ALIMTA pemetreksed 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, natrijev 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 zdravjenja 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 injekcje vitamina B12 lahko dajemo isti dan kot pemetreksed. Kontraindikacije: Preobčutjivost 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 nevtropenja, trombocitopenija 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 kombinaciji s cisplatinom za prebavila so opažali hudo dehidracijo, 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 zdravljenja s pemetreksedom uporabljati učinkovito kontracepcijo. Poročali so o primerih radiacijske 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 sekrecijo (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 koagulacijskem 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 nevtrofilci/granulociti, znižani levkociti, znižan hemoglobin, znižani trombociti, nevropatija-senzorna, diareja, bruhanje, stomatitis/faringitis, slabost, anoreksija, zaprtje, izpuščaj, alopecija, 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/faringitis, slabost, anoreksija, izpuščaj/luščenje, utrujenost Pogosti: znižani trombociti, zaprtje, povišanje SGPT (ALT), povišanje SGOT (AST), srbenje, alopecija, povišana telesna temperatura. Klinične študje nedrobnoceličnega pljučnega karcinoma - ALIMTA v kombinacji s cisplatinom, zdravljenje 1. zbora: Zelo pogosti: znižan hemoglobin, znižani nevtrofilci/granulociti, znižani levkocitit, znižani trombociti, slabost, bruhanje, anoreksja, 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, anoreksja, 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 pjučnice 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 radiacijske pljučnice pri bolnikih, ki so jih zdravili z radiacijo pred, med ali po njihovem zdravljenju s pemetreksedom, primerih radiacijskega izpuščaja pri bolnikih, ki so se v preteklosti zdravili z radioterapijo, o primerih periferne ishemije, 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 Eli Lilly Farmacevtska družba, d.o.o. Brnčičeva 41G, 1231 Ljubljana - Črnuče, Slovenija Telefon: +386 (0)1 5800 010 Faks: +386 (0)1 5691 705 ADIOLQGY AND NCOLOGY March 2012 Vol. 46 No. 2 Pages 89-178 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Publisher Association of Radiology and Oncology Affiliated with Slovenian Medical Association - Slovenian Association of Radiology, Nuclear Medicine Society, Slovenian Society for Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medical Association - Croatian Society of Radiology Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups of S.I.R.M. Italian Society of Medical Radiology Aims and scope Radiology and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chief Gregor Serša Ljubljana, Slovenia Executive Editor Viljem Kovač Ljubljana, Slovenia Deputy Editors Andrej Cör Izola, Slovenia Igor Kocijančič Ljubljana, Slovenia Mirjana Rajer Ljubljana, Slovenia Karmen Stanič Ljubljana, Slovenia Editorial Board Karl H. Bohuslavizki Hamburg, Germany Maja Čemažar Ljubljana, Slovenia Christian Dittrich Vienna, Austria Metka Filipič Ljubljana, Slovenia Tullio Giraldi Trieste, Italy Maria Godény Budapest, Hungary Vassil Hadjidekov Sofia, Bulgaria Nyström Hakan Uppsala, Sweden Marko Hočevar 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 Primož Strojan Ljubljana, Slovenia Borut Štabuc Ljubljana, Slovenia Ranka Štern-Padovan Zagreb, Croatia Justin Teissié Toulouse, France Gillian M. Tozer Sheffield, UK Andrea Veronesi Aviano, Italy Branko Zakotnik Ljubljana, Slovenia Advisory Committee Marija Auersperg Ljubljana, Slovenia Tomaž Benulič Ljubljana, Slovenia Božo Casar Ljubljana, Slovenia Jure Fettich Ljubljana, Slovenia Valentin Fidler Ljubljana, Slovenia Berta Jereb Ljubljana, Slovenia Vladimir Jevtič Ljubljana, Slovenia Maksimilijan Kadivec Ljubljana, Slovenia Stojan Plesničar Ljubljana, Slovenia Uroš Smrdel Ljubljana, Slovenia Živa Zupančič Ljubljana, Slovenia Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217 SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369 Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. 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Indexed and abstracted by: Science Citation Index Expanded (SciSearch®) Journal Citation Reports/Science Edition Scopus EMBASE/Excerpta Medica DOAJ Open J-gate Chemical Abstracts Biomedicina Slovenica Summon by Serial Solutions (ProQuest) This journal is printed on acid- free paper On the web: ISSN 1581-3207 http://versitaopen.com/ro http://www.radioloncol.com http://versita.com/science/medicine/ro/ http://www.onko-i.si/radioloncol/ contents experimental radiology 89 Double BioDisk: a new bioprosthetic device for transcatheter closure of atrial septal defects - a feasibility study in adult sheep Dusan Pavcnik, Kurt Tekulve, Barry T. Uchida, Zhong-Huo Luo, Miran Jeromel, William G. Van Alstine, Frederick S. Keller, Josef Rösch radiology 97 Outcome of MRI-guided vacuum-assisted breast biopsy - initial experience at Institute of Oncology Ljubljana, Slovenia Marta Zebic-Sinkovec, Kristijana Hertl, Maksimiljan Kadivec, Mihael Cavlek, Gasper Podobnik, Marko Snoj 106 Differentiation of malignant and benign lung lesions with diffusion-weighted MR imaging Sevtap Gümüstas, Nagihan Inan, Gür Akansel, Ercüment Çiftçi, Ali Demirci, Sevgiye Kaçar Özkara 114 Evaluation of penile erection rigidity in healthy men using virtual touch tissue quantification Xiaozhi Zheng, Ping Ji, Hongwei Mao, Jing Wu experimental oncology 119 Lucifer Yellow uptake by CHO cells exposed to magnetic and electric pulses Leila Towhidi, Seyed Mohammad P Firoozabadi, Hossein Mozdarani, Damijan Miklavcic 126 The optimization of needle electrode number and placement for irreversible electroporation of hepatocellular carcinoma Oyinlolu O. Adeyanju, Haitham M. Al-Angari, Alan V. Sahakian clinical oncology 136 Improved survival after introduction of chemotherapy for malignant pleural mesothelioma in Slovenia: population-based survey of 444 patients Viljem Kovac, Matjaz Zwitter, Tina Zagar 145 Long term outcome after combined modality treatment for anal cancer Irena Oblak, Primoz Petric, Franc Anderluh, Vaneja Velenik, Peter Albert Fras 153 The diffuse large B-cell lymphoma - where do we stand now in everyday clinical practice Brigita Gregoric, Vesna Zadnik, Barbara Jezeršek Novakovic 160 Effectiveness of L-thyroxine treatment on TSH suppression during pregnancy in patients with a history of thyroid carcinoma after total thyroidectomy and radioiodine ablation Blaz Krhin and Nikola Besic 166 What sampling device is the most appropriate for vaginal vault cytology in gynaecological cancer follow up? Del Pup Lino, Canzonieri Vincenzo, Serraino Diego, Campagnutta Elio radiophysics 170 Dosimetric characterizations of GZP6 60Co high dose rate brachytherapy sources: application of superimposition method Mohammad Taghi Bahreyni Toossi, Mahdi Ghorbani, Ali Asghar Mowlavi, Ali Soleimani Meigooni Slovenian abstracts 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, Summon by Serial Solutions (ProQuest) Double BioDisk: a new bioprosthetic device for transcatheter closure of atrial septal defects - a feasibility study in adult sheep Dusan Pavcnik1, Kurt Tekulve2, Barry T. Uchida1, Zhong-Huo Luo1, Miran Jeromel14, William G. Van Alstine3, Frederick S. Keller1, Josef Rösch1 1 Dotter Interventional Institute, Oregon Health Sciences University, Portland, Oregon, USA 2 Cook Medical Incorporated, Bloomington, Indiana, USA 3 School of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA 4 Institut of Radiology, University Clinical Center, Ljubljana, Slovenia Radiol Oncol 2012; 46(2):89-96. Received 15 April 2012 Accepted 2 May 2012 Correspondence to: Prof. Dusan Pavcnik, M.D., Ph.D., Dotter Interventional Institute, Oregon Health Sciences University, L342, 3181 SW Sam Jackson Park Road, Portland, Oregon, 97201, USA. Phone: (503) 494-3669; Fax: (503) 494-4258; E-mail: pavcnikd@ohsu.edu Conflict of interest: D.P. reports receiving research grants from Cook Medical. K.T. is Cook Medical employee. W.G.V.A. reports receiving consulting fees from Cook Medical. Background. To evaluate the long-term effectiveness and safety of a new Double BioDisk (DBD) device for closure of atrial septal defect (ASD). Materials and methods. ASD was created with transeptal needle (TS) followed by balloon dilatation in 12 sheep weighing 40.1 to 64 kg (mean 55.2 ± 7.1). The ASD diameters were measured after creation and two weeks later before DBD implantation. The DBDs consists of two nitinol rings 18 to 28 mm in diameter connected with small cannulas and covered with a porcine small intestinal submucosa (SIS). They were implanted via a 10 Fr sheath. DBD effectiveness was evaluated by angiocardiography and by intra-cardiac echogram (ICE) with Doppler studies. Two animals were acute, two were followed for 6 weeks, three for 3 months, three for 6 months and two for 12 months. Results. TS punctures were successful in 10 sheep. In two sheep ASD was created by existing PFO dilation. The ASD size ranged from 13-15 mm (mean 14.1± 0.73 mm) after initial balloon dilation and from 9-13 mm (mean 10.06 ± 1.37 mm) after two weeks. In all animals none of the successfully implanted DBDs spontaneously embolized on release or on follow up. ICE demonstrated no shunting around the DBDs during follows ups. Macroscopic and histologic evaluation of the 6, 12, 24 and 52 weeks animals showed that DBDs were well incorporated in the atrial septum with complete shunt closure. The SIS showed progressive remodeling with the host cells, including endothelization of the DBD devices. Conclusions. ASD closure with the Double BioDisk is safe and effective in adult sheep. Key words: atrial septal defect; transcatheter closure; small intestinal submucosa; biomaterial; embolism; heart septal defects Introduction Since King and Mills reported percutaneous treatment of an atrial septal defect (ASD) in the 1970s, many transcatheter ASD closure devices have been developed.1-5 These ASD closure devices have been also used for patent foramen ovale (PFO) closure. We developed and tested a single disk device cov- ered with porcine small intestinal submucosa (SIS) -BioDisk (BD) - for the closure of PFO in a piglet model.6 For closure of ASD in large animals we developed a Double BioDisk (DBD) covered with SIS. We report on the feasibility, long-term effectiveness and safety of DBD application in adult sheep with percutane-ously created ASD. Sheep were used for testing, since the DBD biological cover is of a porcine origin. FIGURE 1. The double BioDisk (DBD) ASD occlusive device 18 mm in diameter. A. Right atrial side with delivery bar. B. Oblique projection. C. Deployment of the left atrial DBD disk. D. Deployment of DBD across ASD with delivery bar still attached to the delivery wire. E. X-ray of DBD at 3 months. F. Intracardiac echocardiogram 6 months after DBD deployment shows device with thickened discs. No shunting was seen. Material and methods The study protocol was approved by the institutional Animal Care and Use Committee. Twelve adult weighing 40.1 to 64 kg (mean 55.2 ± 7.1 kg) were used for testing the DBD device. A cardiac mobile system (GE/OEC 9800; GE Medical Systems, OEC, Salt Lake City, UT) with digital imaging was used for fluoroscopy and angiocardiography. Angiocardiography was performed with an injector (Medrad Mark Plus, Medrad, Inc. Warrendale, PA). For intracardiac echocardiography (ICE) studies, the AcuNav System (Acuson, Siemens Inc., Mountain View, California) was used. Percutaneous transcatheter creation of ASD Preparation of animals and their anesthesia were described in previous paper.7 Electro cardiogram (EKG), heart rate, oxygen saturation and end tidal CO2 were monitored during the procedure. After induction of general endotracheal anesthesia, the sheep were secured with their back on the radiographic table with their hind limbs in moderate abduction. The neck and the right groin were shaved and prepped. 6 F vascular sheath was percutane-ously placed into the right jugular vein and a short 14 F sheath into the right femoral vein. The jugular vein sheath was used for physiologic monitoring, the femoral vein sheath for procedure performance. The ASDs were created percutaneously with modified transeptal needles (TS) followed by 14 mm balloon dilatation (Cook Medical, Bloomington, IN) as previously described.8 The stretched ASD diameter was then measured with a Coda balloon catheter (Cook Medical). The animals were then recovered and returned to the Department of Comparative Medicine (DMC) for monitoring by veterinarians. Fourteen days after ASDs creation, the sheep were restudied by angiocardiography, ICE study and Coda balloon measurement of the shunts. DBDs were then implanted. Double BioDisk (DBD) device The DBD were constructed as a joint effort between Cook Medical and the Dotter Institute specifications. The DBD consisted of two nitinol rings covered with platinum coil. Both flexible rings were connected with small cannulas and covered with SIS. The cross bar of the right atrial disk was the delivery bar. SIS was sutured with Prolene 6.0 to the radio-opaque rings (Figure 1AB). The DBD was lyophilized and then preloaded by the manufacturer into a 10 Fr cartridge. The delivery system was similar to the system used in the jugular Tulip filter delivery system (Cook Medical). The DBD is self-expanding and self-centering device. The DBD sizes for this study were 18 mm, 23 mm and 28 mm in diameter. Device to defect ratio of 1.8 or larger was used. DBD closures of ASD Preparation of animals, their anesthesia and sheaths placements were similar as for ASD creation. The animals received heparin in dose of 100 IU/kg of body weight. Intravenous saline was administered as needed and respiration rate, expired carbon dioxide, oxygen saturation and EKG were monitored during the procedure. A 5 Fr multipurpose catheter (Cook Medical) was introduced through the femoral vein sheath into the right atrium to catheterize the ASD. A 0.035" Road Runner guide wire (Cook Medical) was used to advance the catheter under fluoroscopy into the left atrium. The Road Runner was then replaced with a long, 0.035" stiff Amplatz wire (Cook Medical). To measure diameter of the ASD, a Coda balloon (Cook Medical) was used. An hourglass appearance of the Coda balloon was indicative of ASD size. No attempts were made to dilate the ASD. After the Coda balloon was removed, a10F Flexor sheath (Cook Medical) was advanced across the ASD into the left atrium. The preloaded DBD was first rehydrated with injection of 10-20 ml of heparinized saline into the cartridge. The delivery bar of the right atrial disk of the DBD was then connected to a hook of a stiff introducing wire. The DBD was introduced into the Flexor delivery sheath and advanced through the ASD into the left atrium. The left atrial disk was then delivered by holding the DBD and by withdrawing the delivery sheath (Figure 1CD). Since the disk is self expanding, it assumed its original rounded shape in the left atrium after exiting the sheath. The DBD was then pulled back against the septum while the other disk connected to the delivery bar was still inside the Flexor sheath in the right atrium. Before releasing the device, the sheath was pulled back to expose the right atrial disk with delivery bar still connected to the hook. Prior to deployment, the DBD position was assessed by contrast injection into the right atrium in the RAO view. When no reflux into the left atrium was found, delivery bar of the DBD was released from the holding catheter. The DBD sizes used for ASD closure were 18 mm in 5 sheep, 23 mm in 4 sheep and 28 mm in one sheep. After DBD deployments angiocardiograms in left anterior oblique and lateral views followed to document the position of the DBD and its effectiveness in occluding the ASD. The delivery catheter and 10 Fr sheath were then removed and replaced with an ICE catheter to evaluate DBD effectiveness with Doppler studies. Two animals were acute; two were followed for six weeks, three for 3 months, three for 6 months and two for 12 months. Acute studies In two acute animals, DBD repositioning and removal were tested prior to delivery catheter removal. First the DBD was expanded and then retracted into the delivery sheath. Then the entire DBD was deployed, but not released. Afterwards it was pulled back into the sheath and redeployed into the ASD. To test its retrievability, the DBD was removed from its ASD position and intentionally embolized into the right atrium. Both the Amplatz gooseneck snare and vascular forceps were used for the retrieval of the embolized DBDs. After retrieval a new DBD was deployed and after 3 hours observation, the acute animals were euthanized. The harvested hearts were cut longitudinally and photographs of the atrial septums were made. Follow-up studies Ten chronic animals were recovered after the procedure and returned to the DMC where veterinarians checked them on a daily basis. Follow-up angiocardiograms of the right atrium and ICE with Doppler studies were done at 6 weeks (n=2), 3 months (n=3), 6 months (n=3) and 12 months (n=2) after DBD placement under the same procedure protocol as in initial studies. After satisfactory views of the DBD effectiveness for ASD closure were obtained, the animals were euthanized and their hearts harvested. After photographs of the atrial septa, the hearts was preserved in buffered formalin and were sent for detailed histological evaluation. Each DBD specimen was embedded in methylmethacrylate blocks. Four cross sections of each device were processed using plastic embedding. All slides were stained with hematoxylin and eosin (H and E) stain using routine methods. Results The ASDs were created safely in all 12 sheep and did not lead to any complications. In 10 sheep ASD RA LA FIGURE 2. Gross specimens of the deployed double Biodisks into the adult sheep ASDs. A. Three hours after deployment, thin layers of early thrombus cover right and left atrium disk of the 28 mm device. B. At 3 months, the 18 mm DBD is almost completely incorporated into myocardium. Glistening disk surfaces indicate complete endothelization. C. At 6 months, the DBD is almost completely incorporated into myocardium of the right and left atrium. D. At 12 months, the DBD is completely incorporated into myocardium of the right and left atrium. were created by puncture and balloon dilation at the fossa ovalis, in the other 2 sheep by balloon dilation of the existing PFO. The stretched ASD size immediately after balloon dilation ranged from 13 to 15 mm (mean 14.1 ± 0.73 mm). At 2 weeks prior to DBD placement the ASD size ranged from 9 -13 mm with a mean 10.06 ± 1.37 mm (Table1). All DBDs were successfully implanted and none spontaneously embolized on release or on follow up. In the first two acute animals, the DBDs were easily retracted into the delivery sheath after their partial or full deployment and redeployed into the ASD. The two intentionally embolized DBDs into the right atrium were safely retrieved and new DBDs were placed into the ASD. Angiocardiograms after DBD deployment showed good placement and absence of shunting. ICE evaluation with Doppler studies did not reveal any shunting around the DBDs. EKGs did not demonstrate any arrhythmias. In the chronic studies, the DBDs were highly visible on x-ray (Figure 1E). There were no fractures of the device frame. Follow up angiocardiograms and ICE Doppler studies documented complete closures of the ASDs and no shunting in all 8 sheep (Figure 1F). Gross examination The implanted DBDs in all animals, acute and chronic, were well self-centered and attached to the ASDs with good apposition to the interatrial septum and adjacent myocardium (Figure 2). Both discs were flattened against the intra-atrial septum and held in place by the connection between the DBD. The DBDs did not obstruct blood flow in to coronary sinus or pulmonary veins nor did it compromise mitral or tricuspid valves. In two acute animals euthanized 3 hours after DBD placement both discs were covered by thin red layer of early thrombus (Figure 2A). In two animals euthanized at 6 weeks, the SIS surfaces on both disks were shiny, transparent and without thrombus. Disks were partially incorporated into the myocardium. In three animals sacrificed at 12 weeks, DBD rings were almost completely incorporated into the myocardium wall. The SIS of the left and right atrium disks were well apposed to the myocardium excluding the ASD opening. The SIS was glistering and shiny indicating endothelization (Figure 2B). The SIS was less transparent than that at 6 weeks. At 24 weeks and 52 weeks DBDs were free from adjacent important cardiac structures in all of the specimens. The SIS membranes of the DBDs were positioned with a good apposition across the ASD FIGURE 3. Histologic cross sections of the deployed double BioDisks into the adult sheep ASDs. A. At 3 months, the right atrial disk of the 18 mm device is apposed and the left atrial disk is incorporated into myocardium. The crossbar is incorporated into the right disk. The disk thickness ranges between 0.1 to 0.25 mm at the right atrial disk and between 1 to 2 mm at the left atrial disk. (Low magnification Hand E) B. At 3 months the center of the left disc is partially remodeled into neointima/connective tissue and contains no inflammatory cells. Remnant SIS is visible in the core of the leaflet. (H and E stain, 40x) C. At 6 months, the 18 mm disks are completely imbedded into myocardium and the distance between them ranges from 2 to 3 mm. The crossbar is incorporated into the right disk. Right atrial disk thickness ranges from 0.4 to 0.8 mm and left atrial disk thickness from 1.5 to 3 mm. (Low magnification Hand E) D. At 6 months the center of the right atrial