, Češka; Odgovoren za trženje v Sloveniji: PharmaSwiss d.o.o., Ljubljana, tel: 01 236 4 700, faks: 01 236 4 705; MGS-120609. Pred predpisovanjem preberite celoten povzetek glavnih značilnosti zdravila!
Reference: 1. Povzetek glavnih značilnosti zdravila Megace - 12. junij 2009; 2. Register zdravil Republike Slovenije XII - leto 2010; 3. Beller, E., 1997. Ann Oncol 8: 277-283; 4. Čufer, T, 2002. Onkologija 9(2): 73-75; 5. Yavuzsen, T., 2005. J Clin Oncol 23(33): 8500-8511; 6. Bilten Recept 8(2), 8.12.2010
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Tantum Verde 1,5 mg/ml oralno pršilo, raztopina
Kakovostna in količinska sestava
1 ml raztopine vsebuje 1,5 mg benzidaminijevega klorida, kar ustreza 1,34 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,255 mg benzidaminijevega klorida, kar ustreza 0,2278 mg benzidamina. En razpršek vsebuje 13,6 mg 96 odstotnega etanola, kar ustreza 12,728 mg 100 odstotnega etanola, in 0,17 mg metilparahidroksibenzoata (E218).
Terapevtske indikacije
Samozdravljenje: lajšanje bolečine in oteklin pri vnetju v ustni votlini in žrelu, ki so lahko posledica okužb in stanj po operaciji. Po nasvetu in navodilu zdravnika: lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa.
Odmerjanje in način uporabe
Uporaba 2- do 6-krat na dan (vsake 1,5 do 3 ure). Odrasli: 4 do 8 razprškov 2- do 6-krat na dan. Otroci od 6 do 12 let: 4 razprški 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 4 kg telesne mase; do največ 4 razprške 2 do 6-krat na dan.
Kontraindikacije
Znana preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi
Pri manjšini bolnikov lahko resne bolezni povzročijo ustne/žrelne ulceracije. Če se simptomi v treh dneh ne izboljšajo, se mora bolnik posvetovati z zdravnikom ali zobozdravnikom, kot je primerno. Zdravilo vsebuje aspartam (E951) (vir fenilalanina), ki je lahko škodljiv za bolnike s fenilketonurijo. Zdravilo vsebuje izomalt (E953) (sinonim: izomaltitol (E953)). Bolniki z redko dedno intoleranco za fruktozo ne smejo jemati tega zdravila. Uporaba benzidamina ni priporočljiva za bolnike s preobčutljivostjo za salicilno kislino ali druga nesteroidna protivnetna zdravila. Pri bolnikih, ki imajo ali so imeli bronhialno astmo, lahko pride do bronhospazma. Pri takih bolnikih je potrebna previdnost.
Medsebojno delovanje z drugimi zdravili in druge oblike interakcij
Pri ljudeh raziskav o interakcijah niso opravljali.
Nosečnost in dojenje
Tantum Verde z okusom mentola 3 mg pastile se med nosečnostjo in dojenjem ne smejo uporabljati.
Vpliv na sposobnost vožnje in upravljanja s stroji
Uporaba benzidamina lokalno v priporočenem odmerku ne vpliva na sposobnost vožnje in upravljanja s stroji.
Neželeni učinki
Bolezni prebavil Redki: pekoč občutek v ustih, suha usta. Bolezni imunskega sistema Redki: preobčutljivostna reakcija. Bolezni dihal, prsnega koša in mediastinalnega prostora Zelo redki: laringospazem.
Bolezni kože in podkožja Občasni: fotosenzitivnost. Zelo redki: angioedem. Rok uporabnosti
4 leta. Zdravila ne smete uporabljati po datumu izteka roka uporabnosti, ki je naveden na ovojnini. Posebna navodila za shranjevanje Za shranjevanje pastil niso potrebna posebna navodila. Plastenko z raztopino shranjujte v zunanji ovojnini za zagotovitev zaščite pred svetlobo. Shranjujte pri temperaturi do 25°C. Shranjujte v originalni ovojnini in nedosegljivo otrokom.