disk is remodeled into mature neointima/connective tissue and contains no inflammatory cells. Remnant SIS is not visible. All luminal surfaces are lined by endothelium (H and E stain, 40x). TABLE 1. Results of percutaneous atrial septal defect closure Animal Weight No kg Stretched ASD Placement diameter mm DBD size mm successful Follow Up Weeks ICE Follow Up day 1 day 14 Yes or No 1 49.8 15 acute 2 57.6 13 acute 3 60.7 14 11 4 61.6 14 10 5 44.3 15 9 6 47.4 14 9.5 7 53.2 14 9 8 46.3 13 10 9 64.0 15 13 10 46.2 14 9 11 40.1 14 9.2 12 52.2 15 9 ASD=atrial septal defect, DBD=Double BioDisk, Y=yes, ICE= Intra-cardiac Echogram 28 Y acute no shunt 23 Y acute no shunt 23 Y 6 no shunt 23 Y 6 no shunt 18 Y 12 no shunt 18 Y 12 no shunt 18 Y 12 no shunt 18 Y 24 no shunt 23 Y 24 no shunt 18 Y 24 no shunt 18 Y 52 no shunt 18 Y 52 no shunt and fused together with myocardium (Figure 2C). At 12 months, the disks apposition and incorporation into adjacent myocardium was complete, in the right and in the left atrium (Figure 2D). Glistening SIS surfaces were seen in all specimens. The delivery crossbars of the DBDs were covered with shiny tissue and partially fused with device. Histological evaluation Histological sections through the center of the DBDs exhibited well covered ASDs by the device disks. At follow up, there was progressive device apposition and incorporation of disks with adjacent myocardium, progressive SIS remodeling into neointima-connective tissue and progressive SIS endothelization. At six weeks follow up, almost 85% of the rings on the right and 50% on the left atrial side were incorporated into the myocardium and surrounded by fibrous tissue less than 200 microns thick. The residual ring parts not embedded in the myocardium were surrounded by fibrous connective tissue and lined by endothelium. The SIS thickness ranged from 0.1 mm in the center to 2.5 mm near the rings and was covered by spindle shaped cells forming a neointima. Flat endothe-lium covered most of the SIS. The crossbars were surrounded by fibrous tissue. Only minimal inflammatory changes with a few lymphocytes were seen. At 3 months, approximately 95% of the rings on the right side and 60% on the left side were embedded into adjacent myocardium and surrounded by fibrous tissue less than 0.1 mm thick. The SIS showed progressive remodeling into fibrous connective tissue. The SIS disks were 0.1-0.5 mm thick at their centers and 1.5- 3 mm at their periphery. The right disk was thinner than the left disk (Figure 3AB). All discs surfaces were covered by mature endothelium. Crossbars were surrounded by neointima and incorporated into disks. At 6 months, the disks apposition and incorporation into adjacent myocardium was almost complete, 96% in the right atrium and 85% in the left atrium (Figure 3C). The ring wires not embedded into the myocardium were surrounded by neointima lined by endothelium. The SIS discs were composed of mature neointima consisting of fibrous connective tissue. SIS collagen fibers were not observed indicating complete remodeling (Figure 3D). The disks were 0.4-0.9 mm thick at the center and 1.5-3mm at the periphery. The crossbars were incorporated into the disk (Figure 3C). All lumen surfaces of the discs and crossbars were covered by flat mature endothelium. Discussion Adult sheep were used to test the DBD for several reasons. We reported several minimally invasive studies in sheep and we are familiar with this model.910 The size of their heart size approximates the size of the human heart and allows large closure devices to be tested. Adult sheep have a stable body size and when compared to swine that continue to grow and put on weight.611 They allow for long-term follow-up. In addition, the sheep coagulation and fibrinolytic systems are closer to that of humans compared to those in canine or swine.12 Since the DBD cover is composed of swine origin, SIS is a xenogenic biomaterial to sheep.13 The cardiac anatomy of sheep with a more anteriorly positioned heart has a significantly steeper interatrial septum compared to humans that can be challenging for TS puncture. A long TS needle was developed corresponding to the length of sheep body with a curved needle tip allowing TS punctures to be easily and safely accomplished in all of our animals.8 We always punctured and established the ASDs in the area of the fossa ovalis because it allows for a mature stable ASD size. Two weeks after its creation, the ASD size in our animals decreased slightly from a mean 14.1 ± 0.73 mm to a mean 10.06 ± 1.37 mm. The ASD creation was not attempted in the septum secundum because in sheep this structure is significantly thicker than it is in humans. Previous experience showed us that punctures followed by dilations at the septum secundum do not lead to stable ASDs. They heal in two weeks.8 This is our third generation device for cardiac septal closure. The first one, the Monodisk, was developed in the early nineties.4 It consisted of a stainless steel ring covered with two layers of nylon mesh that was positioned on the left atrial ASD side. Three coiled stainless steel wires on the back side of the Monodisk served for its delivery and anchoring at the right atrial ASD side. A 9F introducing sheath was used for its delivery. Six months follow-up studies in canines showed it was effective for closure of ASD 8-10 mm in size. However problems with the Monodisk were its rigid frame, complex delivery, difficult post delivery retrieval and our inability to find a FDA approved nylon mesh alternative. The second generation of the disk closure device - the Biodisk - was developed about 15 years later after we gained experience with nitinol and SIS. The nitinol that was used as a pliable material for the disk frame and the biomaterial SIS was used for the covering. The Biodisk was intended for closure of PFO. It consisted of one disk inserted into the left atrial ASD side. Nitinol wires of the disk were covered by platinum coil and a crossbar attached to the disk was used for delivery and for anchoring on the right atrial ASD side. An 8F sheath was used for Biodisk delivery. Initial testing in piglets showed its easy delivery, good retrievability and long-term effectiveness for closure of PFO measured with the 10-14 mm balloons.6 The DBD with two disks was developed for closure of ASD. The Biodisk with only one disk would not be sufficient for the occasional complex anatomy and large size of ASDs. The DBD feasibility study demonstrated that this new biopros-thetic device has excellent potential for ASD closures. The DBD was easy to deploy, it self-centered during deployment and covered the entire ASD without encroachment on structures in both atria. When needed, the DBD could be repositioned or retrieved if released inappropriately or lost as shown after its intentional embolization. The DBD showed excellent effectiveness for ASD closures and no residual shunting was seen at ICE examinations with color Doppler studies. The gross studies showed good apposition of the disks to the septum and myocardium without compromise of valves and other cardiac structures. At 3 hours after DBD placement, a thin layer of early thrombus covered the SIS disks as initial the phase of neointi-mal formation. Otherwise, no thrombus was found on the follow-up studies as the DBDs rapidly en-dotheliolized. The histologic follow-up studies then revealed progressive apposition leading to full incorporation of the DBD disks with adjacent myocardium, progressive SIS remodeling into connective tissue and complete DBD endothelization. These healing processes were well demonstrated on the histologic DBD cross sections. The space between the disks, originally separated by thick septum secundum and residual septum primum progressively decreased by neointimal formation and some atrophy of residual septum. The neointimal formation started at the disks periphery, extended to the center of the disks. It was more pronounced on the left atrial SIS disks. The 6 and 12 months follow-up showed complete SIS remodeling into the heart connective tissue. The DBD feasibility study did not compare the biomaterial SIS with septal occluders covered with synthetic fabrics. This was done in detail by Jux et al. in 2006.14 They compared the first septal occluder device with biodegradable matrix, the Biostar covered with the purified intestinal collagen layer (ICL) with the Starflex covered with a knitted pol- yester fabric. The ICL, similar to SIS, originated from porcine small intestinal mucosa and both had similar thickness between 150-200 microns. A 10F sheath was used for deployment of both the Biostar and DBD. The study by Jux et al. in young sheep that from 7 days to 2 years showed distinct advantages of the biomaterial matrix. Biostar had decreased thrombogenicity, particularly when the device was heparin coated. It showed accelerated healing with early endothelization and low immune response with fast ICL remodeling into connective cardiac tissue. Because of these positive results, the Biostar has already been applied successfully in treatment of ASD in children and adults.151617 Conclusions Long term both right and left atrial SIS disks were remodeled into the heart connective tissue, so that only a minimal amount of metal spring material has been left behind. ASD closure with the Double BioDisk is safe and effective in adult sheep. Acknowledgments This study was sponsored by Cook Medical, Bloomington, Indiana. The authors thank William E. Schoenlein and the team from Purdue University, West Lafayette, Indiana and Garry Pressler and the team from MED Institute, Inc., West Lafayette, Indiana for their assistance with the animal experiments and the histology. The authors thank Sheri Imai-Swiggart for her assistance. References 1. King TD, Mills NL. Nonoperative closure of atrial septal defect. Surgery 1976; 75: 383-8. 2. Rao PS. History of atrial septal occlusion devices. In Rao PS and Kern MJ: Catheter based devices: for the treatment of non-coronary cardiovascular disease in adults and children. Philadelphia: Lippincott Williams&Wikins; 2003. p. 3-9. 3. Majunke N, Sievert H. ASD/PFO Devices: What Is in the Pipeline? J Intervent Cardiol 2007; 20: 517-23. 4. Pavcnik D, Wright KC, Wallace S. Monodisk: device for percutaneous transcatheter closure of cardiac septal defects. Cardiovasc Intervent Radiol 1993; 16: 308-12. 5. Kim MS, Klein AJ, Carroll JD. Transcatheter closure of intracardiac defects in adults. J Interv Cardiol 2007; 20: 524-45. 6. Pavcnik D, Tekulve K, Uchida B, Pavcnik Arnol M, Van Alstine WG, Keller FS, et al. Biodisk: a new device for closure of patent foramen ovale. Catheter Cardiovasc Interv 2010; 75: 861-7. 7. Ni RF, Kranokpiraksa P, Pavcnik D, Kakizawa H, Uchida BT, Keller FS, et al. Testing percutaneous arterial closure devices: An animal model. Cardiovasc Intervent Radiol 2009; 32: 313-6. 8. Uchida BT, Pavcnik D, Shimohira M, Choi YH, Jeromel M, Keller FS, et al. New coaxial transseptal needle for creation of atrial septal defects in adult sheep. Cardiovasc Intervent Radiol 2011; 34: 620-5. 9. Kranokpiraksa P, Pavcnik D, Kakizawa H, Uchida BT, Jeromel M, Keller FS, et al. Hemostatic efficacy of chitosan-based bandage for closure of percutaneous arterial access sites: An experimental study in heparinized sheep model. Radiol Oncol 2010; 44: 86-91. 10. Wei Lu, Pavcnik D, Uchida B, Park WK, Liu L, Timmermans HA, et al. The ovine jugular vein as a model for interventional radiology procedures. Radiol Oncol 2008; 42: 59-65. 11. Jeromel P, Pavcnik D. Infrahepatic caudal/inferior vena cava interruption with azygos/hemiazygos continuation. Vascular anomaly in swine. Radiol Oncol 2010; 44: 149-52. 12. Narayanawamy M, Wright KC, Kandarpa K. Animal models for atherosclerosis, restenosis, and endovascular graft research. J Vasc Interv Radiol 2000; 11: 5-17. 13. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007; 28: 3587-93. 14. Jux C, Bertram H, Wohlsein P, Bruegmann M, Paul T. Interventional atrial septal defect closure using a totally bioresorbable occluder matrix: development and preclinical evaluation of the BioSTAR device. J Am Coll Cardiol 2006; 48: 161-9. 15. Hoehn R, Hesse C, Ince H, Peuster M. First experience with the biostardevice for various applications in pediatric patients with congenital heart disease. Catheter Cardiovasc Interv 2010; 75: 72-7. 16. Morgan G, Lee KJ, Charturvedi R, Benson L. A biodegradable device (Biostar) for atrial septal defect in children Catheter Cardiovasc Interv 2010; 76: 241-5. 17. Mullen MJ, Hildick-Smith D, De Giovanni JV, Duke C. BioSTAR evaluation study (BEST): a prospective, multicenter, phase I clinical trial to evaluate the feasibility, efficacy, and safety of the BioSTAR bioabsorbable septal repair implant for the closure of atrial-levels shunts. Circulation 2006; 114:1962-7. Outcome of MRI-guided vacuum-assisted breast biopsy - initial experience at Institute of Oncology Ljubljana, Slovenia Marta Zebic-Sinkovec1, Kristijana Hertl1, Maksimiljan Kadivec1, Mihael Cavlek1, Gasper Podobnik1, Marko Snoj2 1 Department of Radiology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Surgery, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2012; 46(2): 97-105. Received 13 October 2011 Accepted 28 November 2011 Correspondence to: Marta Zebič-Šinkovec, MD, Department of Radiology, Institute of Oncology Ljubljana, Zaloška 2, 1000 Ljubljana, Slovenia. Phone: +386 1 5879 940; Fax: +386 1 5879 407; E-mail: mzebic@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Like all breast imaging modalities MRI has limited specificity and the positive predictive value for lesions detected by MRI alone ranges between 15 and 50%. MRI guided procedures (needle biopsy, presurgical localisation) are mandatory for suspicious findings visible only at MRI, with potential influence on therapeutic decision. The aim of this retrospective study was to evaluate our initial clinical experience with MRI-guided vacuum-assisted breast biopsy as an alternative to surgical excision and to investigate the outcome of MRI-guided breast biopsy as a function of the MRI features of the lesions. Patients and methods. In 14 women (median age 51 years) with 14 MRI-detected lesions, MRI-guided vacuum-assisted breast biopsy was performed. We evaluated the MRI findings that led to biopsy and we investigated the core and postoperative histology results and follow-up data. Results. The biopsy was technically successful in 14 (93%) of 15 women. Of 14 biopsies in 14 women, core histology revealed 6 malignant (6/14, 43%), 6 benign (6/14, 43%) and 2 high-risk (2/14, 14%) lesions. Among the 6 cancer 3 were invasive and 3 were ductal carcinoma in situ (DCIS). The probability of malignancy in our experience was higher for non-mass lesion type and for washout and plateau kinetics. Conclusions. Our initial experience confirms that MRI-guided vacuum-assisted biopsy is fast, safe and accurate alternative to surgical biopsy for breast lesions detected at MRI only. Key words: breast cancer; MRI; MRI guided vacuum assisted biopsy Introduction Magnetic resonance imaging (MRI) is a method for the detection of many cancers.12 Contrast-enhanced magnetic resonance imaging (CE-MRI) is currently the most sensitive additional imaging method for the detection of invasive breast carcinoma and seems to be able to detect ductal carcinoma in situ (DCIS), especially high grade DCIS without necrosis.3 Compared with studies of MRI performance published in the 1990s, the specificity of breast MRI has gradually improved, mostly be- cause of improved technology and increased reader experience.4 Suspicious MRI detected lesions are not always detectable by mammography and ultrasound. Positive predictive value for lesions detected by MRI alone ranges between 15% and 50% and it depends upon patient selection and MR interpretation algorythm.5-8 MRI guided procedures (needle biopsy, presurgical localisation) are mandatory for lesions visible at MRI only, when they look suspicious and have potential influence on therapeutic decision.9 MRI-guided vacuum assisted biopsy O (D FIGURE 1 A,B. Biopsy coil device. Photographs show a four-channel breast biopsy coil with positioning device (A) and a grid-positioning device (B). (MR-VAB) was pioneered by Sylvia H. Heywang-Köbrunner and first described in 1999.10 Since then, the technique has been optimized and its reproducibility proven in an European multicenter study.11 Some other authors have published their experience with this method and also with other vacuum devices.1213 In 2006 there was an interdisciplinary consensus conference in Nordestedt, Germany.14 The purpose of this meeting was to determine technique and optimize quality assurance protocols. MR-VAB was first time performed at our institution in 2008 and since then it has become routinely used in our practice. The aim of the present retrospective study was to evaluate the initial clinical experience with MRI-guided vacuum-assisted breast biopsy as an alternative to surgical excision and to investigate the outcome of MRI-guided breast biopsy as a function of the MRI features of the lesions. Patients and methods Patients and lesions The selection criteria for the MR-guided biopsy were the presence of Breast Imaging Reporting and Data System (BI-RADS) 5, BI-RADS 4 and BIRADS 3 lesions that were visible by CE-MRI only. Initially, fifteen patients with 15 lesions had been referred for MR-VAB. In 1 patient the lesion could not be reproduced on the preinterventional planning MRI because of the technical problem. The median age of patients was 51 years (range, 35-71 years). In 6 patients mammography performed before biopsy showed dense breast or benign changes, in 2 patients asymmetry was described, in three cases there was architectural distortion only in one projection, in two cases mammographic and MRI findings were discordant and one patient was too young to perform mammography. In 7 patients sonography performed before biopsy failed to reveal a sonographic correlate and in 4 others sonography and MRI findings were discordant. In 3 patients sonography was not performed at the discretion of the radiologist because the possibility of identifying a sonographic lesion was presumed to be very low. MRI findings before biopsy Except the MRI examinations of 2 women who underwent MRI at another institution, breast MRI was performed using a 1.5-T magnet (Magnetom Avanto, Siemens Medical Solution, Erlangen, Germany) with a dedicated bilateral breast surface coil with a prone position. The imaging protocol and parameters were as follows: axial T1-weighted image (TR/TE, 593/13) and short time inversion recovery (STIR) (TR/TE/TI, 12390/76/130) of both breasts were obtained with 3 mm slice thickness. Next, T1-weighted images were acquired using a 3D fast low-angle shot pulse sequence (FLASH) through both breasts (TR/TE 7, 8/4, 72, flip angle 25°). Precontrast images were obtained over a 313 x 448 matrix in the axial plane with a slice thickness of 1.0 mm with distance factor 20% before administration of the contrast agent. Then, contrast-enhanced dynamic imaging was performed with an injection of 0.1 mmol per kilogram of body weight of gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany); five se- quential contrast-enhanced images were acquired at every 1 min 23 s. The precontrast images were then subtracted from the corresponding postcon-trast images on a pixel-by-pixel bias with the use of the standard software subtraction function available on our console. Two radiologists reviewed all imaging studies in consensus. The BI-RADS MRI lexicon was used. MRI-guided biopsy MRI-guided vacuum-assisted breast biopsy was performed using a 1.5-T magnet (Magnetom Avanto, Siemens, Erlangen, Germany) with MRI-supported Breast Immobilization and Biopsy System with the 4-channel breast coil (Noras MRI products GmbH, Höchberg, Germany) in prone position. Marker Block filled with diluted gadolinium contrast agent was used for reference point. The positioning device has medial and lateral compression plates for moderate compression. An axial T1- weighted images were acquired using a 3D FLASH through both breasts (TR/TE 7, 6/4, 72, flip angle 25°). Precontrast images were FIGURE 2. 1.5 T Magnetom Avanto (Siemens, Erlangen, Germany). Patient lie face down on the exam table. Breast is moderately compressed in the mediolateral direction. obtained over a 256 x 320 matrix in the axial plane with a slice thickness of 1.0 mm with distance factor 20% before administration of the contrast agent. Twenty seconds after contrast agent had been in- lfi.11 CyiVLEit'MifAEL FIGURE 3A-D. MRI-guided biopsy using axial, contrast-enhanced FLASH 3D T1W images. The pre-biopsy fat-suppressed image (A) shows an enhancing lesion in the right breast. The biopsy needle (B) is seen after localization, along with the lesion. Following the biopsy (D) the small magnetic susceptibility artefact due to the clip in situ is seen. ® CD FIGURE 4. MRI-guided biopsy device (a) is inserted into breast to acquire tissue specimens (b) jected; another axial T1-3D FLASH sequence was performed. 