u m U
Imetnik dovoljenja za promet CSC Pharma d.o.o. Jana Husa 1a 1000 Ljubljana
<£> A
CSC ANGELINI
* - s kemoterapijo povzročena navzea in bruhanje
Preprečuje CINV*
a začetka
EMEND 80 mg trde kapsule EMEND 125 mg trde kapsule SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila, ki ga dobite pri naših strokovnih sodelavcih! Sestava: Ena
EMEND 125 mg trda kapsula vsebuje 125 mg aprepitanta in 125 mg saharoze. Ena EMEND 80 mg trda kapsula vsebuje 80 mg apneprtarrta in 80 mg saharoze. Terapevtske indikacije: Preprečevanje akutne in zapoznele navzee in bruhanja povezanih z zelo emetogerio kemoterapijo raka s cisplatinom pri odraslih. Preprečevanje navzee in bruhanja, povezanih z zmerno emetogerio kemoterapijo raka pri odraslih. Zdravilo EMEND 125 mg/k) mg se daje v sklopu kombiniranega zdravljenja. Odmerjanje in način uporabe: Zdravilo EMEND se daje 3 dni po shemi zdravljenja, ki vključuje kortikosteroid in antagonist 5-HT3. Priporočeno odmerjanje zdravila EMEND je 125 mg peroralno(p.o.) enkrat dnevno eno uro pred pričetkom kemoterapije prvi dan ter 80 mg (p,o.) enkrat na dan drugi in tretji dan. Fosaprepitant 115 mg, liofilizirano predzdravilo aprepitanta v obliki 15-minutne infuzije, lahko prvi dan, 30 minut pred kemoterapijo nadomesti uporabo zdravila EMEND (125 mg). Zdravilo EMEND se lahko jemlje s hrano ali brez. Trdo kapsulo je treba pogoltniti celo. Pri starostnikih, bolnikih z okvaro ledvic in pri bolnikih s končno ledvično odpovedjo, ki se zdravijo s hemodializo, bolnikih z blago okvaro jeter ter glede na spol odmerka ni treba prilagajati, Pri bolnikih z zmerno okvaro jeter je število podatkov omejeno, pri bolnikih s hudo okvaro jeter pa podatkov ni, Pri teh bolnikih je treba aprepitant uporabljati previdno. Uporabe pri bolnikih, ki so mlajši od 18 let, zaradi nezadostnih podatkov o varnosti in učinkovitosti ne priporočamo, Kontraindikacije: Preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. Sočasno jemanje s pimozidom,ter1enadinom, zastemizolom ali s cisapridom. Posebna opozorila In previdnostni ukrepi: Zdravilo EMEND je treba uporabljati previdno pri bolnikih, ki sočasno jemljejo peroralne zdravilne učinkovine, ki se primarno presnavljajo s CYP3A4 in z ozkim terapevtskim območjem, kot so ciklosporin, takrolimus, siroli-mus, everolimus, alfentanil, diergotamin, ergotamin, tentanil in kinidin. Previdnost je še posebej potrebna pri sočasnem dajanju irinotekana, saj lahko kombinacija poveča toksični učinek. Pri sočasni uporabi zdravila EMEND z alkaloidi rženega rožička (ergot alkaloidi) svetujemo previdnost zaradi morebitnega tveganja za pojav z ergot alkaloidi povezanih toksičnih učinkov. Sočasna uporaba zdravila EMEND z varfarinom zmanjša protrombinski čas, izražen kot INR. Pri bolnikih, ki se kontinuirano zdravijo z vrarfarinom, je treba INR skrbno spremljati med zdravljenjem z zdravilom EMEND in še 2 tedna po vsakem 3-dr>evnem ciklusu zdravljenja navzee in bruhanja zaradi kemoterapije z zdravilom EMEND. Med jemanjem zdravila EMEND in še 28 dni po koncu jemanja se lahko zmanjša učinkovitost hormonskih kontraceptivov, Med zdravljenjem z zdravilom EMEND in 2 meseca po zadnjem odmerku zdravila EMEND je treba uporabljati alternativno ali dodatno kontracepcijsko metodo. Sočasnemu jemanju zdravila EMEND in zdravilnih učinkovin, ki močno inducirajo aktivnost CYP3A4 (npr. rifampicin, fenitoin, karbamazepin, fenobarbital), seje treba izogibati, ker kombinacija povzroči zmanjšanje plazemskih koncentracij aprepitanta. Sočasna uporaba zdravila EMEND in zeliščnih pripravkov, ki vsebujejo šentjanževko, ni priporočljiva, Potrebna je previdnost pri sočasni uporabi zdravila EMEND in zdravilnih učinkovin, ki zavirajo aktivnost CYP3A4 (npr. ketokonazol, rtrakonazol, vorikonazol, posakonazol, klaritromicin, telitromicin, nefazodon in zaviralci proteaz), ker se zaradi kombinacije pričakuje zvišanje plazemskih koncentracij aprepitanta. Zdravilo EMEND vsebuje saharozo. Bolniki z redkimi dednimi motnjami - fruktozno intoleranco, malabsorpcijo glukoze in galaktoze ali insuficienco saharaze-izomaltaze - ne smejo jemati tega zdravila. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Aprepitant (125 mg/80 mg) je substrat, zmerni zaviralec in induktor CYP3A4. Aprepitant je tudi induktor CYP2C9, Med zdravljenjem se CYP3A4 inhibira. Po koncu zdravljenja pa zdravilo EMEND povzroči blago indukcijo CYP2C9, CYP3A4 in glukuronidacije. Aprepitant nima medsebojnega vpliva z digoksinom, zato verjetno ne irrtereagira s P-glikoproteinskim prenašalcem. Kot blag induktor CYP2C9, induktor CYP3A4 in glukuronidacije lahko aprepitant zniža plazemske koncentracije substratov, ki se izločajo po teh poteh. Ta učinek se lahko pokaže šele po koncu zdravljenja z zdravilom EMEND, Za substrate CYP2C9 in CYP3A4 je indukcija prehodna, največji učinek pa je dosežen v 3-5 dneh po koncu 3 dnevnega zdravljenja z zdravilom EMEND. Učinek traja nekaj dni, potem pa počasa upada in je klinično nepomemben v dveh tednih po koncu zdravljenja. V tem obdobju svetujemo previdnost pri dajanju peroralnih zdravilnih učinkovin, ki se presnavljajo s CYP2C9. Kortikosteroid!: Pri sočasnem jemanju je treba običajni peroraini odmerek deksametazona zmanjšati za približno 50 %, običajni intravenski odmerek metilprednizolona zmanjšati za približno 25 % in
običajni peroraini odmerek metilprednizolona zmanjšati za približno 50 %. Kemoterapevtiki: Pri bolnikih, ki poleg zdravila EMEND peroralno prejemajo kemoterapevtike, ki se primamo ali delno presnavljajo s CYP3A4 (npr. etopozld, vlnorelbln), svetujemo previdnost,. Pri takih bolnikih bo morda potreben dodatni nadzor. ImunosupresM: Zmanjšanja odmerka imunosupresivov, ki se presnavljajo s GYP3A4 (npr. ciklosporin, takrolimus, everolimus in sirolimus), ne priporočamo. Midazolam: Pri sočasni uporabi z zdravilom EMEND (125mg / 80 mg) je treba upoštevati možne učinke zvišanih plazemskih koncentracij midazolama in drugih benzodiazepinov, ki se presnavljajo predvsem s CYP3A4 (alprazolam, triazolam). Tolbutamid:Zdravilo EMEND je pri jemanju po shemi 125 mg prvi dan ter 80 mg/dan drugi in tretji dan zmanjšal AUC tolbutamida (ki je substrat za CYP2C9), ki so ga bolniki prejemali v enkratnem odmerku 500 mg per os pred začetkom 3 dnevne sheme odmerjanja zdravila EMEND ter 4., 8. In 15. dan. Antagonisti 5 HT^V kliničnih raziskavah medsebojnega delovanja aprepitant nI Imel klinično pomembnih učinkov na fermakokinetiko ondansetrona, granisetrona in hidrodolasetrona. Ketokonazol: Pri enkratnem odmerku 125 mg aprepitanta 5. dan 10 dnevnega zdravljenja s ketokonazolom (ki je močan zaviralec CYP3A4) 400 mg na dan, se je AUC aprepitanta povečal za približno 5 krat, srednji končni razpolovni čas aprepitanta pa se je podaljšal za približno za 3 krat. RHamptin: Pri enkratnem odmerku 375 mg aprepitanta 9. dan 14 dnevnega zdravljenja z rifampicinom (ki je močan induktor CYP3A4) 600 mg na dan, se je AUC aprepitanta zmanjšal za 91 %, srednji končni razpolovni čas aprepitanta pa se je skrajšal za 68 %. Neželeni učlnld: Pri bolnikih, zdravljenih z aprepitantom, so opazili naslednje neželene učinke, ki so se pojavljali pogosteje kot pri standardni terapiji: Pogosti (>1/100, <1/10): anorekslja, glavobol, omotica, kolcanje, konstipacija, driska, dispepsija, spahovanje, astenija/utrujenost, zvišanje ALT, zvišanjeAST. Občasni (>1/1.000, <1/100): kandidoza, okužbe s stafilokoki, anemija, febrilna nevtropenija, povečanje telesne mase, polidipsija, dezorientacija, evfbrija, anksioznost, neobičajne sanje, motnje mišljenja, letargija, zaspanost, konjunkbvttis, tinitus, bradikardija, palpitacije, bolezen srca in ožilja, zardevanje/navali vročine, faringitis, kihanje, kašelj, zatekanje izcedka