0.1 mmol per kilogram of body weight of gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany) was injected with a rate of 2 ml/s using an automated injector (CT/MRI injector Mississippi, Ulrich medical, Ulm, Germany). The volume of interest was selected to include the compression device and a Marker Block. Biopsies were performed with a 9-gauge MRI compatible vacuum-assisted biopsy device (ATEC, Suros Surgical Systems, Indianapolis, USA). CAD stream diagnostic and interventional guidance tool, Version 4.1 (Confirma, Washington, USA) was used to target coordinates for biopsy. An axial T1-3D FLASH sequence was performed to control the needle placement. The biopsy device was then placed into the breast through the coaxial needle. Multiple samples were obtained by turning the needle clockwise and around its axis. The biopsy site was marked with a titanium clip (ATEC TRI mark TD 13 - MR Biopsy Site Marker). "Postclip" axial 3D FLASH was performed to assess clip deployment. After biopsy, the breast is compressed with ice, sterile strips are applied, and a sterile gauze bandage is applied. MRI-guided wire localization The procedure for wire placement was the same as that for breast biopsy. After localizing the lesion, a cannula with a wire loaded into it was inserted to the required depth. After confirming the position on axial images, the wire was placed into the lesion and the position was verified with a repeat axial scan. The wire gives a thin linear magnetic susceptibility artefact, and the lesion can be seen near the tip of the wire. It was then fixed with a lock at the skin surface to prevent dislodgement. The same positioning device for lesion localization with an MRI-compatible needle with a hooked wire, made of a special alloy for easy and safe penetration of the solid tumour tissue (TULOC, Somatex, Germany) were used. It has a diameter of 0.95 mm and it is 90 mm long. The wire was fixed into the lesion with the hooks at the end of the wire and onto the skin with a lock which prevents any dislodge-ment of the wire. Indication for MRI and MRI-VAB The indications for MRI were classified into a screening setting and a diagnostic setting. The indications for (MR-VAB) were: MRI only seen lesions - targeted » second look« ultrasound and/or mammography showed normal or benign findings or findings that were not concordant with MRI findings. Ultrasound was not performed at the discretion of radiologist, because the probability that lesion would be seen by ultrasound was very low. Management, follow-up, data collection, analysis The MR-VAB was presumed to be adequate if imaging performed immediately after biopsy showed TABLE 1. MRI findings of 14 targeted lesions and the probability of malignancy Features No. of Lesions/ Total No. of Lesions (%) No. of Malignant Lesions/ No. of Lesions (%) Lesion type Focus 0 0 Mass 5/14 (36) 1/5 (20) Nonmass 9/14 (64) 5/9 (55) Total 14/14 (100) 6/14 (43) Morphology Margin of mass Smooth 2/14 (14) 0/2 (0) Nonsmooth 3/14 (21) 1/3 (33) Distribution of Nonmass Regional 1/14 (7) 1/1 (100) Segmental 1/14 (7) 1/1 (100) Ductal 6/14 (43) 2/6 (33) Focal areas 1/14 (7) 1/1 (100) Total 9/14 (64) 5/9 (55) Kinetic feature Persistent Plateau Washout Total 1/14 (7) 6/14 (43) 7/14 (50) 14/14 (100) 0/1 (0) 2/6 (33) 4/7 (57) 6/14 (43) Signal intensity T2-weigted images High 4/14 (23) 2/4 (50) Intermediate 8/14 (57) 3/8 (37) Low 2/14 (14) 1/2 (50) Total 14/14 (100) 6/14 (43) a cavity that unequivocally included the area of highest suspicion. Two view mammograms were also performed after biopsy to confirm clip localization. Surgical follow-up was recommended for all malignant lesions, for one high-risk lesion, and in one case, when the benign lesion was judged to have pathology result that was discordant with imaging findings. 6 of the 6 malignant lesions and 1 of the 2 high-risk lesions were operated upon at our institution and confirmed by operative histology. In 4 cases breast ablation and in 3 cases sentinel node and occult lesion localization (SNOLL) under X-ray guidance and surgical excision was performed. The reference standards were core biopsy results and postoperative histology. Medical records were reviewed for patient age, personal history of breast cancer, indication for MR mam-mography, radiologic results, histopathologic results and follow-up results. Histopathologic re- sults were examined on the basis of pathologic report of MR-VAB and postoperative histology. The probability of malignancy for an MRI abnormality was calculated as the ratio between the number of lesions with pathologically proven malignancy and the number of biopsied lesions. We investigated the probability of malignancy for MRI abnormalities according to the MRI features of the lesions. The retrospective study was carried out according to the Helsinki Declaration. Results The biopsy was technically successful in 14 (93%) of 15 women. Median age was 51 years (range, 3571 years). In 1 woman the biopsy was deferred because of a technical problem. O TABLE 2. The characteristics of the malignant lesions Site of the lesion Right 2 Left 4 Type of the lesion MLE 1 NMLE 5 T2-weighted images Signal intensity High 2 Intermediate 3 Low 1 Kinetics Rapid-plateau 2 Wash-out 4 Size 2.6cm ( range; 0.8 - 6 ) Histologic type Of cancer Invazive lobular 2 Invazive tubular 1 DCIS 3 MLE = masslike enhancemnt; NMLE = nonmasslike enhancement; DCIS = ductal carcinoma in situ ® FIGURE 5A-C. 65 year old woman with a history of nipple-discharge in her left breast. Mammographic asimmetry. Ultrasound images showed dilated empty ducts, ductography was suspicious for papilloma. Axial T1-weighted subtracted image after Gadolinium injection( 2nd minute) .(a) small round sharply circumscribed masses within a duct sistem-papillomas.(b) segmental, clumped, asymmetric enhancement, fast initial increase and postinitial wash out. (c) high signal intensity on T2 -weighted image - BI-RADS 5. Pathologic diagnosis through MRI-guided vacuum-assisted biopsy was massive DCIS with foci of microinvasion. Fourteen lesions underwent biopsy, among them 4 lesions were categorized as BI-RADS 5, 8 lesions as BI-RADS 4 and 2 as BI-RADS 3. The median size of these 14 lesions was 2 cm (range, 0.8-6 cm). Among those 14 lesions, 4 were located between lower quadrants, 2 in lower outer quadrant, one between outer quadrants, 3 in upper out- er quadrant, 3 between upper quadrants and one in upper inner quadrant. For 13 of the14 lesions a single skin incision was made; for one lesion, a second incision was required for repositioning the stylet before biopsy. The median number of specimens obtained per lesion was 8 (range, 4-17). In 11 lesions, only a single round of tissue acquisition was necessary, in 3 lesions the MRI after the first round of tissue acquisition did not ensure lesion sampling, and a second round of tissue acquisition was performed. Clip placement was attempted in 14 lesions and was successful in 13 (93%). The median time to perform MRI-guided vacuum-assisted biopsy, from the original axial localizing images to the final images obtained after clip deployment was 39 min (range, 28-60 min). A complication was encountered in 1 of 14 patients (7%). The complication was a clinical haematoma. Cancer was found in 6 of 14 lesions and in 6 of 14 women, based on the review of vacuum-assisted biopsy and surgical histology. Six were benign lesions and 2 were high-risk lesions. In two cases MR-VAB was considered uncertain according to the correlation of imaging and histopathology, therefore surgical biopsy and re-biopsy were performed. Among these 6 cancers (Figures 1-6) 3 were invasive cancers (2 invasive lobular carcinoma and 1 invasive tubular carcinoma) and other 3 were DCIS (1 massive DCIS with foci of well differentiated invasive carcinoma). Three of 6 cancers were found in women with personal history of breast carcinoma. The median size of the MRI lesions in these 6 cancers was 2.6 cm (range 0.8-6.0 cm). MRI-guided vacuum-assisted biopsy revealed 2 high-risk lesions, in one case there was atypical ductal hyperplasia and in other papilloma. Papilloma was operated on and was proved to be benign. MRI review suggested that the MRI target might have been excised at vacuum-assisted biopsy in one of these cancers and was sampled in five. Surgery was performed in our institution on 6 of the 6 malignant lesions on 1 of the 2 high-risk lesions and on 1 lesion with discordant result. Finally, 6 of the 14 biopsy lesions were malignant the overall probability of malignancy for an MRI abnormality was 43% (6/14). The MRI features of the lesions and the probabilities of malignancy according to lesion features are summarized in Table 1. The characteristics of malignant lesions are summarized in Table 2. Discussion MRI-guided-vacuum assisted biopsy has many advantages compared with other biopsy methods for the diagnosis of MRI-detected lesions. In our initial experience with this method, the technical success rate was 93%. In 57% of women MR-VAB yielded benign results and spared most women with MRI-detected lesions the need for the surgical excision. With MR-VAB a continuous suction and acquisition of larger tissue volume is possible. Only MR-VAB allows good visualisation of the cavity and/or direct visualisation of the size reduction of the enhancing lesion. It retrieves larger volume of tissue, so we have fewer inadequate specimens. If a lesion is deemed to be not accessible to MR-VAB, MR-guided needle localization followed by surgery can be performed. Our results are in agreement with previous findings in literature.15-19 MR-VAB can be performed quickly.20 Average time to perform biopsy of a single lesion was 39 min in our study. Lesion visibility decreases rapidly over time during the biopsy, due to wash out of contrast in the lesion. The lesion must be identified immediately after the contrast injection. Sampling is dependent on identification and immobilizing the lesion. Lesions that underwent MR-VAB were rated by 2 experienced radiologists in consensus. The BI-RADS MRI lexicon was used. The publication of BI-RADS MRI lexicon in 2003 was an important step toward a standardized approach on the lesion description, but should be regarded as work in progress. Since then some studies have been published about the value of the BI-RADS lexicon ® ® FIGURE 6AB. 47 year old woman with history of invasive breast carcinoma 10 years ago in her left breast. Retraction of mammila on the right, mammograpic dense breast and benign calcifications. First look and second look ultrasound images showed a cyst. Axial T1-weighted subtracted image after Gadolinium injection (2nd minute).(A) 8x3 mm ductal - linear homogenous, asymmetric enhancement, fast initial increase with postinitial plateau (B) high signal intensity on T2-weighted images, categoriaztion BI-RADS 4. MRI-guided vacuum-assisted biopsy revealed high-grade DCIS withouth microcalcification. FIGURE 7. 64-year old woman with history of invasive breast carcinoma in her right breast 10 years ago. Mammography and ultrasound images showed discrete architectural distortion in the left breast. US-guided core biopsy was inconclusive. Axial T1-weighted subtracted image after Gadolinium injection (2nd minute) shows regional, heterogeneous, asymmetric, non-mass like enhancement, fast initial increase, post initial wash-out, intermediate signal intensity on T2-weighted images - BI-RADS 5. Pathologic diagnosis through MRI-guided vacuum-assisted biopsy was DCIS. FIGURE 8. 57-year old woman, US-guided fine needle biopsy performed at another institution was suspicious for pappilary carcinoma. Mammographically dense breast, first and second look ultrasound images at institute of oncology showed simple cyst. Axial T1-weighted subtracted image after. Gadolinium injection (2nd minute) shows ductal, reticular- dendritic, asymmetric, non-mass like enhancement, fast initial enhancement with post initial plateau. Intermediate signal intensity on T2-weighted images. BI-RADS 4. MRI-guided vacuum-assisted biopsy revealed invasive tubular carcinoma. FIGURE 9. 63-year old woman with history of invasive ductal carcinoma in her right breast 8 years ago. Clinical exam revealed enlarged lymph node in the left axilla. Fine needle aspiration biopsy showed metastatic lymph node. Mammographically discrete architectural distortion only in one projection in the left and postoperative changes in the right breast. Ultrasound was not performed at the discretion of the radiologist. Axial T1-weighted subtracted image after Gadolinium injection (2nd minute) shows focal, heterogeneous, asymmetric, non-mass like enhancement in the left breast, the kinetic curve shows fast initial enhancement with post-initial wash out, low signal intensity on T2-weighted images - BI-RADS 4. MRI -guided vacuum-assisted biopsy revealed invasive lobular carcinoma. and some new predictors for the differentiation of benign from malignant non-mass lesions have been suggested.21-24 Most of the patients in our series underwent mammography and sonography before MRI-VAB. Only in 3 patients sonography was not performed at the discretion of radiologist. This criteria increased the relative number of non-mass lesions in our study (9/14, 64%) and the number of lesions that are 1cm or smaller in size (6/14, 43%). These lesions are by their nature not well shown by sonography or mammography. Among the cancers, the lesions had non-mass like enhancement characteristics in 5 of 6 cases (83%) and 2 of 6 cancers were smaller than 1cm. Among 5 patients with non-mass like enhancement pattern and proved malignancy, 3 had personal history of breast carcinoma - should be MRI screening a part of an annual follow-up for patients diagnosed with breast cancer.25 On T2-weighted images one cancer showed low signal intensity, three intermediate and 2 high signal intensities. Among 5 non-mass like enhancing lesions there was one lesion with low signal intensity on T2 weighted image, 2 lesions with intermediate and 2 lesions with high signal on T2 weighted images. Both lesions with high-signal were DCIS, one with ductal, homogenous enhancement smaller than 1 cm and the other one with segmental clumped enhancement 6 cm in size. As published in literature, the permeability of the basement membrane of DCIS-containing ducts, is increased, allowing gadolinium chelates to penetrate the membrane and accumulate within the DCIS-filed milk ducts. That is proposed to be the explanation for enhancement of purely DCIS on breast MR Images.26-31 In some cases, after MRI-VAB, benign histology offers no explanation for a contrast-enhancing lesion. The radiologic/pathologic mismatch is more problematic for MRI-VAB than for mammograph-ically-guided interventions because no specimen radiography can be used to verify appropriate core biopsy. If any doubt persists, early post-biopsy breast MRI is performed in our practice but not earlier than after 6 months.32 There were 2 cases, where after 6-months follow up the lesions were FIGURE 10. 71-year old woman, MR mammography performed in the other institution. Axial Tl-weighted subtracted image after Gadolinium injection (2nd minute) shows 3 round masses in her left breast, with spiculated margins, rim like enhancement, intermediate signal intensity on T2-weighted images. Kinetic curve shows fast initial enhancement with post-initial wash out. Lesions were categorized as BI-RADS 5. Pathologic diagnosis through MRI- guided vacuum-assisted biopsy was invasive lobular carcinoma. unchanged in size and morphology. In one case the surgical excision revealed radial scar. In the other one re-biopsy was done and the lesion proved to be benign. Our study has some limitations. Our series is small and represents our initial experience. Because this study is retrospective, the lesion descriptions in a few cases did not use terms from the BI-RADS MRI lexicon, thus, we modified them as much as possible. Two patients underwent MRI before biopsy at other institution and we could not analyze morphologic and kinetics, only the reports were available. 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MRI follow-up after concordant, histologically benign diagnosis of breast lesions sampled by MRI-guided biopsy. AJR Am J Roentgenol 2009; 193: 850-5. Differentiation of malignant and benign lung lesions with diffusion-weighted MR imaging Sevtap Gümüstas1, Nagihan Inan1, Gür Akansel1, Ercüment Çiftçi1, Ali Demirci1, Sevgiye Kaçar Özkara2 1 Department of Radiology, 2 Department of Pathology, School of Medicine, University of Kocaeli, Turkey Radiol Oncol 2012; 46(2): 106-113. Received: 1 August 2011 Accepted: 8 January 2012 Correspondence to: Sevtap Gümüstas, Kocaeli Uni. Tip Fak Radyoloji AD, 41380 Umuttepe Kocaeli, Turkey. Phone: +90 262 3037126; Fax: +90 262 303 80 03; E-mail: svtgumustas@hotmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to evaluate the role of diffusion-weighted magnetic resonance imaging in the differential diagnosis of lung lesions. Patients and methods. Sixty-seven patients with lung lesions (48 malignant, 19 benign) were included in this prospective study. Signal intensities (SIs) were measured in diffusion-weighted MR images that were obtained with b=0, 500 and 1000 s/mm2 values. Apparent diffusion coefficient (ADC) maps were calculated by using images with b=0 and 1000 s/mm2 values. The statistical significance was determined using the Student-t test. Results. The SIs of malignant lesions were significantly higher than those of benign lesions (p<0.004 for b=0 s/mm2 and p<0.000 for the other b values). Using b=500 s/mm2, SI>391 indicated a malignant lesion with a sensitivity of 95%, specificity of 73% and positive predictive value of 87%. Using b=1000 s/mm2, SI>277 indicated a malignant lesion with a sensitivity of 93%, specificity of 69% and positive predictive value of 85%. There was no significant difference between malignant and benign lesions regarding ADC values (p=0.675). There was no significant difference in SIs or ADC values between small cell carcinoma and non-small cell carcinoma. When comparing undifferentiated with well- partially differentiated cancers, SIs were higher with all b values, but the difference was statistically significant only with b=1000 s/mm2 (p<0.04). Conclusions. Diffusion-weighteted MR trace image SI is useful for the differentiation of malignant versus benign lung lesions. Key words: pulmonary lesions; diffusion-weighted imaging; apparent diffusion coefficient; magnetic resonance imaging Introduction Lung cancer is one of the leading causes of death.1 It usually arises as a solid nodule or mass on chest radiography or computed tomography (CT). Although many well known characteristics have been described for nodule differentiation on CT, it remains a challenge for radiologists to differentiate lesions as malignant or benign.2-5 In recent years, fluorine-18 fluorodeoxyglucose positron emission tomography (PET) has been used for this purpose. Both CT and PET deliver high doses of radiation. In addition PET has been known to give false-positive results in inflammatory masses.6-9 For these reasons, an accurate and safe alternative method is still desirable for the determination of malignant versus benign pulmonary lesions. Recent advances in fast imaging techniques like echo-planar imaging, makes magnetic resonance imaging (MRI) more suitable for chest applications.10-12 There are reports using dynamic contrast MRI of lung masses.1314 Diffusion-weighted magnetic resonance imaging (DWI), initially used in the central nervous system, has been increasingly applied in other body areas, such as the mediasti-num15, pancreas16, and liver.1718 The aim of our study was to determine whether quantitative analysis of DWI could be helpful in the differentiation of malignant and benign pulmonary lesions. Udri 10H4 SumlamJ "Jflj u * «3D Pon-^KT R.Snwr Un Kfe-mt Stu* w Wi S-IHiUit -Willi- f USA tum 3TIH r*r2 f.Hy "Lj □ rri"i rnn Kts-TJT liti/ r* A 10 mm in diameter in view of the limited planar resolution of DWI; (c) presence of a specific proven diagnosis of the lesion either histopathologically or by using clinical, radiologi- cal and laboratory data or based on at least 1-year radiological follow-up; (d) no current administration of chemotherapeutic or radiotherapeutic treatment; (e) absence of any contraindications for MRI; and (f) ability of patients to lie still and hold their breath approximately 26 seconds in the MRI unit. Biopsies of lung lesions were carried out by the radiologist (EÇ) in the interventional radiology department of the same hospital. MRI was performed on these patients on the same day but before the percutaneous biopsy in order to avoid hemorrhage-related distortions. The study was carried out according to the Helsinki Declaration. MR Imaging All patients were examined with a 1.5 Tesla MR unit (Gyroscan Intera; Philips Medical Systems, Eindhoven, The Netherlands) using a four-element phased-array body coil. This system had a maximal gradient strength of 30 mT/m and a slew rate of 150 mT/m/msec. All patients were examined initially with the routine MRI protocol for the thorax that included: precontrast axial T1-weighted (W) breath-hold spoiled gradient echo (fast field echo: FFE) (TR/TE/FA/NEX:169/4.6/80/1) and coronal and axial T2-W single-shot turbo spin-echo (TR/TE/ NEX/TSE factor: 700/80/1/72). Subsequently, three series of axial single-shot spin-echo echo-planar (SS-SE-EPI) DWI (1,000/81; echo-planar imaging (EPI) factor, 77; sensitizing gradients in x, y, and z directions) were acquired using b = 0, 500 and 1000 s/mm2 values. ADC maps were reconstructed from the b = 0 and b = 1000 s/mm2 images. MRI, including DWI, consisted of a multisection acquisition with a slice thickness of 6 mm, an intersection gap of 1 mm, and an acquisition matrix of 128 x 256. All sequences were acquired using a partially parallel imaging acquisition and SENSE (sensitivity encoding) reconstruction with a reduction factor (R) of 2. The scanning time of the acquisition of each DWI series during a single breath-hold was 26 seconds. Quantitative analysis Quantitative measurements were made using a dedicated Workstation (Dell Workstation Precision 650, with the View Forum software platform provided by Philips Healthcare). All images were assessed by two radiologists (SG, NI) who were blinded to the clinical history of the patients. First, CT images were evaluated in order to assess the calcification, necrosis and GGO components. CT TABLE 1. Histopathological and clinical diagnosis of lesions Diagnosis Chronic inflammatory changes (n=9) Sarcoidosis (n=3) Acute bacteriel pneumonia (n=2) Tuberculosis pneumonia (n=2) Romatoid nodule (n=1) Granuloma (n=1) Hamartoma (n=1) SCLC (n=6) NSCLC (n=42) NSCLC subgroup; Adenocarcinoma (n=13 ) Squamous cell carcinoma (n=5 ) Large cell carcinoma (n=2 ) Nonidentified NSCLC (n=22 ) SCLC = small cell lung cancer, NSCLC = non-small cell lung cancer scans were also evaluated for contour characteristics of the lesions (irregular or smooth) and concomitant interstitial findings were recorded. Those findings were compared with the DWI findings. The range of interval between the CT and MRI examinations was 0-10 days (mean, 5. 