iz nosu v žrelo, draženje žrela, perforirajoč duodenalen ulkus, navzea, bruhanje, refluks kisline, motnje okusa, neugodje v epigastriju, obstipacija, gastroezofagalna refluksna bolezen, bolečine v trebuhu, suha usta, enterokolitis, vetrovi, stomatitis, napihnjen trebuh, trdo blato, nevtropenlčnl kolltls, Izpuščaji, akne, fotosenztttvnost, prekomerno znojenje, mastna toža, srbenje, lezlje kože, srbeči Izpuščaj, mišični krči, bolečine v mišicah, mišična oslabelost, poliurija, disurija, pdakisurija, edem, nelagodje v prsnem košu, splošno slabo počutje, žeja, mrzlica, motnja hoje, zvišanje alkalne fosfataze, hiperglikemija, mikrohematurija, hiponatriemija, zmanjšanje telesne mase, zmanjšano število nevtrofilcev. Poročali so o enem primeru angioedema in urtikarije. Pri enem bolniku, ki je dobival aprepitant ob kemoterapiji zaradi raka, so poročali o pojavu Stevens-Johnsonovega sindroma, V obdobju trženja zdravila so poročali še o (pogostnost je neznana): pruritus, izpuščaj, ur-tikarija, preobčutljivostne reakcije, vključno z anafilaktičnimi reakcijami. EMEND 80mg trde kapsule EMEND 125 mg trde kapsule Imetnik dovoljenja za promet Merck Sharp & Dohme Ltd., Hertford Road, Hoddesdon, Hertfordshire EN 11 9BU, Velika Britanija Način in režim izdaje zdravila: Predpisovanje in izdaja zdravila je le na zdravniški recept. Datum zadnje revizije besedila: 01/2010
Ei\®\o IVEI\ieNDe
(aprepitant, MSD) (fosaprepitant dimeglumin, MSD)
Merck Sharp & Dohme, inovativna zdravila d.o.o.
Smartinska cesta 140,1000 Ljubljana; telefon: 01/5204 201, faks: 01/5204 349 Tiskano v Sloveniji, junij 2011.
Preventivno od začetka
Prvi na poti individualnega zdravljenja bolnikov z napredovalim nedrobnoceličnim pljučnim rakom.
Iressa je prva in edina tarčna monoterapija, ki dokazano podaljša preživetje brez napredovanja bolezni v primerjavi z dvojno kemoterapijo kot zdravljenje prvega reda pri bolnikih z napredovalim nedrobnoceličnim pljučnim rakom z^mutadjo EGFR.
IRESSA® (GEFITINIB)
SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA
Sestava: Filmsko obložene tablete vsebujejo 250 mg gefitiniba.
Indikacije: zdravljenje odraslih bolnikov z lokalno napredovalim ali metastatskim nedrobnoceličnim pljučnim rakom z aktivacijskimi mutacijami EGFR-TK
Odmerjanje in način uporabe: Zdravljenje z gefitinibom mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil proti raku. Priporočeno odmerjanje zdravila IRESSA je ena 250-mg tableta enkrat na dan. Tableto je mogoče vzeti s hrano ali brez nje, vsak dan ob približno istem času.
Kontraindikacije: preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov, dojenje
Opozorila in previdnostni ukrepi: Pri 1,3 % bolnikov, ki so dobivali gefitinib, so opažali intersticijsko bolezen pljuč (IBP). Ta se lahko pojavi akutno in je bila v nekaterih primerih smrtna. Če se bolniku poslabšajo dihalni simptomi, npr. dispneja, kašelj in zvišana telesna temperatura, morate zdravljenje z zdravilom IRESSA prekiniti in bolnika takoj preiskati. Če je potrjena IBP, morate terapijo z zdravilom IRESSA končati in bolnika ustrezno zdraviti. Opažene so bile nepravilnosti testov jetrnih funkcij, občasno zabeležene kot hepatitis. Opisani so bili posamezni primeri odpovedi jeter. Zato so priporočljive redne kontrole delovanja jeter. V primeru blagih do zmernih sprememb v delovanju jeter je treba zdravilo IRESSA uporabljati previdno. Če so spremembe hude, pride v poštev prekinitev zdravljenja. Zdravilo IRESSA vsebuje laktozo. Bolniki z redko dedno intoleranco za galaktozo, laponsko obliko zmanjšane aktivnosti laktaze ali malabsorpcijo glukoze/galaktoze ne smejo jemati tega zdravila. Bolnikom naročite, da morajo takoj poiskati zdravniško pomoč, če se jim pojavijo kakršnikoli očesni simptomi, huda ali dolgotrajna driska, navzea, bruhanje ali anoreksija, ker lahko vse te posredno povzročijo dehidracijo.
Medsebojno delovanje zdravil: Sočasna uporaba močnih zaviralcev CYP3A4 lahko poveča koncentracijo gefitiniba v plazmi. Močni zaviralci CYP2D6 lahko pri izrazitih metabolizatorjih CYP2D6 povečajo koncentracijo gefitiniba v plazmi za približno 2-krat. Induktorji CYP3A4 lahko povečajo presnovo gefitiniba in zmanjšajo njegovo koncentracijo v plazmi. Zato lahko sočasna uporaba induktorjev CYP3A4 zmanjša učinkovitost zdravljenja in se ji je treba izogniti. Snovi, ki občutno in dolgotrajno zvišajo pH v želodcu, lahko zmanjšajo koncentracijo gefitiniba v plazmi in tako zmanjšajo njegovo učinkovitost. Veliki odmerki kratkodelujočih antacidov, uporabljenih blizu časa jemanja gefitiniba, imajo lahko podoben učinek. Pri nekaterih bolnikih, ki so jemali varfarin skupaj z gefitinibom, so se pojavili zvišanje internacionalnega normaliziranega razmerja (INR) in/ali krvavitve. Bolnike, ki sočasno jemljejo varfarin in gefitinib, morate redno kontrolirati glede sprememb protrombinskega časa (PČ) ali INR. Neželeni učinki: V kumulativnem naboru podatkov kliničnih preskušanj III. faze so bili najpogosteje opisani neželeni učinki, ki so se pojavili pri več kot 20 % bolnikov, driska in kožne reakcije (vključno z izpuščajem, aknami, suho kožo in srbenjem). Neželeni učinki se ponavadi pojavijo prvi mesec zdravljenja in so praviloma reverzibilni. Ostali pogostejši neželeni učinki so: anoreksija, konjunktivitis, blefaritis in suho oko, krvavitev, npr. epistaksa in hematurija, intersticijska bolezen pljuč (1,3 %), navzea, bruhanje, stomatitis, dehidracija, suha usta, nepravilnosti testov jetrnih funkcij, bolezni nohtov, alopecija, asimptomatično laboratorijsko zvišanje kreatinina v krvi, proteinurija, cistitis, astenija, pireksija. Vrsta in vsebina ovojnine: škatla s 30 tabletami po 250 mg gefitiniba Način izdajanja zdravila: samo na recept Datum priprave besedila: januar 2011
Imetnik dovoljenja za promet: AstraZeneca AB, S-151 85, Sodertalje, Švedska Pred predpisovanjem, prosimo, preberite celoten povzetek glavnih značilnosti zdravila. Dodatne informacije so na voljo pri:
AstraZeneca UK Limited, Podružnica v Sloveniji, Verovškova 55, 1000 Ljubljana, telefon: 01/51 35 600.
Samo za strokovno javnost. Informacija pripravljena: avgust 2011
AstraZeneca
ONKOLOGIJA

IRESSÄ
gefitinib
Instructions for authors
The editorial policy
Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers, professional papers, review articles, case reports and varia (editorials, short communications, professional information, book reviews, letters, etc.) pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Therefore, the scope of the journal is to cover beside radiology the diagnostic and therapeutic aspects in oncology, which distinguishes it from other journals in the field.
The Editorial Board requires that the paper has not been published or submitted for publication elsewhere; the authors are responsible for all statements in their papers. Accepted articles become the property of the journal and, therefore cannot be published elsewhere without the written permission of the editors.
Submission of the manuscript
The manuscript written in English should be submitted electronically to: gsersa@onko-i.si. In the case the figures are too big to be submitted electronically, authors are asked to send along the printed copy of the manuscript, together with all the files on CD, to the editorial office. The type of computer and word-processing package should be specified (Word for Windows is preferred).
Editorial Office Radiology and Oncology
Zaloska cesta 2
P.O. Box 2217
SI-1000 Ljubljana
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Phone: +386 (0)1 5879 434, Tel./Fax: +386 (0)1 5879 434, E-mail: gsersa@onko-i.si
All articles are subjected to the editorial review and the review by independent referees. Manuscripts which do not comply with the technical requirements stated herein will be returned to the authors for the correction before peer-review. The editorial board reserves the right to ask authors to make appropriate changes of the contents as well as grammatical and stylistic corrections when necessary. Page charges will be charged for manuscripts exceeding the recommended page number, as well as additional editorial work and requests for printed reprints. All articles are published printed and on-line as the open access. To support the open access policy of the journal, the authors are encouraged to pay the open access charge of 500 EUR.