6 days). Then, the lesion was visualized once more on the conventional T1-W and T2-W MRI in terms of location, size and content of cystic-necrotic parts to detect interval changes since the time of the CT scan. These conventional images were only used for the lesion identification and not for the analysis. Afterwards, signal intensity (SI) of the lesion was measured for each b value (0, 500 and 1000 s/mm2) on DWI using a round or elliptical region of interest (ROI). The ROI was placed centrally, and the size of the ROI was kept as large as possible, covering at least two-thirds of the lesion, yet avoiding the interference from the surrounding lung tissue, necrotic parts and major blood vessels. ADCs were then calculated from the ADC maps that were reconstructed from b = 0 and b = 1000 s/mm2 values. Statistical analysis SIs and ADCs were compared between the groups. The fitness of numeric data set to normal distribution was determined using the Kolmogorov-Smirnov test. The data were normally distributed, so the differences in SIs and ADCs were analyzed using the Student-t test. A p value of less than 0.05 was considered statistically significant. To evaluate the diagnostic performance of the quantitative tests for differentiating malignant and benign lesions and to describe the sensitivity and specifi- Benign lesions (n=19) Malignant lesions FIGURE 2. A 32-year-old woman with bacterial pneumonia. (A) Axial T2-weighted MR image shows ill-defined peripheral mass in the superior segment of the lower lobe of the left lung (arrows). (B) diffusion-weighted magnetic resonance image with b value of 500 sec/mm2 shows minimal hyperintense mass (SI = 203). (C) diffusion-weighted magnetic resonance image with b value of 1000 sec/mm2 shows hyperintensity of the mass is less remarkable (SI = 162). (D) ADC map shows ADC value is (2.76x10-3) sec/mirG city of the tests, receiver operating characteristic (ROC) analysis was performed. The optimum cutoff value was determined as the value that best discriminates between the two groups in terms of maximum sensitivity and minimum number of false-positive results. All statistical analyses were performed using the statistical software SPSS.15 Results The mean age of all patients was 64 ± 21 years (range: 41-83 years). The mean age of the patients in the benign group was 61 ± 19 years (range: 41-72 years) and in the malignant group was 66 ± 21 years (range: 44-83 years). The difference in age between groups was not statistically significant (p = 0.5). The mean diameter of masses for the entire group was 33 ± 18 mm. The mean diameter of malignant lesions was 37 ± 19 mm (range: 20-130 mm) and of benign lesions was 25 ± 15 mm (range: 1560 mm), respectively. The difference in diameter between groups was statistically significant (p = 0.005). Except for one patient who had two nodules (the primary tumor and its pulmonary metastasis), one lesion was measured in the remaining patients. Of the 67 lesions, 48 were malignant and 19 were benign (Table 1). The malignant lesions consisted of 42 non-small cell lung cancers (NSCLCs) and 6 small cell lung cancers (SCLCs). NSCLC subgroups TABLE 2. Quantitative analysis of diffusion-weighted magnetic resonance imaging (mean ± SD) of malignant and benign lesions (b=0, 500, 1000 sec/mm2 and ADC values) b and ADC values malignant lesions (n=48) benign lesions (n=19) P b 0 (sec/ mm2) 1500.40 ± 820 960.31 ± 550 0.004 b 500 (sec/ mm2) 611.32 ± 343 247.10 ± 124 0.000 b 1.000 (sec/ mm2) 371.00 ± 194 175.68 ± 94 0.000 ADC (x10-3) mm2/sec 1.65 ± 0.90 1.75± 0.70 0.675 TABLE 3. Quantitative analysis of diffusion-weighted magnetic resonance imaging (mean ± SD) of poorly-differentiated and medium- well differentiated malignant lessions (b=0, 500, 1000 sec/mm2 and ADC values) b and ADC values poorly-differentiated (n=11) medium-well differentiated (n=31) P b 0 (sec/ mm2) b 500 (sec/ mm2) b 1.000 (sec/ mm2) ADC (x10-3 ) mm2/sec 1646 ± 787 751.4 ± 440 517.27 ± 273 1.5 ± 0.5 1453.3 ± 837 568.87 ± 304 323.67 ± 135 1.8 ± 0.8 0.497 0.245 0.044 0.240 TABLE 4. Quantitative analysis of diffusion-weighted magnetic resonance imaging (mean ± SD) of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) (b=0, 500, 1000 sec/mm2 and ADC values) b and ADC values NSCLCs (n=42) SCLCs (n=A) P b 0 (sec/ mm2) b 500 (sec/ mm2) b 1.000 (sec/ mm2) ADC (x10-3 ) mm2/sec 1110.8 ± 209 468.6 ± 238 366 ± 177 1.9 ± 0.8 1705 ± 873 646.9 ± 350 379.1 ± 197 1.5 ± 1.0 0.001 0.186 0.881 0.464 included 13 adenocarcinomas, 5 squamous-cell carcinomas, 2 large-cell carcinomas, and 22 unidentified NSCLCs that could not be subgrouped in any of these. Eleven of the malignant lesions were poorly-differentiated, the others were medium- or well- differentiated. The benign lesions consisted of 9 chronic inflammatory changes, 3 sarcoidosis, 2 acute bacterial pneumonias, 2 tuberculosis pneumonias, 1 rheumatoid nodule, 1 granuloma, and 1 hamartoma. In 51 patients, the final diagnosis was made by histopathological confirmation on the basis of percutaneous biopsy, and included 48 primary lung cancers, 2 nonspecific inflammatory changes and 1 hamartoma. The diagnosis was confirmed with laboratory, radiological and clinical parameters in 3 sarcoidosis, 2 bacterial pneumonias, and 2 tuberculosis pneumonias. In sarcoidosis patients, the lesions regressed with a specific treatment. Two tuberculosis (Mycobacterium tuberculosis) and 2 bacterial pneumonia cases were diagnosed bacte-riologically. In 9 patients (1 granuloma, 1 rheumatoid nodule and 7 chronic inflammatory changes) lesions remained stable on follow-up CT for 12 months or more. We could obtain DWI SI and an ADC value for all lesions. The results of the quantitative analysis of SIs and ADC values are reviewed in Table 2. The mean SI of malignant lesions was higher than that of benign lesions (Figure 1, 2). The difference between malignant and benign lesions was significant for all b values (p < 0.004 for b = 0 s/mm2 and p < 0.000 for the other b values) (Figure 3). The area under the ROC curve was 0.933 ± 0.031 for the SI on images with b = 500 s/mm2. Using b = 500 s/ mm2, a SI >391 indicated a malignant lesion with a sensitivity of 95%, specificity of 73% and positive predictive value of 87%. Six among 48 malignant lesions (4 NSCLCs and 2 SCLCs) revealed SIs <391, which could be confused as benign. For the SI on images with b = 1000 s/mm2, the area under the ROC curve was 0.831 ± 0.055. A SI >227 with b = 1000 s/mm2 indicated a malignant lesion with a sensitivity of 93%, specificity of 69% and positive predictive value of 85%. Seven among 48 malignant lesions (6 NSCLCs and 1 SCLCs) re- vealed SIs <227 which could be confused as benign. Among the benign lesions, 1 sarcoidosis and 1 acute bacterial pneumonia had SI higher than the cut-off value on DWI with both b = 500 and b = 1000 s/ mm2. A patient with a chronic inflammatory lesion had a SI higher than the cut-off value only for b = 1000 s/mm2. Although the mean ADC of the malignant lesions (1.5 x10-3 mm2/sec) was lower than of the benign group (1.9 x10-3 mm2/sec), the difference was not statistically significant (p < 0.675). The results of the subgroup quantitative analysis are reviewed in Table 3. When we analyzed the malignant lesions in accordance with the histologic differentiation, the SI of poorly-differentiated cancers was higher for all b values, but statistically a significant difference was observed only with b = 1000 s/mm2 (p < 0.04). Although the ADC of poorly-differentiated lesions was lower than of the medium-well differentiated lesions, the difference was not statistically significant (p < 0.240). A comparison of the NSCLCs and the SCLCs demonstrated that the SIs of SCLCs were higher than those of NSCLCs. Although the ADC value of the SCLCs was lower than the ADC value of the NSCLCs, the difference was not statistically significant (p < 0.464) (Table 4). When we compare CT images with the DWI, all of the malignant lesions had irregular contours on CT images. Of the benign lesions 16 had also irregular contours but 3 (1 rheumatoid nodule, 1 granuloma, and 1 hamartoma) had smooth contours. The difference was not statistically significant (p = 0.3). Lymphangitic tumoral spread as irregular septal thickening was detected as a concomitant interstitial finding in 6 of the malignant masses on CT images. Discussion The aim of DWI is to evaluate the diffusion process in vivo. ADC values are quantitative expressions of the diffusion characteristics of tissues. These characteristics are related to several factors such as tissue cellularity, cell density and extracellular-intra-cellular components.16 DWI has been an important diagnostic tool for neuroradiology, especially for ischemic events of the brain.19 Although DWI has been used to differentiate malignant and benign lesions in several other locations, there are few studies about the intrathoracic lesion characteriza-tion.20,21-27 In our study, we found SIs of malignant masses on diffusion trace imaging were significantly high- FIGURE 3. Comparison of SI with b value of 1000 s/mm5 in malignant and benign lesions. The SIs of malignant lesions were significantly higher than those of benign lesions. er than of benign masses with low (b = 500 s/mm2) and high (b = 1000 s/mm2) b values. In the study of Uto et al. the SI ratio of malignant lesions to the spinal cord was found higher than that of benign lesions.21 Satoh et al. also found a higher qualitative score in SI of malignant versus benign masses but they did not measure quantitatively.22 In our study, the patient population was larger than in these two studies. Although malignant lesions showed lower ADC values than benign masses in our study, the difference was not significant. One reason may be the use of fixed TRs, due to which the DW series were acquired in different phases of the cardiac cycle. Therefore, DW images of different patients may have been affected differently by the pulsatile motion. Another reason may be the distortion artifacts, which limited the reliability of the ADC measurements for small and low-lateral segmental lesions. In the literature, some studies did not evaluate ADC values probably because of susceptibility artifacts.2225 Uto et al. found no significant difference between malignant and benign lesions by means of ADC.21 Mori et al. found a significant difference between malignant and benign lesions by using an ADC cut-off value of 1.1x10-3 mm2/sec.23 The large number of patients may be the explanation for this result. Liu et al. reported that ADC values in malignant lesions were significantly lower than in benign lesions.24 In the same study, there was no significant difference between malignant and benign lesions in DWI SIs. Histopathologically, tumoral cellularity of SCLC is high, and these tumoral cells have very large nuclei and almost no cytoplasm.28 All these features were expected to restrict the tissue diffusion and reduce ADC values. In our study, the SI of SCLC lesions was higher than of the NSCLC subgroup but the difference was significant only with b = 0 s/mm2. Thus, hyperintensity of lesions may be related to the T2 shine-through effect. Although ADCs were lower in the SCLC subgroup, the difference was not statistically significant. This may be because of the limited number of patients (n=6). In their study of tissue characterization in lung cancers, Abdel Razek et al. found significantly lower ADC values for SCLC when comparing with NSCLC groups in a similar patient population.26 Liu et al. also found lower ADC values for the SCLC than the NSCLC group.24 However, Koyama et al. found no significant differences between subtypes of lung cancers.27 In our study, SI of poorly- differentiated malignant masses was higher than of medium-well differentiated masses on all trace images and the difference with b value of 1,000 s/mm2 was statistically significant. ADC values were lower in the poorly-differentiated subgroup although the difference was not statistically significant. Histologically, tumor cellularity is higher in the poorly-differentiated cancers which could explain the low ADC values.29 Similar results of significantly lower ADCs in poorly-differentiated adenocarcinomas compared medium-well- differentiated cancer types are reported in the literature and are in accordance with our findings.20263031 When compared with CT images, contour characteristics of the lesions cannot be assessed with DWI but it does not seem to be a significant disability because contour characteristics are not reliable in differentiating malignant and benign lesions. On CT images lymphangitic tumoral spread was detected as a concomitant interstitial finding in some of the malignant masses. This finding is very helpful to estimate the malignant character of the lesion. The sensitivity and specificity achieved by DWI suggest it could be used for malignant versus benign differentiation. However, the inability of MRI to properly assess the interstitium and lymphangitic tumoral spread is a limitation for predicting malignancy. In addition, although just primary lung cancers are involved into our study, it must be kept in mind that calcified metastases such as those from osteosarcoma may be difficult to detect because of relatively lower proton density resulting in low signal intensity. Our study had some technical limitations. The use of DWI in the thorax was hindered by certain limitations such as physiologic motion artifacts (respiration and cardiac motion), low signal-to-noise ratio (SNR) of the low lung proton density and the susceptibility artifacts caused by air-tissue interfaces.18 We used the EPI sequence with high b values, which had a lower SNR, thus resulting in greater image distortion. In addition, we obtained DW images using a breath-hold echo-planar sequence with SENSE and this made the measurements vulnerable to susceptibility effects. We did not use pulse-triggered DWI, known to reduce the accuracy of ADC measurements.18 Finally, the patient population, especially the benign subgroup, was relatively small, which might compromise the accuracy of the results. In conclusion, DWI may be used to differentiate malignant and benign lung lesions in addition to other radiologic imaging techniques even with high b values such as b = 1000 s/mm2. DW trace image SI, together with ADC measurements is useful for the differentiation of malignant versus benign lung lesions. References 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer statistics. CA Cancer J Clin 2006; 56:106-30. 2. Erasmus JJ, Connolly JE, McAdams HP, Roggli VL. Solitary pulmonary nodules. Part 1. Morphologic evaluation for differentiation of benign and malignant lesions. RadioGraphics 2000; 20: 43-58. 3. Swensen SJ, Viggiano RW, Midthun DE, Müller NL, Scherrick A, Yamashita K, et al. Lung nodule enhancement at CT: multicenter study. Radiology 2000; 214: 73-80. 4. Henschke CI, Yankelevitz DF, Naidich DP, McCauley ID, McGuinness G, Libby MD, et al. CT screening for lung cancer: suspiciousness of nodules according to size on baseline scans. Radiology 2004; 231: 164-8. 5. Jeong YJ, Lee KS, Jeong SY, Chung MJ, Shim SS, Kim H, et al. Solitary pulmonary nodule: characterization with combined wash-in and washout features at dynamic multidetector row CT. Radiology 2005; 237: 675-83. 6. Gould MK, MacLean CC, Kuschner WG, Rydzak CE, Owens DK. Accuracy of positron emission tomography for diagnosis of pulmonary nodules and mass lesions. JAMA 2001; 285: 914-24. 7. Yi CA, Lee KS, Kim B, Choi JY, Kwon JO, Kim H, et al. Tissue characterization of solitary pulmonary nodule: comparative study between helical dynamic CT and integrated PET/CT. JNuclMed 2006; 47: 443-50. 8. Cheran SK, Nielsen ND, Patz EF Jr. False-negative findings for primary lung tumors on FDG positron emission tomography: staging and prognostic implications. AJR 2004; 182: 1129-32. 9. Nomori H, Watanabe K, Ohtsuka T, Naruke T, Suemasu K, Uno K. Evaluation of F-18 fluorodeoxyglucose (FDG) PET scanning for pulmonary nodules less than 3cm in diameter, with special reference to CT images. Lung Cancer 2004; 45: 19-27. 10. Tanaka R, Horikoshi H, Nakazato Y, Seki E, Minato K, lijima M, et al. Magnetic resonance imaging in peripheral lung adenocarcinoma; correlation with histopathologic features. J Thorac Imaging 2009; 24: 4-9. 11. Ohno Y, Sugimura K, Hatabu H. MR imaging of lung cancer. EJR 2002; 44: 172-81. 12. Podobnik J, Kocijancic I, Kovac V, Sersa I. 3T MRI in evaluation of asbestos-related thoracic diseases - preliminary results. Radiol Oncol 2010; 44: 92-6. 13. Schaefer JF, Vollmar J, Schick F, Vonthein R, Seemann MD, Aebert H, et al. Solitary pulmonary nodules: dynamic contrast-enhanced MR imagingperfusion differences in malignant and benign lesions. Radiology 2004; 232: 544-53. 14. Ohno Y, Hatabu H, Takenaka D, Adachi S, Kono M, Sugimura K, et al. Solitary pulmonary nodules: Potential role of dynamic MR imaging in management: initial experience. Radiology 2002; 224: 503-11. 15. Abdel Razek A, Elmorsy A, Elshafey M, Elhadedy T, Hamza O. Assessment of mediastinal tumors with diffusion-weighted single-shot echo-planar MRI. J Magn Reson imaging 2009; 30: 535-40. 16. Ichikawa T, Erturk SM, Motosugi U, Sou H, Iino H, Araki T, et al. High-b value diffusion-weighted MRI for detecting pancreatic adenocarcinoma: Preliminary results. AJR 2007; 188: 409-14. 17. Inan N, Kilinc F, Sarisoy T, Gumustas S, Akansel G, Demirci A. Diffusion weighted MR imaging in the differential diagnosis of haemangiomas and metastases of the liver. Radiol Oncol 2010; 44: 24-9. 18. Murtz P, Flacke S, Traber F, van den Brink JS, Gieseke J, Schild HH. Abdomen: diffusion weighted MR imaging with pulse-triggered singleshot sequences. Radiology 2002; 224: 258-64. 19. Ozsunar Y, Sorensen AG. Diffusion- and perfusion- weighted magnetic resonance imaging in human acute ischemic stroke: technical considerations. Top Magn Reson imaging 2000; 11: 259-72. 20. Matoba M, Tonami H, Kondou T, Yokota H, Higashi K, Toga H, et al. Lung carcinoma: diffusion weighted MR imaging—Preliminary evaluation with apparent diffusion coefficient. Radiology 2007; 243: 570-7. 21. Uto T, Takehara Y, Nakamura Y, Naito T, Hashimoto D, Inui N, et al. Higher sensitivity and specificity for diffusion- weighted imaging of malignant lung lesions without apparent diffusion coefficient quantification. Radiology 2009; 252: 247-54. 22. Satoh S, Kitazume Y, Ohdama S, Kimula Y, Taura S, Endo Y. Can malignant and benign pulmonary nodules be differentiated with diffusion-weighted MRI? AJR 2008; 191: 464-70. 23. Mori T, Nomori H, Ikeda K, Kawanaka K, Shiraishi S, Katahira K, et al. Diffusion-weighted magnetic resonance imaging for diagnosing malignant pulmonary nodules/masses. Comparison with positron emission tomography. J Thorac Oncol 2008; 3: 358-64. 24. Liu H, Liu Y, Yu T, Ye N. Usefulness of diffusion-weighted MR imaging in the evaluation of pulmonary lesions. Eur Radiol 2010; 20: 807-15. 25. Qi LP, Zhang XP, Tang T, Li J, Sun YS, Zhu GY. Using diffusion-weighted MR imaging for tumor detection in the collapsed lung: a preliminary study. Eur Radiol 2009; 19: 333-41. 26. Abdel Razek A, Fathy A, Abdel Gawad T. Correlation of apparent diffusion coefficient value with prognostic parameters of lung cancer. JCAT 2011; 35: 248-52. 27. Koyama H, Ohno Y, Aoyama N, Onishi Y, Matsumoto K, Nagomi M, et al. Comparison of STIR turbo SE imaging and diffusion-weighted imaging of the lung: capability for detection and subtype classification of pulmonary adenocarcinomas. Eur Radiol 2010; 20: 790-800. 28. Cotran RS, Kumar V, Robbins SL: Robbins Pathologic Basis of Disease, 5th edition. Philadelphia, W.B. Saunders, 1994, pp. 720-25. 29. Clayton F. Bronchioloalveolar carcinoma: Cell types, patterns of growth, and prognostic correlates. Cancer 1986; 57: 1555-64. 30. Kanauchi N, Oizumi H, Honma T, Kato H, Endo M, Suzuki J, et al. Role of diffusion-weighted magnetic resonance imaging for predicting of tumor invasiveness for clinical stage IA non-small cell lung cancer. Eur J Cardiothorac Surg 2009; 35: 706-11. 31. Henzler T, Schmid-Bindert G, Schoenberg SO, Fink C. Diffusion and perfusion MRI of the lung and mediastinum. Eur J Radiol 2010; 76: 329-36. Evaluation of penile erection rigidity in healthy men using virtual touch tissue quantification Xiaozhi Zheng, Ping Ji, Hongwei Mao, Jing Wu Department of Ultrasound, The Fourth Affiliated Hospital of Nantong University (The First People's Hospital of Yancheng), 14 Yuehe Road, Yancheng 224006, Jiangsu Province, P.R. China. Radiol Oncol 2012; 46(2): 114-118. Received 1 August 2011 Accepted 19 December 2011 Correspondence to: Jing Wu, Department of Ultrasound, The Fourth Affiliated Hospital of Nantong University (The First People's Hospital of Yancheng), 14 Yuehe Road, Yancheng 224006, Jiangsu Province, P.R. China; Phone: +86 0515 88508717; Fax: +86 0515 88500299; E-mail: wujingzxz@163.