Preparation of manuscripts
Radiology and Oncology will consider manuscripts prepared according to the Uniform Requirements for Manuscripts Submitted to Biomedical Journals by International Committee of Medical Journal Editors (http://www.icmje.org/). The manuscript should be typed double-spaced with a 3-cm margin at the top and left-hand side of the sheet. The manuscript should be written in grammatically and stylistically correct language. Abbreviations should be avoided. If their use is neccessary, they should be explained at the first time mentioned. The technical data should conform to the SI system. The manuscript, including the references, must not exceed 15 typewritten pages, and the number of figures and tables is limited to 8. If appropriate, organize the text so that it includes: Introduction, Materials and methods, Results and Discussion. Exceptionally, the results and discussion can be combined in a single section. Start each section on a new page, and number each page consecutively with Arabic numerals.
The Title page should include a concise and informative title, followed by the full name(s) of the author(s); the institutional affiliation of each author; the name and address of the corresponding author (including telephone, fax and E-mail), and an abbreviated title. This should be followed by the abstract page, summarizing in less than 250 words the reasons for the study, experimental approach, the major findings (with specific data if possible), and the principal conclusions, and providing 3-6 key words for indexing purposes. Structured abstracts are preferred. Slovene authors are requested to provide title and the abstract in Slovene language in a separate file. The text of the research article should then proceed as follows: Introduction should summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study.
Materials and methods should provide enough information to enable experiments to be repeated. New methods should be described in detail.
Results should be presented clearly and concisely without repeating the data in the figures and tables. Emphasis should be on clear and precise presentation of results and their significance in relation to the aim of the investigation.
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SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA
Ime zdravila ALIMTA 100 mg prašek za koncentrat za raztopino za infundiranje in ALIMTA 500 mg prašek za koncentrat za raztopino za infundiranje Kakovostna in količinska sestava ALIMTA 100 mg: vsaka viala vsebuje 100 mg pemetrekseda (v obliki dinatrijevega pemetrekseda). Po pripravi vsebuje vsaka viala 25 mg/ml pemetrekseda. Pomožne snovi: Vsaka viala vsebuje približno 11 mg natrija, manitol, klorovodikova kislina, natrjev hidroksid. ALIMTA 500 mg: vsaka viala vsebuje 500 mg pemetrekseda (v obliki dinatrijevega pemetrekseda). Po pripravi vsebuje vsaka viala 25 mg/ml pemetrekseda. Pomožne snovi: Vsaka viala vsebuje približno 54 mg natrija, manitol, klorovodikova kislina, natrijev hidroksid. Terapevtske indikacije: ALIMTA je v kombinaciji s cisplatinom indicirana za zdravljenje bolnikov z neresektabilnim malignim plevralnim mezoteliomom, ki jih še nismo zdravili s kemoterapijo. ALIMTA je v kombinaciji s cisplatinom indicirana kot zdravljenje prvega izbora za bolnike z lokalno napredovalim ali metastatskim nedrobnoceličnim karcinomom pljuč, ki nima pretežno ploščatocelične histologije. ALIMTA je indicirana kot monoterapija za zdravljenje lokalno napredovalega ali metastatskega nedrobnoceličnega pljučnega karcinoma, ki nima pretežno ploščatocelične histologije pri bolnikih, pri katerih bolezen ni napredovala neposredno po kemoterapiji na osnovi platine. ALIMTA je indicirana kot monoterapija za zdravljenje drugega izbora bolnikov z lokalno napredovalim ali metastatskim nedrobnoceličnim pljučnim karcinomom, ki nima pretežno ploščatocelične histologije. Odmerjanje in način uporabe: Odmerjanje: ALIMTO smemo dajati le pod nadzorom zdravnika, usposobljenega za uporabo kemoterapije za zdravljenje raka. ALIMTA v kombinaciji s cisplatinom Priporočeni odmerek ALIMTE je 500 mg/m2 telesne površine (TP), dan kot intravenska infuzija v 10 minutah prvi dan vsakega 21-dnevnega ciklusa. Priporočeni odmerek cisplatina je 75 mg/m2 TP infundiran v dveh urah približno 30 minut po zaključku infuzije pemetrekseda prvi dan vsakega 21 dnevnega ciklusa. Bolniki morajo prejeti zadostno antiemetično zdravljenje, pred in/ali po prejemanju cisplatina jih moramo tudi ustrezno h id ri rati. ALIMTA kot samostojno zdravilo Priporočeni odmerek ALIMTE je 500 mg/m2 TP, dan kot intravenska infuzija v 10 minutah prvi dan vsakega 21 dnevnega ciklusa. Režimpremedikacije Da zmanjšamo incidenco in resnost kožnih reakcij, dajemo kortikosteroid dan pred dajanjem pemetrekseda, na dan dajanja pemetrekseda in naslednji dan. Kortikosteroid naj ustreza 4 mg deksametazona, danega peroralno dvakrat dnevno. Za zmanjšanje toksičnosti morajo bolniki dnevno jemati tudi peroralno folno kislino ali multivitaminski pripravek, ki jo vsebuje (350 do 1000 mikrogramov). V sedmih dneh pred prvim odmerkom pemetrekseda morajo vzeti vsaj pet odmerkov folne kisline, odmerjanje pa morajo nadaljevati ves čas zdravljenja in še 21 dni po zadnjem odmerku pemetrekseda. Bolniki morajo prejeti tudi intramuskularno injekcijo vitamina B12 (1000 mikrogramov) v tednu pred prvim odmerkom pemetrekseda in enkrat vsake tri cikluse zatem. Kasnejše injekcije vitamina B12 lahko dajemo isti dan kot pemetreksed. Kontraindikacije: Preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. Dojenje. Sočasno cepljenje proti rumeni mrzlici. Posebna opozorila in previdnostni ukrepi: Pemetreksed lahko zavre delovanje kostnega mozga, kar se kaže kot nevtropenija, trombocitopenja in anemija (ali pancitopenija). Mielosupresija običajno predstavlja toksičnost za omejitev odmerka. Pri bolnikih, ki pred zdravljenjem niso prejemali kortikosteroidov, so poročali o kožnih reakcijah. Uporabe pemetrekseda pri bolnikih z očistkom kreatinina < 45 ml/min ne priporočamo. Bolniki z blagim do zmernim popuščanjem delovanja ledvic naj se izogibajo jemanju nesteroidnih protivnetnih zdravil (NSAID), denimo, ibuprofena in acetilsalicilne kisline 2 dni pred dajanjem pemetrekseda, na dan dajanja in še 2 dni po dajanju pemetrekseda. Vsi bolniki, 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 resnih 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 kombinacji s cisplatinom za prebavila so opažali hudo dehidracjo, zato moramo bolnike pred prejemanjem terapije in/ali po njej ustrezno hidrirati, prejeti morajo zadostno antiemetično zdravljenje. Občasno so v kliničnih študijah pemetrekseda, običajno ob sočasnem dajanju z drugo citotoksično učinkovino, poročali o resnih srčnožilnih dogodkih, vključno z miokardnim infarktom in možganskožilnimi dogodki. Odsvetujemo uporabo živih oslabljenih cepiv Spolno zrelim moškim odsvetujemo zaploditev otroka v času zdravljenja in še 6 mesecev zatem. Priporočamo ukrepe proti zanositvi ali vzdržnost. Zaradi možnosti, da zdravljenje s pemetreksedom povzroči trajno neplodnost, naj se moški pred začetkom zdravljenja posvetujejo o shranjevanju semena. Ženske v rodni dobi morajo v času zdravjenja s pemetreksedom uporabljati učinkovito kontracepcijo. Poročali so o primerih radiacjske pljučnice pri bolnikih, ki so jih zdravili z radiacijo pred, med ali po zdravljenju s pemetreksedom. Poročali so o radiacijskem izpuščaju pri bolnikih, ki so se zdravili z radioterapijo pred tedni ali leti. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Sočasno dajanje nefrotoksičnih zdravil (denimo, aminoglikozidov, diuretikov zanke, spojin platine, ciklosporina) lahko potencialno povzroči zakasnjeni očistek pemetrekseda. Sočasno dajanje snovi, ki se tudi izločajo s tubulno sekrecjo (denimo, probenecid, penicilin), lahko potencialno povzroči zakasnjeni očistek pemetrekseda. Pri bolnikih z normalnim delovanjem ledvic lahko visoki odmerki nesteroidnih protivnetnih zdravil (NSAID-i, denimo, ibuprofen) in acetilsalicilna kislina v visokih odmerkih zmanjšajo eliminacijo pemetrekseda in tako lahko povečajo pojavnost neželenih