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to describe the shear wave velocity (SWV) values of the penis by virtual touch tissue quantification (VTTQ) and to examine the clinical usefulness of this procedure in evaluation of the rigidity changes in penile erection. Patients and methods. VTTQ was performed in 37 healthy volunteers. In the course of erection, SWV values of glans penis, corpus penis and radix penis were quantified and grades of erection were documented. The SWV values at different grades of erection were compared. Results. The axial and radial SWV values of glans penis, corpus penis and radix penis all significantly decreased from Grade 0 to Grade 4 of erection. At Grade 4, they were less than one-third of that at Grade 0 (axial direction: 0.79 ± 0.13 vs. 2.79 ± 0.32 for glans penis, P<0.001 ; 0.77 ± 0.19 vs. 2.84 ± 0.30 for corpus penis, P<0.001 and 0.76 ± 0.15 vs. 2.81 ± 0.34 for radix penis, P<0.001 ; radial direction: 0.82 ± 0.15 vs. 2.83 ± 0.31 for glans penis, P<0.001 ; 0.79 ± 0.18 vs. 2.81 ± 0.27 for corpus penis, P<0.001 and 0.81 ± 0.16 vs. 2.82 ± 0.33 for radix penis, P<0.001 ). Conclusions. VTTQ can provide numerical measurements of penile rigidity and can effectively and sensitively indicate the axial and radial rigidity changes in penile erection, which provide a new approach to assessing the erectile function. Key words: virtual touch tissue quantification; axial and radial rigidity; erection; shear wave velocity. Introduction Penile erection rigidity is one of the key factors for successful sexual intercourse, as well as an important index in the diagnosis and treatment of erectile dysfunction (ED). Ideally, the evaluation of ED should include the measurement of axial rigidity and radial rigidity. This requires special devices, such as RigiScan, Digital Inflexion Rigidometer. However, each device has its pros and cons, related to availability, convenience, validity and costs. For example, RigiScan is the most widely utilized device for measuring penile radial rigidity1-5, but this device does not directly determine axial rigidity.367 On the contrast, Digital Inflexion Rigidometer is a useful device for measuring penile axial rigidity389, but it does not directly determine radial rigidity. Complete erection rigidity assessment needs a combinatorial use of RigiScan and Digital Inflexion Rigidometer, which increased the cost and inconvenience. Alternative methods for penile erection rigidity assessment are needed. Ultrasound is widely used for clinical imaging1011, and virtual touch tissue quantification (VTTQ) is a new, promising implementation of the ultrasound acoustic radiation force impulse (ARFI) imaging, which can effectively and objectively detect the tissue rigidity by measuring the shear wave velocity (SWV) values.12-14 Due to the non-invasive and easily accessible nature of VTTQ, this tech- nology makes it possible to conduct a thoroughly evaluation of erectile rigidity at any portion and any direction in the penis without any discomfort and special preparation. Recently, VTTQ has been used to quantify the rigidity of the liver, kidneys, pancreas, spleen, prostate and breast.12-19 In our recent study, in order to provide a supplementary approach to assess the penile erection rigidity, we described the normal axial and radial SWV values of the penis by VTTQ and examined the clinical usefulness of this procedure in evaluation of the rigidity changes in penile erection. TT I 11 ■ iu ■ ; r : Vi «I iT mi* D»(rtii-" 3 crtï * ■Jfct ® Patients and methods Study Population Our study was approved by the local human research ethics committee and free informed consent was obtained from all the subjects. 37 healthy men with a mean age of 34.6 years (range: from 18 to 63 years) were recruited. All the subjects were evaluated by means of clinical and physical assessment, detailed sexual history, laboratory data (glucose, cholesterol, and triglycerides serum levels), and endocrine assays (testosterone, prolactin, follicular stimulation hormone, and luteinizing hormone), electrocardiogram, radiology, ultrasonographic (US) examinations and computer tomography. The inclusion criteria were: absence of any history of focal or diffuse disease at any of the examined organs. The subjects with risk factors for ED, such as diabetes mellitus, hypertension, ischemic heart disease, neurogenic injury to the spinal cord, and with psychogenic factors were excluded from the study. Examination protocol Two urologists with 10 years of experience in diagnosing and treating ED performed clinical assessments. Erectile response was induced by masturbation one hour after 100 mg sildenafil citrate (Pfizer, New York, NJ, USA) administration, and it was evaluated and judged by the urologist and subject at the same time. Penile erection was categorized into four grades using the following crite-ria.2021 Grade 0 (G0), flaccid; Grade 1(G1), mild tumescence; Grade 2(G2), moderate tumescence but inadequate rigidity for vaginal penetration; Grade 3(G3), full tumescence with moderate rigidity allowing vaginal penetration with some difficulty; and Grade 4(G4), full tumescence and full rigidity allowing vaginal penetration without difficulty. n - - ■ ülEMLNS 1 In--rn 1 -Itiltl ■ 1 n MC'J I Kïl fSÏ '"■r : V ■ ■i Vi-2 »111 ftiplh1' D cm ■ He "st ® ® FIGURE 1. Shear wave velocity measurement in the penis with virtual touch tissue quantification during the erection (A and C: Grade 0; B and D: Grade 4) from the axial direction(A and B) and radial direction (C and D), respectively. O CD ® 4n £31 o o o > « 2- ) = 0 [1] a represents tissue conductivity and ^ represents the electric potential. The electric field was calculated from the electric potential. E = -V0 [2] E represents the electric field. The boundaries of the cube were also set to be electrically insulating: -n-J = 0 [3] n represents the unit outward normal vector and J represents the current density. The tissue density was set to be 1050 kg/m3, 28 and the electrode conductivity was set to be 4x106 S/m.29 The active electrode(s) were set to an electric potential ^ = and the electrode(s) that were not active were set to ground. A bounding box around the tumor was used in the FEM simulations to improve the quality of the meshing and computations. The mesh consisted of 29,505 elements. The electrodes modeled in the study were plat-inum-iridium (90%/10%) electrodes represented by cylinders with 2.0 cm exposed length and 1 mm diameter with insulated portions above the exposed regions of the electrode. The normal liver was given a relative permittivity of 8.2x104, while the tumor relative permittivity was set to 9.9x10.430 We modeled the nonlinear change in the electrical conductivity due to the process of membrane per-meabilization with a sigmoid relationship depending upon the electric field magnitude as according to Sel et al.31 for the liver and Ivorra et al.32 for the tumor. d2 Tumor 'hemiline -electrodes FIGURE 1. Depiction of the electrode configurations. (left) The purple circles represent active electrodes, and the lighter shaded electrodes are set to ground. The distance (d1) between electrodes and the depth of insertion (d2) was varied in the study. The fit equation for the liver was: a( E) = 0.075 + (0.27 - 0.075) * ( 1 l + 10e( ;-l/3000»(£-58000)) The fit equation for the tumor was: a(E) = 0.166(2.154 * exp(-e(^m4*(|M00|-150°») +1) E represents the electric field in V/m. Since the liver model from Sel et al. was determined from rabbit data, we changed the baseline value to 0.075 S/m to coincide with human data derived from Haemmerich et al. 2009.33 The fit equation for the tumor was derived from mouse data, so the baseline conductivity was selected from Laufer et al.30 to be 0.166 S/m. The value from Laufer et al. was closer to the tumor fit equation's original baseline than the value from Haemmerich et al. for tumors, and conversely the chosen baseline value for the normal liver was closer to the original fit equation value from Sel et al. than that found in Laufer et al. It is well known that liver tumor conductivities tend to be higher than normal liver conductivities, and the ratio of 0.166/0.075^2.21 is a reasonable ratio between the tumor and the liver.1828, 34 Mainly three different parameters of electrode configurations were analyzed for the study: 1) distance between electrodes 2) depth of electrode insertion 3) the number of electrodes. Basically, the parameter space for the distance between electrodes and the depth of electrode insertion was explored for three different electrode numbers: two, three, and four electrodes. The means of optimizing this was to increment the applied voltage at the active electrode(s) by 100 V intervals to the minimum value necessary to cover 95% of the tumor with an electric field sufficient to irreversibly electroporate the tumor tissue: VIRE. This electric field was set at est VIRE values consistently occur at the largest irreversible electroporation (tissue ablation) using an isosurface plotted in the COMSOL® 3D environment. VIRE was determined at several electrode distances and depths, and the results were tabulated for two, three, and four electrodes. The seven different distances between the electrodes that were measured were 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 4 cm, and 5 cm. A distance of 1.9 cm was used for three electrodes as opposed to 2 cm to allow meshing with the electrodes close to the tumor border. The depths of insertion of the electrodes, as measured from the tumor's spherical hemiline to the bottom of the electrodes, were 0.5 cm, 1 cm, 1.25 cm, and 1.5 cm (Figure 1). In previous studies (data not shown), we tested shallower depths, but they had exorbitantly large VIREs and thus were not included for these experiments. The applied pulse duration was selected to be 20 |js, which is at the lower end of the spectrum of pulse durations.35 This minimizes computational time and would also be useful for minimizing tissue resistive heating, which is directly related to pulse duration. A shorter pulse was not selected also because there should be an adequate amount of time to charge the membrane for IRE to be effective, and the membrane has a charge time of about 1 |js.36 There has been no rigorous study as of yet examining the use of shorter versus longer pulses in the irreversible electroporation ablation of liver cancer cells as far as monitoring the size of the ablation zone. However, ultra-short pulses have been employed with very high electric fields (up to 300 kV/cm) to induce apoptosis by irreversibly electro-porating inner organelles of the cell.37 The volume of normal (non-cancerous) tissue that was irreversibly electroporated—the Volume of Ablated Normal Tissue (VANT)—was calculated in COMSOL® by integrating the volume of normal liver tissue in the simulated environment at or above 680 V/cm. VIRE and VANT were plotted versus distance at several depths, and some summary statistics were determined (mean, min, max). Results v y IRE The VIRE results for two, three, and four electrodes are graphically depicted in Figure 2 A-C. The high- 680 V/cm, as derived from Davalos et al..5 The voltage was iterated, and the criterion for tumor ablation was determined via the MATLAB™ environment. Also, we were able to visualize the region of distances for all three sets of electrodes. The two electrode data show a parabolic minimum at a distance of 2 cm. We can observe two regions from the results in Figure 2A, the first where the electrodes V-IRE versus Distance at Several Depths, Two Electrodes V-ANTs versus Distance at Several Depths, Two Electrodes V-IRE versus Distance at Several Drptts, Three Electrodes V-ANTs versus Distance at Several Depths, Three Electrodes V-IRE rrreue Distance at Several Depths, Ftur Electrodes V-ANTe versus Vietatdf at Several Vrptte, Ftur Eledtrtree FIGURE 2 A-C. Graphical depiction of VIRE values upon distance at several depths. There appears to be ta parabolic minima at 2 cm distance between the electrodes for the two electrode set, whereas the VIRE values for both three and four electrodes decrease linearly with the distance between the electrodes. D-F Graphical depiction of the dependence of the VANT values upon distance at several depths. The VANT values decrease with distance for all electrode setups. Two electrodes overall have the lowest VANT values, followed by four electrodes and then three electrodes. TABLE 1: Summary statistics from the three VIRE tables (V) Number of Electrodes Minimum VIRE Mean VIRE Maxim Two 3700 5296 8500 Three 7500 10761 18700 Four 4700 7279 13600 summed VIRE standard deviations while varying either distance or depth (V) Parameter varying Two electrodes Three electrodes Four electrodes Distance 6343 16036 12873 Depth 2133 1616 1762 TABLE 2: Summary statistics from the three VANT tables (cm3) Electrode # Minimum Mean Maximum Two 3.53 12.1 35.1 Three 13.2 60.5 171.3 Four 11.5 60.7 178.6 summed VANT Standard Deviations while varying either Distance or Depth Parameter varying Two electrodes Distance 41.2 Depth 11.4 Three electrodes Four electrodes 221 236 12.5 20.0 are placed inside the tumor and the second where the electrodes are outside the tumor. The two-electrode data show a drop in the voltage as the distance increases from 1 cm, before it rises again near the tumor-tissue boarder at a distance of 2.5 cm. This behavior is observed for all electrode depths. The lowest VIRE values occur at depths of 1 cm and 1.25 cm, which correspond to the center of the electrode overlapping with the center of the tumor. The higher VIRE values generally occur at a depth of 1.5 cm and then 0.5 cm, indicating the highest VIRE values are at the shallowest depth of insertion, and the deepest insertions (lower overlap) produce lower VIRE values. The lowest VIRE values for three and four electrodes appear to occur at the shortest distances between the electrodes. However, the three-electrode configuration showed an increase in the voltage as the distance increased, before it dropped again near the tumor-tissue boarder. Again this observation is independent of the electrode depth. For the four-electrode configuration, there was either no change in the voltage (with 1 cm and 1.25 cm depth), an increase (0.5 cm depth), or a decrease in the voltage (with 1.5 cm depth) as a function of distance. When the electrodes were placed outside of the tumor, there was a monotonic increase in the required voltage for ablation as a function of distance for all of the electrode configurations and with all choices of depth. Overall, for all the different electrode configurations, there appears to be a smaller dependence of the VIRE values upon the depth of electrode insertion as compared to the distance between electrodes. To visualize the differing ablation zones for the different electrode configurations, we examined a cross section of the center of the tumor, demarcating regions that were greater than or equal to 680 V/cm for two, three, and four electrodes at a distance of 2.5 cm and a depth of 1 cm and plotted the ablation zones in Figure 3. Due to the asymmetric ablation shape with three-electrode configuration we tried moving the center of the electrode array and checked whether this would lower VIRE and VANT (Figure 3D). In fact this, lowered VIRE and VANT from 9200 V and 38.4 cm3 to 4600 V and 16.3 cm3 respectively when moving the center of the electrode array 1 cm to the right of the center of the tumor. Summary statistics for VIRE and influence of parameters (distance, depth) on VIRE Overall, the VIRE values were lowest for two electrodes, followed by four electrodes and then three electrodes. Table 1 below shows some summary statistics concerning the three VIRE tables as well as information about the influence of the different parameters upon the VIRE. The minimum, mean, and maximum VIRE were lowest for two electrodes followed by four electrodes and then three electrodes. The summed standard deviations for varying the distance were consistently greater for two, three, and four electrodes as compared to the summed standard deviations while varying the depth. VANT results The VANT results for two three, and four electrodes are depicted in Figure 2 D-F. The VANT values increase monotonically with distance for all electrode configurations and show the same variation with depth of electrode insertion as with the VIRE values where the depths of 1 cm and 1.25 cm consistently have the lower VANT values relative to depths of 0.5 cm and 1.5 cm. The two electrode setups have the lowest VANT values, followed by four electrodes and three electrodes. The summary statistics are shown for VANT values in Table 2. As with the VIRE values, the summed standard deviations are greater, when varying distance as opposed to depth. The best configuration for the a) two b) three and c) four electrode setups, as according the lowest VIRE are: a) a distance of 2 cm and a depth of 1 cm (VIRE =3700 V) b) a distance of 1 cm and a depth of 1 cm (VIRE =7500 V) and c) a distance of 2 cm and a depth of 1 cm (VIRE =4900 V). The overall, global best choice for all electrode configurations was: the two-electrode configuration with a distance of 2 cm and a depth of 1 cm with VIRE 3700 V. Discussion The overall goal of our study was to find the optimal distance, depth, and number of electrodes for a simulated irreversible electroporation therapy of a subcutaneous HCC tumor. We systematically varied the distance and depth for three different sets of electrodes (two, three, and four electrodes) and looked at the outcome measures VIRE and VANT. The currently used Angiodynamics™ system uses only two electrodes at a time, but our study helps explore the potential benefits of different electrode ® FIGURE 3. Cross section of the tumor (circle) and its ablation zone (blue) at an applied voltage of 5.05 kV, which corresponds to the VIRE for two electrodes at a distance of 2.5 cm and depth of 1 cm for A) two electrodes B) three electrodes C) four electrodes D) three electrodes with the center of the electrodes shifted to the right 1 cm Note the asymmetry in B and the narrowing of the ablation zone in C. A bounding box around the tumor was used in the FEM simulations to improve the quality of the meshing and computations. configuration patterns, particularly with the three electrode configurations.35 Our study builds upon findings from Zupanic et al.16 in that we include the possibility of electrodes within the tumor in our 3D simulations, which is advantageous, because the electric fields are highest near the electrodes and rapidly drop off with distance (i.e., the greatest amount of therapy occurs near the electrodes). It must be considered, however, that for some tumor cases it may not be possible to have the electrodes within the tumor.22 In looking at VIRE, two electrodes produced the lowest VIRE, at a value of 3700 V. It was unexpected that the two-electrode configuration produced the lowest outcome measure values (VIRE and VANT) whereas the three electrode configurations produced the highest, and the four electrode configuration outcome measures were in the middle. We expected that the greater surface area provided by a higher number of electrodes would cause the four-electrode configuration to have the lowest VIRE values. This observation could be explained by a number of factors that played a role in the simulated tumor ablation process: a) the number of electrodes clearly plays a role as exhibited in Figure 3 B) the high threshold of 95% tumor abla- tion c) the symmetry of electrode positioning and d) the distribution of the electric fields between the electrodes that occurred in the study. The number of electrodes alters the shape of the ablated zone, as exhibited in Figure 3 where we visualize the difference in the ablation zone for two, three, and four electrode setups at a distance of 2.5 cm and depth of 1 cm with the same voltage of 5050 V. For example, the two-electrode configuration in Figure 3A has an elliptic ablation shape while the three-electrode has the most asymmetric shape. Another factor to consider is the different rise in the conductivity of the tissue and tumor due to the different nonlinear response to the rise in the electric field. As the electrodes are place outside and away from the tumor, the ablation shape has less effect as the whole tumor becomes more encompassed by the overall ablated zone, while the applied pulse has less effect on raising the tumor conductivity and more effect on raising the tissue conductivity. The three-electrode configuration was the most asymmetric of the ones studied (relative to the tumor), as we used a symmetrical spherical tumor. Thus, it is reasonable to deduce that having an outcome whereby the tumor must be 95% ablated rather than 80% or 90% ablated can lead to using high voltages for configurations that use more than two electrodes. If one uses a threshold of 100% tumor ablation, the VIRE values increase significantly. For example, the VIRE becomes 4300 V instead of 3700 V for two electrodes with a distance of 2 cm and depth of 1 cm, and VIRE becomes 16450 V instead of 4900 V for four electrodes with 2 cm distance and 1 cm depth. The asymmetric distribution of electrodes for three electrodes can more easily lead to incomplete tumor coverage (Figure 3B). This would explain why four electrodes, with greater symmetry of electrode patterning, would have lower outcome measures than that for three electrodes. The effect of moving the electrode center with respect to the tumor center in improving VIRE and VANT make us consider this feature when searching for the optimum electrode position, especially for configurations that result in asymmetric ablation zones. We noted the appearance of slightly higher VIRE values for three electrodes at 1.5 cm relative to the other nearby distances and reasoned that it may be caused by some effect due to both asymmetry and the nonlinear distribution of electric fields due to electrode positioning. As for two electrodes versus four electrodes, we visualized the differing ablation zone shapes in Figure 3A,C. We saw that the four electrodes had a narrowing of the ablation zone near the center of the tumor, which would account for the higher VIRE values for the four electrodes as compared with the two electrodes. Also, the best choice for depth between 1 and 1.25 cm can be explained as more electrode surface area is facing the tumor with these depths. These findings suggest that clinicians applying IRE for subcutaneous tumors may find the use of two electrodes more beneficial. This information is also useful, because an increase in the number of electrodes can also lead to increased pain and discomfort for the patient and a greater risk of tumor seeding.38, 39 The lowest VANT values occur roughly at the same distances and depths as the lowest VIRE values. The monotonic increase in the VANT values as a function of distance is expected as more normal tissue volume experiences higher electric field intensities as the electrodes are placed further from the tumor. It is desirable in the application of IRE to minimize both the ablated normal tissue as well as the thermally ablated tissue, for the sake of preserving tissue structural elements (e.g., extracellular matrix, blood vessels, etc.). There appears to be a curvilinear relationship between the VANT values and the distance between the electrodes, and thus clinicians should be careful to minimize the distance between the electrodes when applying the electric field across the tumor tissue to avoid the destruction of normal tissue. We found that the distance between the electrodes held greater significance for determining what the optimal electrode configuration was, as compared with the depth of electrode insertion. This is apparent visually in Figure 2. Noting the greater influence of distance upon VIRE and the volume of normal and thermally ablated tissue could allow for more leeway in the electrode positioning as far as the depth of electrode insertion as compared to the distance between electrodes. It appeared that the dependence of the outcome measures with depth was greater for two and four electrodes as compared with three electrodes. Also, it seemed that the optimal depth was to have the center of the electrodes inserted close to or deeper than the center of the tumor. This finding corroborates what Zupanic et al.16 However, we build upon Zupanic et al. in that we include configurations where the electrodes were inserted into the tumor, and we simulated a greater tumor size (Zupanic et al. used sizes: 2 mm, 4 mm, 8 mm radii). It would be interesting to conduct similar studies with different tumor sizes and to explore the effects of tumor size on the results. However, a tumor with a diameter of 2.5 cm is within the desired size range of <3.0 cm preferred by clinicians who apply the therapy40, and it could be considered an average size.27 Also, it may not be practical to record results for every feasible tumor size. However, these findings and similar studies, besides serving as a reference for clinicians, has the value of better informing the use of dedicated optimization algorithms. Rather than having one sole output from an optimization algorithm, one gains a better understanding of how the fitness function (VIRE) changes over the parameter space. In other words, the plots of VIRE over parameter space provide a better understanding of how the desired optimal value can vary with designated parameters. Thus, one can determine an optimization algorithm that best fits the fitness landscape, whether it is a gradient descent or genetic algorithm. Each algorithm has its strengths and weaknesses, and one could evaluate what the best optimization algorithm would be based upon these results. Also, by plotting the value of the objective function over the parameter space, one can know with greater certainty where the global minima versus local minima are. This is opposed to the use of an optimization method that would only output one or a limited number of values for the parameters, and one may not know if it is a local versus a global minima. In the use of a dedicated optimization algorithm, it may be beneficial to relax the stringent standard of 95% ablation to 80-90% ablation to increase the diversity of potential solutions and utilize overlapping ablation zones. In clinical practice it may be better to attempt to ablate the tumor all at once to prevent future seeding. It would also be difficult to have overlapping IRE ablation zones due to the difficulty in visualizing the development of the ablation zone with high resolution in real time. Research in visualizing the ablation zone in real time has been done via electrical impedance tom-ography41, but significant advances must be made for this to become a reality. Some researchers have suggested that MRI may be a viable route for visualizing the ablation zone during IRE therapy.42-44 Limitations The liver and tumor are assumed to be isotropic and homogenous. Future studies could also implement liver and tumor geometries from medical images. We do not account for the dependence of per-meabilization on cell size, shape, and interaction with surroundings.31 Also, we did not study different electrode pulse sequence patterns or shapes, which can have a significant impact on the efficacy of the therapy.45 If electrode sequence activation patterns had been used, the four-electrode configurations could have been more efficacious, due to improved tumor coverage.46 Adjustments of the voltage to reach VIRE were done in increments of 50 V, and thus there is the possibility that some of the measurements were overestimated. Also, although we thought VIRE to occur at 680 V/cm, it is possible that it occurs at a different cutoff value. However, the results would be expected to scale accordingly with a higher or lower threshold value for VIRE. The voltage amplitudes recorded from the study are higher than the maximum voltage supplied by NanoKnife (3000 V)35, but the information gleaned from the efficacy of different positions would still be useful to clinicians, and hopefully as technology improves, the observed voltages would become more feasible. Our optimum electrode position with two electrodes and a distance of 2.5 cm and 1 cm depth would ablate 82% of the tumor at the maximum system voltage of 3000 V (data not shown). It would also be useful to know thermal information about the electrode configurations used, but IRE pulse sequences use somewhere on the order of 90-100 pulses47, which would be impractical to examine for each electrode configuration with current computational capabilities. Conclusion We demonstrated that the optimal electrode configuration for applying IRE therapy for the ablation of HCC tumors could be determined using numerical modeling. We determined that it may be better to use two electrodes rather than three or four electrodes and that it is more important to be mindful of the distance between the electrodes rather than the depth of insertion of the electrodes to minimize the thermally ablated volume of tissue and the affected normal tissue. All of this information could serve as useful guidelines for physicians attempting to employ irreversible electroporation for the treatment of liver cancers. Although the model parameters we employed were specifically for liver cancer, the findings could be similar for other types of cancer e.g. kidney, breast, lung, etc. Our results corroborated previous findings that the distribution of the electric field in the tissue is highly dependent upon the electrode configuration, and future studies could further explore different electrode configurations with patient-generated tumor geometries and tissue properties. Acknowledgements This work was supported by F31 CA150658 fellowship for PhDs from the National Cancer Institute of the NIH. References 1. World health organization: Cancer. World Health Organization. 2012. Accessed: March 12, 2012. Available from: http://www.who.int/mediacen-tre/factsheets/fs297/en/index.html 2. Gomaa AI, Khan SA, Toledano MB, Waked I, Taylor-Robinson SD. Hepatocellular carcinoma: Epidemiology, risk factors and pathogenesis. World J Gastroenterol 2008; 14: 4300-8 3. Medicine Net: Liver cancer. Stoppler, M. 2011. Accessed: March 12, 2012. Available from: http://www.medicinenet.com/liver_cancer/article.htm 4. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012; Epub ahead of print. 5. Davalos RV, Mir IL, Rubinsky B. Tissue ablation with irreversible electroporation. Ann Biomed Eng 2005; 33: 223-31. 6. Daniels C, Rubinsky B. Electrical field and temperature model of nonthermal irreversible electroporation in heterogeneous tissues. 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Power Modulator Symposium, 2002 and 2002 High-Voltage Workshop. Conference Record of the Twenty-Fifth International 2002 38. Cabibbo G, Craxi A. Needle track seeding following percutaneous procedures for hepatocellular carcinoma. World J Hepatol 2009; 1: 62-6. 39. Tezcan Y, Mehmet, K. Hepatocellular carcinoma with subcutaneous metastasis of the scalp. Radiol Oncol 2011; 45: 292-5. 40. Narayanan G. Irreversible electroporation for treatment of liver cancer. Gastroenterol Hepatol (N Y) 2011; 7: 313-6. 41. Granot Y, Ivorra A, Maor E, Rubinsky B. In vivo imaging of irreversible elec-troporation by means of electrical impedance tomography. Phys Med Biol 2009; 54: 4927-43. 42. Zhang Y, Guo Y, Ragin AB, Lewandowski RJ, Yang GY, Nijm GM, et al. Mr imaging to assess immediate response to irreversible electroporation for targeted ablation of liver tissues: Preclinical feasibility studies in a rodent model. Radiology 2010; 256: 424-32. 43. Kranjc M, Bajd F, Sersa I, Miklavcic D. 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Improved survival after introduction of chemotherapy for malignant pleural mesothelioma in Slovenia: Population-based survey of 444 patients Viljem Kovac1, Matjaz Zwitter1, Tina Zagar2 1 Department of Radiotherapy, 2 Cancer Registry of Republic of Slovenia, Institute of Oncology, Ljubljana, Slovenia Radiol Oncol 2012; 46(2): 136-144. Received 15 January 2012 Accepted 10 February 2012 Correspondence to: Viljem Kovač, MD, PhD, Institute of Oncology, Zaloška 2, 1000 Ljubljana, Slovenia. Phone: +386 1 5879 504; Fax: +386 1 5879 400; E-mail: vkovac@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Malignant pleural mesothelioma is a rare tumour with increasing frequency throughout the world. Due to long latency after exposure to asbestos, restrictions in the production and use of asbestos have not yet alleviated the burden of mesothelioma. During the last decade, several trials confirmed the benefit of systemic treatment with drugs such as doublets with cisplatina and gemcitabine or pemetrexed for carefully selected patients in good performance status. The purpose of this survey was to assess the impact of systemic treatment for the whole national population of patients with mesothelioma. Patients and methods. A retrospective study included all patients in Slovenia with histologically confirmed diagnosis of malignant pleural mesothelioma in the period from 1974 till 2008. Data from the Cancer Registry of Slovenia were supplemented by review of clinical records of the Institute of Oncology in Ljubljana where virtually all non-surgical treatment for mesothelioma was performed. We analysed the incidence, treatment, and survival of patients treated in the era of infrequent chemotherapy (1974-2003, the first period) and after it (2004-2008, the second period). Results. The survey included 444 patients, of whom 325 and 119 were diagnosed in the first and second period, respectively. Joinpoint regression analysis showed that after 1995 the trend in crude incidence rates increased more rapidly; the annual change was 0.03 per 100,000 per year before 1995 and 0.06 per 100,000 per year after. There was clear male predominance (70%) throughout the period covered by the survey. The proportion of patients above 65 years of age increased from 41.8% to 54.6% for the first and second period, respectively (p = 0.02). With a total of 52 (11.7%) operated patients, surgical treatment was rare and used only for selected patients with early disease and without comorbidity, leading to their relatively long median survival of 13.6 months. Chemotherapy was applied to 56 (1 7.2%) and to 96 (80.7%) patients during the first and second period, respectively. While a variety of older drugs were used in the first period, the most common regimen in the second period (applied to 91 patients) was doublet of low-dose gemcitabine in prolonged infusion and cisplatin. For the whole population of patients regardless the mode of treatment, median survival was 7.4 and 12.6 months (p-value = 0.037) for the first and second period, respectively. Conclusions. Increasing incidence, male predominance and increased proportion of older patients confirm that the burden of mesothelioma persists in spite of a 15-years old ban in the production of asbestos. Modern chemotherapy, and in particular treatment with low-dose gemcitabine in prolonged infusion and cisplatin significantly prolonged median survival of patients with malignant pleural mesothelioma in Slovenia. Key words: malignant pleural mesothelioma; incidence; survival; chemotherapy; gemcitabine in prolonged infusion Introduction Malignant pleural mesothelioma (MPM) is a rare and highly aggressive tumour arising from mes-othelial surfaces of pleura.12 After recognizing asbestos as the most important factor in the pathogenesis of mesothelioma, the production and use of asbestos were banned in most developed countries. However, due to the long latent period between exposure to asbestos and the development of the disease, the incidence of mesothelioma is expected to increase for at least another decade. Since the risk for development of mesothelioma persists for several decades after professional or environmental exposure, the burden of the disease will shift to the older population.2-6 In spite of all efforts to find an effective treatment, the prognosis of mesothelioma remains poor and over 90% of patients die from the disease.7 For the few patients in good performance status, without significant co-morbidity and with apparently limited disease, magnetic resonance (MR) and positron emission tomography-computerised tomography (PET-CT) are helpful in selecting patients with early stages for surgery and/or multimodal-ity therapy with curative intent.8-12 However, even most aggressive treatment rarely leads to cure. In several series of early-stage mesothelioma treated with multi-modality approach including surgery, median survival rarely exceeds two years. The optimal selection of the surgical procedure remains to be defined and the standard treatment for early stage of MPM remains unclear.12-15 At the time of diagnosis, most patients have advanced disease. In this situation, systemic treatment is the only modality with a potential to influence survival. Due to scepticism regarding clinical benefit of chemotherapy for patients with mesothe-lioma, a randomised clinical study was conducted in England, comparing chemotherapy with navel-bine to best supportive care alone. While the trial did not confirm a statistical significant difference, it did show a clear trend for an improved survival for patients treated with chemotherapy, in comparison to the control arm.16 During the past 15 years, dozens of trials of chemotherapy for patients with mesothelioma have been reported. Since all these trials were performed on selected populations of patients and none of them included a control arm without chemotherapy, the real value of chemotherapy remains unknown. This is especially true for patients in poor performance status or with significant co-morbidity who are not eligible for large multi-center clinical trials.17 In Slovenia, we had two distinct periods of treatment of mesothelioma. Until 2003, occasional patients with early disease were treated with surgery, some patients received palliative irradiation, and very few patients received any form of systemic treatment. In 2003, we activated a Phase II clinical trial of low-dose gemcitabine in prolonged infusion and cisplatin.18 Due to the national policy of referral of all patients with mesothelioma to the Institute of Oncology Ljubljana, virtually all patients with mesothelioma eligible for treatment with platin-based chemotherapy were included in the trial. So far, we found only one population-based report, which has been published to prove that chemotherapy can improve survival for patients with mesothelioma.19 We therefore did the following survey, aiming to evaluate the role of chemotherapy for pleural mesothelioma on a population basis in Slovenia. Patients and methods The survey included all patients with permanent residence in Slovenia with a diagnosis of malignant pleural mesothelioma in the period from 1974 till 2008 and reported to the Cancer Registry of Republic of Slovenia. Data were derived from individual hospital reports to the Cancer Registry. For patients who received any form of specific anticancer treatment, additional data were obtained from the clinical documentation of the Institute of Oncology Ljubljana. Eligible patients had biopsy-proven malignant mesothelioma regardless the histologic subtype. Almost all patients had thoracoscopy or CT-guided biopsy as well as US-guided needle biopsy.182021 Stage of disease was not consistently recorded in the clinical documentation and was therefore omitted from the analysis. Data on surgery and on the type of chemotherapy were recorded. During the period covered by the survey, radiotherapy was exclusively applied for palliation, using a wide spectrum of fractionation schedules. Since palliative radiotherapy does not influence survival, data on radiotherapy were not included in the analysis. The two periods of treatment were defined as the era of infrequent chemotherapy (1974-2003) and of frequent use of chemotherapy (2004-2008). These were further divided into 5-year periods. December 1, 2011 was the close-out date for data collection. Overall survival time was calculated from the day of diagnosis to the death from all causes or TABLE 1. Demographic data for malignant pleural mesothelioma, Slovenia 1974-2008 1974-2003 2004-2008 Total Males: number (%) Females: number (%) Age: range (mean) Age above 45: number (%) Total 223 (68.6%) 102 (31.4%) 22-89 (61.7) 136 (41.8%) 325 91 (76.5%) 28 (23.5%) 33-87 (64.4)* 65 (54.6%) 119 314 (70.7%) 130 (29.3%) 22-89 (62.4) 201 (45.3%) 444 *p = 0.021 1,6 1,4 1,2 o 8 1,0 O 0 1 0,8 ï 0,6 £ 0,4 0,2 breaking point in 1995 0,0 1974 1979 1984 1989 1994 1999 2004 2009 Incidence year FIGURE 1. The crude incidence rates of patients with malignant pleural mesothelioma with results of trend analysis, Slovenia 1974-2008. when censored. Kaplan-Meier method was used for estimation of survival and log-rank test was used to compare survival distributions between samples. A p-value lower than 0.05 was considered statistically significant. The data were analysed using SPSS statistical package (Release 13.0, SPSS Inc., Chicago, IL). 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). To assess trend in cancer rates, joinpoint regression analysis22 was performed with software Joinpoint Regression Program, Version 3.0. Results Patients The survey included 444 patients, of whom 325 had the diagnosis of malignant pleural mesothe-lioma in the first period (January 1974 - December 2003) and 119 in the second period (January 2004 -December 2008). The incidence increased throughout the analyzed time period (Figure 1, Table 1). Joinpoint regression analysis showed that after 1995, the trend in crude incidence rates increased more rapidly. The annual change in crude rates was 0.03 per 100,000 per year for the 1974-1995 period and 0.06 per 100,000 per year for the 1995-2008 period. Both regression slopes and the difference between the slopes are statistically significant with p-values smaller than 0.05 (Figure 1). Demographic data are presented in Table 1. Male predominance was obvious (70.7%). Difference in gender distribution in two periods 1974-2003 and 2004-2008 was not statistically significant (Pearson Chi-Square test gives p-value of 0.107). However, the mean age was statistically significant different between the two time periods (t-test gave p-value of 0.021, normal distribution for age was confirmed by Kolmogorov-Smirnov Test). Furthermore, the proportion of patients over 65 years of age at the date of diagnosis increased from 41.8% for the first half of the survey period to 54.6% for the later years. Surgery was rarely used, except for one of the 5-year periods (1999-2003) when the surgical procedures were more frequent (Figure 2). Fifty-six (17.2%) patients were treated by systemic therapy in the 1974-2003 period and 96 (80.7%) in the years 2004-2008 (Table 2, Figure 2). Two hundreds and sixty-two patients were treated neither by systemic therapy nor by surgery, while 22 patients were treated by both. The choice of chemotherapy clearly depended on the period. Prior to 2003, only rare patients in unusually good performance status received mono-therapy or doublets with older drugs (cisplatin, doxorubicin, methotrexate, etoposide, or interferon). Exception was 5-year period 1999-2003, when we began with clinical trial and the first 10 patients were treated with a new approach. In the second TABLE 2. Number of newly diagnosed malignant pleural mesothelioma cases, number of patients treated by chemotherapy and by surgery, Slovenia 1974-2008 No. of new cases No. of treated by systemic therapy (%) No. of treated by surgery (%) 1974-1978 18 6 (33.3%) 1 (5.6%) 1979-1983 23 4 (17.4%) 4 (17.4%) 1984-1988 50 1 (2.0%) 6 (12.0%) 1989-1993 47 2 (4.3%) 2 (4.3%) 1994-1998 65 4 (6.2%) 6 (9.2%) 1999-2003 122 39 (32.0%) 25 (20.5%) 2004-2008 119 96 (80.7%) 8 (6.7%) Total 444 152 (34.2%) 52 (11.7%) period, after 2003, 68 patients were enrolled in a Phase II clinical trial and received the doublet of low-dose gemcitabine in prolonged infusion (250 mg/m2/6 hours on days 1 and 8) and cisplatin (75 mg/m2 on day 2). The similar schedule of treatment was given to 4 patients in another clinical trial Agili. Additional, 19 patients with impaired organ function or in poor performance status who did not meet the eligibility criteria for the trial received a modified treatment schedule. For these poor-risk patients, we usually applied gemcitabine at even a lower dose of 200 mg/m2/6 hours and either cisplatin at 60 mg/m2 or carboplatin at AUC 5 and omitted gemcitabine on day 8 of a 3-weekly cycle. Finally, 5 patients treated in the last 5 year of survey received other forms of the first line chemotherapy, the doublet of pemetrexed and cisplatin. Overall survival Median survival increased from 7.4 months (95% confidence interval [CI] was 5.9-23.8) for the period of 1974-2003 to 12.6 months (95% CI 10.7-14.5) for the period 2004-2008. The difference between the two periods was statistically significant (p = 0.037) (Figure 3). Regarding surgery, the median survival for surgical patients was 13.6 months (95% CI 10.6-16.7), as compared to 8.4 months (95% CI 7.0-9.9) for nonsurgical patients (p = 0.000; Figure 4). Patients treated by systemic therapy had significantly longer survival than those who did not receive chemotherapy. Median survival times for patients who did receive or did not receive chemotherapy were 14.5 months (95% CI 11.4-15.8) and 5.6 months (95% CI 3.9-7.3), respectively (p = 0.000; Figure 5). 