učinkov pemetrekseda. Pri bolnikih z blagim do zmernim popuščanjem delovanja ledvic se moramo izogibati sočasnemu dajanju pemetrekseda z NSAID-i (denimo, ibuprofenom) ali acetilsalicilne kisline v visokih odmerkih 2 dni pred dajanjem pemetrekseda, na dan dajanja in še 2 dni po dajanju pemetrekseda. Sočasnemu dajanju NSAID-ov z daljšimi razpolovnimi časi s pemetreksedom se moramo izogibati vsaj 5 dni pred dajanjem pemetrekseda, na dan dajanja in še vsaj 2 dni po dajanju pemetrekseda.Velika različnost med posamezniki v koagulacjskem statusu v času bolezni ter možnost medsebojnega delovanja med peroralnimi antikoagulacijskimi učinkovinami ter kemoterapijo proti raku zahtevata povečano pogostnost spremljanja INR. Kontraindicirana sočasna uporaba: Cepivo proti rumeni mrzlici: tveganje za smrtno generalizirano bolezen po cepljenju. Odsvetovana sočasna uporaba: Živa oslabljena cepiva (razen proti rumeni mrzlici): tveganje za sistemsko, potencialno smrtno bolezen. Neželeni učinki Klinične študije malignega plevralnega mezotelioma Zelo pogosto: znižani nevtrofi lci/granulociti, znižani levkociti, znižan hemoglobin, znižani trombociti, nevropatja-senzorna, diareja, bruhanje, stomatitis/faringitis, slabost, anoreksja, zaprtje, izpuščaj, alopecja, povišan kreatinin, znižan očistek kreatinina, utrujenost. Pogosti: dehidracija, motnje okusa, konjuktivitis, dispepsija. Klinične študije nedrobnoceličnega pljučnega karcinoma - ALIMTA monoterapija, zdravljenje 2. izbora: Zelo pogosti: znižan nevtrofilci/granulociti, znižani levkocitit, znižan hemoglobin, diareja, bruhanje, stomatitis/faring itis, slabost, anoreksija, izpuščaj/luščenje, utrujenost. Pogosti: znižani trombociti, zaprtje, povišanje SGPT (ALT), povišanje SGOT (AST), srbenje, alopecija, povišana telesna temperatura. Klinične študije nedrobnoceličnega pjučnega karcinoma - ALIMTA v kombinaciji s cisplatinom, zdravljenje 1. izbora: Zelo pogosti: znižan hemoglobin, znižani nevtrofilci/granulociti, znižani levkocitit, znižani trombociti, slabost, bruhanje, anoreksija, zaprtje, stomatitis/faringitis, diareja brez kolostomije, alopecija, izpuščaj/luščenje, povišan kreatinin, utrujenost. Pogosti: nevropatija-senzorična, motnje okusa, dispepsija/zgaga. Klinične študije nedrobnoceličnega pljučnega karcinoma - ALIMTA monoterapija, vzdrževalno in nadaljevalno zdravljenje: Zelo pogosti: znižan hemoglobin, slabost, anoreksija, utrujenost. Pogosti: znižani levkociti, znižani nevtrofilci, nevropatija-senzorična, bruhanje, mukozitis/stomatitis, povišanje ALT (SGPT), povišanje AST (SGOT), izpuščaj/luščenje, bolečina. Občasno so v kliničnih študijah pemetrekseda poročali o primerih resnih srčnožilnih in možganskožilnih dogodkov, vključno z miokardnim infarktom, angino pektoris, cerebrovaskularnim insultom in prehodnimi ishemičnimi atakami; primerih kolitisa ter o primerih intersticijske pljučnice z respiratorno insuficience, primerih edema, o ezofagitisu/ radiacijskem ezofagitisu in o primerih sepse. Redkeje pa o primerih potencialno resnega hepatitisa in pancitopenije. Po uvedbi zdravila na trg so poročali o primerih akutne odpovedi ledvic s pemetreksedom samim ali v povezavi z drugimi kemoterapevtiki, primerih radiacjske pljučnice pri bolnikih, ki so jih zdravili z radiacijo pred, med ali po njihovem zdravjenju s pemetreksedom, primerih radiacjskega izpuščaja pri bolnikih, ki so se v preteklosti zdravili z radioterapjo, o primerih periferne ishemje, ki je včasih vodila v nekrozo okončin, redkih primerih buloznih stanj, kot sta Stevens-Johnsonov sindrom in toksična epidermalna nekroliza, ki so bila v nekaterih primerih usodna in o redkih primerih hemolitične anemije. Imetnik dovoljenja za promet Eli Lilly Nederland B.V., Grootslag 1 5, NL 3991 RA, Houten, Nizozemska. Datum zadnje revizije besedila 24.10.2011. Način izdaje zdravila: H. SAMO ZA STROKOVNO JAVNOST.
Podrobnejše informacije o zdravilu Alimta, so dostopne na spletni strani Evropske agencije za zdravila EMA http://www.ema.europa.eu in na lokalnem predstavništvu.	SIALM00025
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