140 -120 -100 - jj 60 -E □ No. of new cases □ No. of treated by chemotherapy □ No. of treated by surgery 1974-1978 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Period FIGURE 2. Number of newly diagnosed malignant pleural mesothelioma cases, number of patients treated by chemotherapy and by surgery, Slovenia 1974-2008. FIGURE 3. Overall survival of Slovenian patients with malignant pleural mesothelioma by two time periods, 1974-2003 and 2004-2008. P-value refers to log-rank test used to compare survival distributions in the two periods. 8 6 6 4 4 4 2 2 0 0 C 1 2 3 4 Tin« Iftiri) FIGURE 4. Overall survival of patients with malignant pleural mesothelioma with respect to surgery, Slovenia 1974-2008. P-value refers to log-rank test used to compare survival distributions between the two data samples. 0- Tlnti FIGURE 5. Overall survival of patients with malignant pleural mesothelioma with respect to systemic therapy, Slovenia 1974-2008. P-value refers to log-rank test used to compare survival distributions between the two data samples. Discussion Our survey is the first one, worldwide, to confirmed that systematic introduction of chemotherapy leads to longer survival for the national population of patients with malignant pleural mesothelioma. The whole unselected population as the basis of our survey confirms the validity of the data and makes our survey distinct to reports on clinical trials which typically include only patients in good performance status and without significant co-morbidity. Slovenia has the privilege of an excellent national cancer registry with a long tradition covering more than 60 years. Moreover, the country is compact, national health policy is well defined, migration of the population is relatively limited, and vital national statistics are complete and reliable. These circumstances further support the validity of the data presented in this survey. In spite of a ban in the production and use of asbestos implemented in 1996, the incidence of mesothelioma in Slovenia is still rising. Joinpoint regression analysis showed that after 1995 the trend in crude incidence rates increased more rapidly (Figure 1). While better diagnostic possibilities in recent years might contribute to the observation of the rising incidence, we nevertheless believe that the data reflect a real persistent and increasing risk for the disease. Furthermore, our data support the concept of a long latency period between exposure to asbestos and development of mesothelioma. In this respect, we see a persistent and markedly increased risk in the local communities at close proximity to the former asbestos factory.18 Also notable is an increasing proportion of elderly patients with mesothelioma and clear male predominance in recent cohorts covered by our survey. At 73 years after the beginning of production of asbestos in Slovenia and 15 years after the facility closed its production of asbestos, these observations additionally indicate that the latency period from the exposure to asbestos to the development of disease is really long. The other putative aetiological factor, Semian virus 40, was not implicated in pathogenesis of malignant pleural mesothelioma in Slovenia.2324 So far, all efforts to implement screening and early diagnosis of mesothelioma for the high-risk populations have failed, or are still in the investigative phase.2 Our survey proves that very few patients are diagnosed at an early stage when multi-modality treatment with a curative intent is a realistic option. We believe that carefully selected patients do benefit from surgery; indeed, patients treated with surgery had significantly better survival than those who were not operated (p-value = 0.000) (Figure 4). In the interpretation of these data, one should consider that the surgical patients are usually those with good prognostic factors: good performance TABLE 3. Effectiveness of different chemotherapy schemes in the treatment of the patients with malignant pleural mesothelioma Trial Phase of study Drugs N RR, MOS, MPFS, 1-year % months months survival, % Byrne MJ et al.30, 1999 Phase II Gemcitabine + cisplatin 21 48 10.0 NA NA Aversa SM et Favaretto AG31, 1999 Phase II Gemcitabine + carboplatin 20 20 NA 4-21 NA van Haarst JMW et al.32, 2002 Phase II Gemcitabine + cisplatin 32 16 9.6 6.0 36 Nowak AK et al.33, 2002 Phase II Gemcitabine + cisplatin 52 17 11.2 6.4 NA Mikulski SM et al.34, 2002 Phase II Ranpirnase 105 5 8.3 3.4 34 Vogelzang NJ et al.28, 2003 Phase III Pemetrexed + cisplatin cisplatin 226 222 41 17 12.1 9.3 5.7 3.9 50 38 Favaretto AG et al.35, 2003 Phase II Gemcitabine + carboplatin 50 26 14.7 8.9 53 Andreopoulou E et al.36, 2004 Phase II Mitomycin C + vinblastine + cisplatin 150 15 7.0 NA 31 van Meerbeeck JP et al.37, 2005 Phase III Raltitrexed + cisplatin cisplatin 126 124 24 14 11.4 8.8 5.3 4.0 46 40 Castagneto B et al.38, 2005 Phase II Gemcitabine + cisplatin 35 26 13.0 8.0 NA Berghmans T et al.39, 2005 Phase II Epirubicin + cisplatin 69 19 13.3 NA 50 Jänne PA et al.40, 2005 Phase II Pemetreksed+gemcitabine 108 17 10.1 7.4 46 Ceresoli GL et al.41, 2006 Phase II Pemetrexed + carboplatin 102 19 12.7 6.5 52 Obasaju CK et al.42, 2007 Phase IV (EAP) Pemetrexed + cisplatin 728 21 10.8 NA 45 Santoro A et al.43, 2007 Phase IV (EAP) Pemetrexed + cisplatin or carboplatin 861 22 NA NA 64 Castagneto B et al.44, 2008 Phase II Pemetrexed + carboplatin 76 25 14.0 NA NA Kalmadi SR et al45, 2008 Phase II Gemcitabine + cisplatin 50 12 10.0 6.0 30 Hillerdal G et al46, 2008 Phase II Gemcitabine + carboplatin + liposomized doxorubicin 173 32 13.0 8.6 NA Sorensen JB et al47, 2008 Phase II Vinorelbin + cisplatin 54 30 16.8 7.2 61 Muers M et al.16, 2008 Phase III Vinorelbin Mitomycin + vinblastine + cisplatin 136 132 16 10 9.5 7.6 6.2 5.6 41 33 Ralli M et al.48, 2009 Phase II Docetaxel + gemcitabine 25 28 15.0 7.0 NA Sorensen JB et al49, 201 1 Phase II Vinorelbin + carboplatin 47 30 14.6 7.2 55 Kovac V et al.18, 2012 Phase II Gemcitabine* + cisplatin 78 50 17.0 8.0 67 N = number of patients included in the trial; RR = response rate; MOS = median overall survival; MPFS = median progression-free survival, NA = not available; EAP = expanded access program * applied in low dose in 6-hours infusion status, low comorbidity, low stage of disease, low weight loss and epitheloid subtype of mesothelioma.18202526 A bias in the selection for surgery precludes any comparison to other patients.1820 Our survey revealed a statistically superior survival for patients treated after 2004 when we introduced chemotherapy as a standard treatment modality for mesothelioma. Regarding this finding, two possible factors leading to a bias should be discussed. The first one is earlier diagnosis in recent cohorts of patients. This seems unlikely, since there was no program for early diagnostics of me-sothelioma and since the number of patients with early operable stages remained constantly low. The second possible bias is improved supportive care in recent years. While this possibility cannot be entirely rejected27, we believe that better supportive care alone cannot be responsible for a prolongation of the median survival for more than 5 months. Hence, it seems reasonable to link improved survival to the new treatment policy and to introduction of chemotherapy. After the trial conducted in England and discussed in the introduction16 and after our survey18, the question of benefit of chemotherapy for most patients with mesothelioma appears to be solved. However, the choice of a particular chemothera-peutic schedule is a distinct question. The three parameters determining the choice are efficacy; side effects, quality of life and convenience for the pa- tients; and costs. We will first discuss the published experience with other scheduled of chemotherapy and later return to low-dose gemcitabine in prolonged infusion and cisplatin as our preferred combination during the last five years of our survey. In 2003, Vogelzang published experience from a landmark trial which compared pemetrexed and cisplatin against monotherapy with cisplatin and demonstrated a statistically significant advantage for the doublet.28 On the basis of this trial, peme-trexed was the first drug to be specifically registered for the treatment of mesothelioma, leading to its wide acceptance as the standard treatment. A critical look reveals that the superiority of pe-metrexed may be attributable to suboptimal control arm: cisplatin alone is was never the standard treatment for mesothelioma, and certainly not at the turn of the century when several other drugs and their combinations were available. In that period, the doublet of gemcitabine and cisplatin or carboplatin was the most widely used systemic treatment for mesothelioma patients.29 Pooled data of 7 studies lead to an estimated median survival of 11.7 months, which is comparable with median survival of 12.1 months in pemetrexed study.1828 A large spectrum of other combinations from various Phase II clinical trials reported results which are at least comparable to the doublet of pemetrexed and cisplatin, and superior to cisplatin alone (Table 3). Superior survival of the national pool of patients with mesothelioma during the last five years of our survey should be attributable to low-dose gemcit-abine in prolonged infusion and cisplatin as our preferred combination. On the basis of a favourable experience in several trials for non-small cell lung cancer50-52, we decided to use this combination also for patients with mesothelioma and included 78 patients in a Phase II clinical trial (10 patients in the 5-year period 1999-2003 and 68 patients in the 5-year period 2004-2008).18 In the last 5-year period, additional 19 patients in poor performance status or with organ dysfunction who were not eligible for the aforementioned trials received a less intensive modification of the same schedule. After completing a Phase II trial for mesothelioma, our research continues with an on-going randomised Phase II trial which compares this combination to the doublet of pemetrexed and cisplatin (Alimta vs. Gemcitabin In Long Infusion - AGILI trial).53 During the last 5 years of the survey, treatment with low-dose gemcitabine in long infusion and cisplatin was applied to a total of 91 patients. This figure represents 94.8% of the total number of patients who received any form of chemotherapy and 76.5% of the total number of patients with mesothelioma during this period (91 out of 119). Future clinical research on mesothelioma should address several important questions. One of them is to compare different chemotherapy schedules for their efficacy and tolerability, a question already addressed in our on-going AGILI trial.53 The second one is the question of maintenance treatment. This concept got wide acceptance in the treatment of advanced non-small cell lung cancer.5455 Regarding mesothe-lioma, several trials (including our Phase II trial of low-dose gemcitabine in prolonged infusion and cisplatin) showed that patients who responded to first-line treatment have fair chances to benefit either from re-induction of the same treatment, or from a different combination of drugs.185657 Finally, research should focus on genetic polymorphisms which influence DNA damage58, leading to individualised systemic treatment. A key issue in the development of individualized therapy is identification of biomarkers to predict chemotherapeu-tics' efficacy and toxicity.5960 Thus, our research on patients with mesothelioma confirmed that the nucleotide excision repair (NER) pathway polymorphisms influence platinum-treatment efficacy and toxicity26 and that ribonucleotide reductase subunit 1 (RRM1) polymorphisms as well as haplotypes are associated with gemcitabine treatment efficacy and toxicity.60 In conclusion, our survey showed superior survival of patients with mesothelioma during the last five years when a new national policy was implemented and virtually all eligible patients received chemotherapy. Our success should be attributable to our preferred schedule of low-dose gemcitabine in prolonged infusion which proved to be effective, with acceptable toxicity also for patients in poor performance status, and linked to reasonable costs. 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Gemcitabine combined with carboplatin in patients with malignant pleural mesothelioma: a multicentric phase II study. Cancer 2003; 97: 2791-7. 36. Andreopoulou E, Ross PJ, O'Brien ME, Ford HE, Priest K, Eisen T, et al. The palliative benefits of MVP (mitomycin C, vinblastine and cisplatin) chemotherapy in patients with malignant mesothelioma. Ann Oncol 2004; 15: 1406-12. 37. van Meerbeeck JP, Gaafar R, Manegold C, van Klaveren RJ, van Marck EA, Vincent M, et al. Randomized phase III study of cisplatin with or without raltitrexed in patients with malignant pleural mesothelioma: an intergroup study of the European Organisation for Research and Treatment of Cancer, Lung Cancer Group and the National Cancer Institute of Canada. J Clin Oncol 2005; 23: 6881-9. 38. Castagneto B, Zai S, Dongiovanni D, Muzio A, Bretti S, Numico G, et al. Cisplatin and gemcitabine in malignant pleural mesothelioma: a phase II study. Am J Clin Oncol 2005; 28: 223-6. 39. 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S0rensen JB, Frank H, Palshof T. Cisplatin and vinorelbine first-line chemotherapy in non-resectable malignant pleural mesothelioma. Br J Cancer 2008; 99: 44-50. 48. Ralli M, Tourkantonis I, Makrilia N, Gkini E, Kotteas E, Gkiozos I, et al. Docetaxel plus gemcitabine as first-line treatment in malignant pleural mesothelioma: a single institution phase II study. Anticancer Res 2009; 29: 3441-4. 49. S0rensen JB, Bech C, Sorensen P, Frank H. Phase II study of carboplatin and vinorelbine 1st line treatment in advanced malignant pleural mesothe-lioma. [Abstract]. J Thorac Oncol 2011; 6(6 Suppl): S1354-5. 50. Zwitter M, Kovac V, Smrdel U, Kocijancic I, Segedin B, Vrankar M. Phase I-II trial of low-dose gemcitabine in prolonged infusion and cisplatin for advanced non-small cell lung cancer. Anticancer Drugs 2005; 16: 1129-34. 51. Zwitter M, Kovac V, Smrdel U, Vrankar M, Zadnik V. Gemcitabine in brief versus prolonged low-dose infusion, both combined with cisplatin, for advanced non-small cell lung cancer: a randomized phase II clinical trial. J Thorac Oncol 2009; 4: 1148-55. 52. Zwitter M, Kovac V, Rajer M, Vrankar M, Smrdel U. Two schedules of chemotherapy for patients with non-small cell lung cancer in poor performance status: a phase II randomized trial. Anticancer Drugs 2010; 21: 662-8. 53. Kovac V. Zwitter M. Cisplatin with Alimpta or gemcitabine in long infusion for mesothelioma (AGILI). Available: http://clinicaltrials.gov/ct2/show/ NCT01281800 54. Brodowicz T, Krzakowski M, Zwitter M, Tzekova V, Ramlau R, Ghilezan N, et al; Central European Cooperative Oncology Group CECOG. Cisplatin and gemcitabine first-line chemotherapy followed by maintenance gemcitabine or best supportive care in advanced non-small cell lung cancer: a phase III trial. Lung Cancer 2006; 52: 155-63. 55. Velez M, Arango BA, Perez CA, Santos ES. Safety and efficacy of pemetrexed in maintenance therapy of non-small cell lung cancer. Clin Med Insights Oncol 2012; 6: 117-24. 56. Ceresoli GL, Zucali PA, De Vincenzo F, Gianoncelli L, Simonelli M, Lorenzi E, et al. Retreatment with pemetrexed-based chemotherapy in patients with malignant pleural mesothelioma. Lung Cancer 2011; 72: 73-7. 57. Zucali PA, Simonelli M, Michetti G, Tiseo M, Ceresoli GL, Collovà E, et al. Second-line chemotherapy in malignant pleural mesothelioma: Results of a retrospective multicenter survey. Lung Cancer 2012; 75: 360-7. 58. Goricar K, Erculj N, Zadel M, Dolzan V. Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage. Radiol Oncol 2012; 46: 46-53. 59. Zimling ZG, S0rensen JB, Gerds TA, Bech C, Andersen CB, Santoni-Rugiu E. Low ERCC1 expression in malignant pleural mesotheliomas treated with cisplatin and vinorelbine predicts prolonged progression-free survival. J Thorac Oncol 2012; 7: 249-56. 60. Erculj N, Kovac V, Hmeljak J, Franko A, Dodič-Fikfak M, Dolzan V. The influence of gemcitabine pathway polymorphisms on treatment outcome in patients with malignant mesothelioma. Pharmacogenet Genomics 2012; 22: 58-68. Long term outcome after combined modality treatment for anal cancer Irena Oblak, Primoz Petric, Franc Anderluh, Vaneja Velenik, Peter Albert Fras Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2012; 46(2): 145-152. Received 23 December 2011 Accepted 25 January 2012 Correspondence to: doc. dr. Oblak Irena, MD, PhD, Department of Radiology, Institute of Oncology Ljubljana, Zaloška 2, 1000 Ljubljana, Slovenia. Phone: +386 1 5879 293; Fax: +386 1 5879 304; E-mail: ioblak@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the retrospective study was to evaluate the effectiveness and toxicity of radiochemother-apy in patients with squamous cell carcinoma of the anal canal treated at a single institution. Patients and methods. Between 1/2003 and 9/2010, 84 patients were treated with radical radiochemotherapy at the Institute of Oncology Ljubljana, Slovenia. The treatment consisted of 3-dimensional conformal external beam radiotherapy with concurrent chemotherapy (5-fluorouracil and mytomycin C), followed by brachytherapy or external beam boost. The toxicity of therapy and its effectiveness were assessed. Results. The treatment was completed according to the protocol in 79.8% of patients. The median follow-up time of 55 survivors was 53 months (range: 16-105 months). The 5-year locoregional control (LRC), disease-free survival (DFS), disease-specific survival (DSS), overall survival (OS) and colostomy-free survival (CFS) rates were 71%, 68%, 81%, 67% and 85%, respectively. No treatment-related mortality was observed. The most frequent acute side-effect of the treatment was radiodermatitis (grade 3-4 in 58.2% of patients). LENT-SOMA grade 3-4 late radiation side effects were observed in 15 (18%) patients. In patients with brachytherapy boost a trend of less late side effects was observed compared to patients with external beam boost (P=0.066). On multivariate analysis, complete clinical disease response was identified as an independent prognostic factor for LRC, DFS and DSS, the salvage surgery for LRC and DFS, whereas Hb below 120 g/l retained its independent prognostic value for OS. Conclusions. Radiochemotherapy provides an excellent disease control and the survival with preserving anal sphincter function in majority of patients. Surgical salvage with abdominoperineal resection for persistent or recurrent disease has curative potential. Key words: anal cancer; radiochemotherapy; salvage surgery; outcome Introduction Anal cancer is a relatively rare tumour, representing 2-4% of all cancers of colorectum and anus.1 Women are more often affected than men. During the past decades, the incidence in developing countries has increased, mostly in young homosexual men, perhaps due to sexual transmission of human papilloma virus (HPV) and human immunodeficiency virus (HIV), which are known causal factors in this and other cancers.12 Anal cancer is predominantly a locoregional disease and distant metastases are found in 5-10% of the patients.13-8 Following publications on organ preserving treatment in the eighties9, the treatment paradigm has shifted from abdomino-perineal resection (APE) with permanent colostomy to radical radiochemo-therapy, resulting in sphincter preservation rates of around 80%, even in cases with locally advanced disease. Surgery is indicated only in cases of residual or recurrent tumour and for complications of radiotherapy.110 Numerous trials have demonstrated complete response rates of 80-90%, with high local control, survival and sphincter preservation rates following radical radiochemotherapy.310-12 The purpose 1.00.8- 2 0.6- £ 0.4- 0.20.0 0 ■LRC ■ DFS 12 48 60 24 36 Time (months) FIGURE 1. Locoregional control (LRC) and disease-free survival (DFS). Patients and methods Patients and tumour characteristics Eighty-six patients with biopsy proven cancer of the anal canal were treated at the Institute of Oncology Ljubljana between January 2003 and September 2010. Two patients with distant metastases were treated with palliative intent and were excluded from present analysis. The remaining 84 patients (48 females and 36 males) were treated with curative intent. Mean age was 63 years (range: 34-87 years). According to the UICC TNM staging criteria, 6 (7.1%) patients had stage I, 48 (57.1%) stage II, 14 (16.7%) stage IIIA and 16 (19%) stage IIIB disease.13 1.0 0.8- > 0.6H čo 0.4- 0.2-1 0.0 ■ DSS ■OS 0 48 60 12 24 36 Time (months) FIGURE 2. Disease-specific survival (DSS) and overall survival (OS). 1.0-» 0.8- O > 0.6- > o V) 0.4- 0.2- 0.0- 0 ■CFS -1-1-1-1-1-1-1-1-1 12 24 36 48 60 Time (months) FIGURE 3. Colostomy-free survival (CFS). of our retrospective study was to evaluate the effectiveness and toxicity of radiochemotherapy in a single-centre prospective cohort of patients with squamous cell carcinoma of the anal canal. Investigations before and during therapy The multidisciplinary approach is the policy of treatment for all cancer patients at the Institute of Oncology Ljubljana14; therefore, all patients were presented to a multidisciplinary advisory team, consisting of a surgeon, radiation oncologist and medical oncologist, in order to assess the prospects of the treatment. All patients underwent a general clinical examination, blood tests, chest radiography and abdominopelvic computed tomography (CT). Locoregional extent of the disease was evaluated with anorectal examination (performed by a surgeon and a radiation oncologist), rectoscopy, endoscopic ultrasound (US) and magnetic resonance imaging (MRI) of the pelvis. In cases, suspicious for inguinal lymph node involvement, fine needle aspiration biopsy was performed. Detailed pre-treatment clinical drawings and photographs were taken and tumour borders tattooed on perianal skin for the purpose of brachytherapy (BT) treatment planning. During the treatment, weekly clinical examination and blood tests were performed. The acute treatment related toxicity was assessed according to National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 2.0.15 Treatment A planned treatment schedule consisted of 3-di-mensional (3D) conformal external beam radiotherapy (EBRT) with concurrent chemotherapy (ChT), followed by brachytherapy (BT) or EBRT boost. 3D conformal EBRT was delivered using a four-field box technique at a 15 MV linear accelerator. Clinical target volume (CTV) included the gross tumour volume with a safety margin of 1.5-2 cm in all directions and the regional lymph node areas. To arrive at a planning target volume (PTV), an additional margin of 0.7 cm in all directions was applied to the CTV. A nominal dose of 45 Gy (1.8 Gy daily fractions, five fractions a week) was prescribed to >95% of the PTV. Prophylactic bilateral inguinal EBRT was given to 45-50 Gy by anterior photon beam and adequate additional electron beam complements of adequate energy to reach the deepest portion of these nodes. In cases with inguinal lymph node metastases, the involved areas were irradiated with separate electron fields to a total dose of 60 Gy. Concurrent ChT was planned in all but stage I patients and patients with significant medical comorbidities. ChT consisted of two cycles of 5-fluorouracil (5-FU) (daily dose of 1000 mg/m2 in 96-hours continuous infusion), given during weeks 1 and 5 of EBRT. Mitomycin C (10 mg/m2 in bolus intravenous injection) was administered on day 1 of the first ChT cycle. After delivery of 45 Gy of EBRT+/-ChT, a boost dose was planned. In tumours, larger than 5 cm or N2-3 disease, the boost was applied with EBRT, whereas in all other cases an interstitial pulseddose rate BT boost was delivered. CTV at the time of BT corresponded to initial tumour extension, as documented by pre-treatment clinical drawings, imaging examinations, photographs and tattooed markings of tumour borders on perianal skin. Metal needles were implanted through a perineal template homogeneously in the CTV, respecting the rules of the Paris system. The distance between needles and ano-rectal mucosa was kept above 5 mm. This was assured by palpation during the needle insertion and by transrectal US. The anal cylinder was inserted to displace uninvolved ano-rectal mucosa from the high dose region. Until 2006, treatment planning was based on two orthogonal radiographs. From then on, CT based treatment planning was introduced. A biologically equivalent dose of 15-30 Gy was prescribed to the reference isodose line, corresponding to the 85% of mean basal dose (linear quadratic model, assuming an a/ß of 10 Gy and 3 Gy for the tumour and late reacting normal tissues, respectively, sublethal damage repair half time of 1.5 hours, reference dose rate of 0.5 Gy per hour). The prescribed dose was chosen depending on initial tumour burden and extent of regression during EBRT. After introduction of CT into treatment planning, subtle individualized 3D-optimization of dose distribution was performed to increase the dose to the CTV while respecting the normal tissues tolerance. In cases of severe treatment-related toxicity, irradiation and/or ChT doses were modified and adapted to the patient's physical condition or laboratory findings. When necessary, ChT application was delayed, or EBRT was temporarily interrupted or terminated. Follow-up after treatment The first post-treatment follow-up visit was performed by a senior radiation oncologist 6 weeks after the completion of radiotherapy. A response to the treatment was evaluated by clinical examination, appropriate imaging studies (MRI, US) and biopsies, when indicated. In patients with clinical complete remission, follow-up investigations were carried out at 3 month-intervals thereafter. In cases of the incomplete response, the clinical evaluation was repeated every 6 weeks until the complete remission was recorded. In cases with evidence of progression or recurrence after the end of the treatment, surgery (APE) was recommended. Late normal tissue side effects (events occurring 3 months or more after the end of the treatment) were assessed at time of each follow-up evaluation, employing the LENT-SOMA scoring system.16 Statistical analysis and ethical consideration A statistical analysis was performed using personal computer and software statistical package SPSS, version 18 (SPSS Inc., USA). The main endpoints of the study were as follows: response to therapy, locoregional control (LRC, the event was local or regional recurrence), disease-free survival (DFS, the event was local, regional or systemic recurrence), disease-specific survival (DSS, the event was death due to the carcinoma of the anal canal), overall survival (OS, the event was death from any cause) and colostomy-free survival (CFS, the event is need for colostomy). The survival of patients was computed from the date of the treatment start to December 1, 2011 (close-out date). Survival probability was calculated using Kaplan-Meier estimate, and log-rank test was used to evaluate the influence of individual prognostic factors (age, performance status, T-, N-and overall stage, radiotherapy and chemotherapy dose), on the analysed endpoints.1718 Independent prognostic values of the factors that appeared statistically significant on the univariate analysis, were tested by the multivariate Cox regression analysis model.19 Two-sided tests were used and TABLE 1. Acute treatment toxicities Toxicity NCI2 grade (%) 0 1 23 4 Total Stomatitis 61 19 12 8 0 100 Nausea, vomiting 82 10 6 2 0 100 Diarrhoea 61 18 11 9 1 100 Radiodermatitis 12 14 16 57 1 100 Infection 55 17 18 7 3 100 Leucocyte count 43 31 18 7 1 100 Haemoglobin level 46 46 7 0 1 100 Platelet count 69 27 3 1 0 100 the differences at P<0.05 were considered as statistically significant. The retrospective study was carried out according to the Declaration of Helsinki. Results Course of treatment Median duration of EBRT and total treatment time was 36 days (range: 29-72 days) and 57 days (range: 30-98 days), respectively. Sixty-seven (79.8%) patients completed the treatment according to the protocol. Total EBRT dose of 45 Gy was applied in 82 (97.6%) patients. In two, due to acute side effects, EBRT was stopped at 18 and 25 Gy, respectively, but the treatment was completed with BT (TD: 30-35 Gy). During EBRT, two cycles of 5-FU were administered as planned in 67 (79.8%) patients. Eight (14%) patients received one cycle only due to adverse side effects during the treatment. Concomitant capecit-abine was administered in one patient who was primarily operated for locoregionally advanced colon carcinoma and in who during preoperative investigations synchronous anal carcinoma was found. Chemotherapy was omitted in 6 (7.1%) and 3 (3.6%) patients due to stage I disease and severe comorbidity, respectively. Boost with the median dose of 14.4 Gy (range: 6-20 Gy) to the primary tumour was applied through reduced photon fields in 33 (39.3%) patients. Interstitial BT boost was performed in 49 (58.3%) patients after EBRT with a mean interval of 27 days (range: 6-57 days). Boost was omitted in two (2.4%) patients because of treatment side effects in one case and the other patient refused the further treatment. Acute side effects The treatment was well tolerated in the majority of patients and no treatment-related mortality was observed. Frequency and intensity of acute adverse side effects are listed in Table 1. The most frequent grade 3 side-effect was radiodermatitis, occurring in 48 (57%) patients during EBRT. One patient developed grade 4 radiodermatitis. All cases of radio-dermatitis healed without consequences. Outcome Median follow-up time was 43 months (range: 8-105 months) in all patients and 53 months (range: 16-105 months) in survivors. At the first follow-up evaluation, which was done 6 weeks after the treatment, the complete clinical remission of the tumour was found in 55 (65.5%) patients. At 18 weeks after the end of the treatment, complete remission, partial response and stable disease were recorded in 67 (79.8%), 12 (14.3%) and 4 (4.8%) patients, respectively. In one patient, there was evidence of tumour progression during the treatment. Five patients with complete response later developed local or locoregional recurrence after a median period of 8 months (range: 4-26 months). In two patients with complete response, distant metastases without local recurrence occurred. Of 24 (28.6%) patients with persistent disease or locoregional recurrence, 12 (14.3%) were treated surgically. In 11 patients, APE was performed and one patient had inguinal dissection due to a recurrence in inguinal lymph nodes. Other 12 (14.3%) patients had unresectable disease. Three of the operated patients died of the recurrent disease, others are alive without evidence of the disease. TABLE 2. Univariate and multivariate analysis of survival 9 111 a ö 0 3 0 0 N 0 M fr Ol l—l N VI N Locoregional control Disease free survival Disease specific survival Overall survival Colostomy free survival Prognostic factor N UVA MVA UVA MVA UVA MVA UVA MVA UVA MVA % p- p-value value % p- p-value value % p- p-value value % P-value P" value % p- p-value value T-stage Tl+2 T3+4 45 39 80.3 55 0.016 80.3 46.6 0.004 92.3 65.2 0.009 75 57.5 0.049 94.4 45.9 0.01 N-stage NO N + 56 28 78.6 50.6 0.028 75 47.8 0.018 90.8 59.4 0.002 73.7 56.9 0.059 89.8 71.7 0.015 Overall stage Stage l+ll Stage III 54 30 78.5 50.8 0.031 74.9 48 0.02 90.8 59.4 0.002 73.6 56.9 0.061 89.8 71.7 0.015 PS 0 1+2 58 26 75.2 54,5 75.2 43.4 81.3 78.9 77.9 39.4 0.041 84.4 84.7 OTT <73 days > 73 days 70 14 73.4 56.3 0.043 69 56.3 0.062 83 67.5 72.3 44.2 0.062 70.6 60.9 0.086 CR Yes No 67 17 91.1 5.9 <0.001 <0.001 85.6 5.9 <0.001 <0.001 95.9 22.4 <0.001 <0.001 80 21 <0.001 66.8 59.3 0.014 Hb < 120 g/l > 120 g/l 47 37 58.5 82.7 0.016 0.061 52.6 80 0.017 68 94 0.015 45.5 90.7 0.001 0.007 78.7 89.6 SS Yes No 13 71 30.8 77.9 <0.001 0.01 30.8 73.3 <0.001 0.003 71.9 82.4 67.3 71.9 10.1 100 <0.001 N, number of patients; UVA, univariate analysis; MVA, multivariate analysis; PS, performance status; OTT, overall treatment time; CR, complete response; Hb, hemoglobin; SS, salvage surgery. 0 ff ö * ID SI r 0 3 10 rrn ID 1 3 t (D On the study close-out date, 55 (65.5%) patients were alive, 51 (92.7%) of them being disease free. Fifteen (17.9%) patients died from the anal canal cancer. One (1.2%) patient, who experienced lo-coregional recurrence, died from metachronous bronchus cancer, four (4.8%) patients died from metastatic breast cancer, metastatic colon cancer, metastatic bronchus cancer and metastatic malignant melanoma, six patients (7.1%) died from vascular events and in three (3.5%) patients the cause of death could not be determined. The 5-year LRC, DFS, DSS, OS and CFS rates for all patients are 71%, 68%, 81%, 67% and 85%, respectively (Figure 1-3). Late side effects Grade 3-4 late radiation side effects according to the LENT-SOMA scoring system13 were observed in 15 (18%) patients. Three (4%) patients experienced post-treatment anal stenosis, requiring repeated dilatations and two (2%) developed chronic non-healing ulcer at the anal verge. Nine (11%) patients had grade 3 incontinence of anal sphincter. In one patient without disease recurrence, colostomy was performed due to severe anal sphincter disfunction. In one patient with anal stenosis, hematuria was observed, as well. Forty-nine (58.3%) patients with BT boost on primary tumour had less late site effects compared to 33 (39.3%) patients with EBRT boost, but the difference was not significant (P=0.066). Prognostic Factors On the univariate analysis, patients with locally advanced disease (T3-4) and incomplete response had worse LRC and all studied survival endpoints when compared to their counterparts. Patients with the involvement of lymph nodes and patients with overall disease stage III had worse LRC, DFS, DSS and CFS in comparison with patients with N0 and overall stage I or II and patients with Hb below 120 g/l had worse LRC, DFS, DSS and OS in comparison with patients with Hb 120 g/l or higher. In addition, patients with poor performance status (WHO 1 or 2) had worse OS and patients with overall treatment time over 73 days had worse LRC. The patients with salvage surgery (APE or nodal dissection) for residual disease or tumour and/or regional lymph node recurrence had worse LRC, DFS and CFS, but not DSS and OS compared to complete responders. For other analysed factors (sex, age, treatment intensity and the method of radiotherapy boost) no impact on the outcome was found. On the multivariate analysis, a complete clinical disease response was identified as an independent prognostic factor for LRC, DFS and DSS, the salvage surgery for LRC and DFS, whereas Hb below 120 g/l retained its independent prognostic value for OS, and for LRC it was on the threshold of statistical significance (P=0.061) (Table 2). Discussion Before Negro et al. in 1974 reported that a complete tumour response can be achieved with radiochem-otherapy, APE was the standard of the treatment in patients with anal cancer.9 Nowadays, radiotherapy with concomitant ChT represents a standard treatment of anal cancer. Complete response rates and 5-year OS in patients with early stage disease range from 80-90% and 95-100%, respectively, and in patients with tumours larger than 5 cm from 5075% and 35-70%.131120 In our study the complete response was recorded in 67 (79.8%) patients, regardless of the stage. Results of our analysis compare favourably to other published studies.131920-22 According to the data of the Cancer Registry of Slovenia, 24 (24%) patients were not referred to the treatment with radiotherapy in the period between 2003 and 2007.4-8 We can only speculate that these patients were treated with local excision and were not presented to multidisciplinary advisory board. It could be debated if all these patients had an appropriate treatment, since it is well known that the local excision should be reserved only for small, well differentiated mucosal or submucosal tumours (<2 cm) and without sphincter involve-ment.1 Although in 24 (28.6%) patients the complete clinical response could not be achieved or they had recurrent disease, in only 12 (50%) patients salvage surgery was possible and only 8 (66.7%) of these operated patients were free of the disease. APE was performed in 11 patients and in one patient bilateral nodal dissection was carried out due to a solitary lymph node involvement. In one patient, APE was necessary due to severe sphincter incontinence after the end of the treatment. Our results on the salvage surgery rate are comparable to results of Ajani et al. and Peiffert et al. with salvage APE rate of 16% and 10%, respectively.2223 In the study of Akbari et al., where salvage surgery was performed in 57 patients with persistent or recurrent disease, the 5-year OS for all patients was 33%, whereas in our study it was 67%.24 As the median follow-up time in Akbari's study was 34.1 months, whereas in ours it was 43 months (range: 8-105 months), the direct comparison of reported results of these two studies could be misleading. However, the observed 75% rate of disease-free patients after salvage surgery is without doubt encouraging. It is well known that patients with a complete tumour response following radiochemotherapy have a better local control and survival, which was demonstrated in our series, as well.32325 We found out that a complete clinical disease response was an independent prognostic factor for LRC, DFS and DSS. The patients who had salvage surgery had worse LRC and DFS but it is encouraging that no statistically significant difference in DSS and OS was found. We can conclude that patients in whom salvage surgery was performed had similar OS as patients with the complete tumour remission. In some, but not all series, they reported that residual or recurrent carcinoma of anal canal after radioche-motherapy was associated with poor outcome after the attempted salvage surgery.25 Furthermore, they found out that APE is successful as salvage therapy in about 50% of patients with local disease only but salvage rate is very poor in patients that have nodal involvement or residual or recurrent carcinoma which is fixed to the pelvic sidewall.26-27 In our study Hb below 120 g/l was identified as an independent prognostic factor for OS. It is not surprising, because anaemia is namely a well known prognostic factor for the lower tumour control and the survival in patients who are treated with radiotherapy.28 It may be related with hypoxia and consequent development of tumour cells' radioresistency.29 At first evaluation at 6 weeks after the end of the treatment, the complete tumour remission was found in only 55 (65.5%) of patients, while another 12 (14.3%) patients reached the complete remission at 18 weeks post treatment. There are several other reports of very slow disease regression, with a complete response observed even up to 6-9 months after the treatment was completed.13031 The evaluation recommendations suggest that if there is no progression of the disease, a careful »wait and see« policy with repeated biopsies may be advocated. However, in cases of persistent disease or tumour progression, APE is recommended following the histological confirmation of the presence of viable malignant cells. In our study, the profile and frequency of acute and late treatment-related toxic side effects were comparable to reports of other researchers.21032 The most frequently reported acute side effect was radiodermatitis with grade 3 or 4 occurring in 58% of patients during EBRT, whereas 15 (18%) patients experienced grade 3 or 4 late radiation side effects. Three (4%) patients experienced post-treatment anal stenosis, two (2%) developed chronic non-healing ulcer at the anal verge, 9 (11%) patients had grade 3 incontinence of anal sphincter and in one patient without disease recurrence, colostomy was performed due to the severe anal sphincter dysfunction. The rate of our late side effects is similar to other reports.3,11223233 The patients with BT boost on primary tumour had less late side effects (P=0.066). We should emphasize that these patients had less advanced tumours and, correspondingly, smaller tissue volumes were irradiated during boost phase of the treatment. Furthermore, it is well known that patients with BT boost have less late toxicity when compared to EBRT boost, because the use of interstitial implant has the advantage of more focused escalation of irradiation dose, resulting in more efficient sparing of the surrounding normal tissues.34 There are several possibilities for the improvement in the disease control and the survival in the future. In locally advanced disease, innovative approaches with 3D image-based BT boost and intensity modulated radiotherapy offer a potential for the individualised escalation of the target dose while respecting normal tissue tolerance.35 For the treatment of unresectable recurrences and distant metastases, the development of more active new anti-cancer drugs, for example epidermal growth factor receptor (EGFR) inhibitors may represent an option. Finally, the majority of the anal cancers are causally connected to the persistent HPV infection, so it can be assumed that the HPV vaccines may become an important prevention measure against anal cancer in the future. To conclude, radiochemotherapy provides an excellent disease control and the survival with preserving anal sphincter function in majority of patients which were evidenced also by our results. Surgical salvage with APE for persistent or recurrent disease should be considered whenever applicable as it can be curative in substantial proportion of such patients. References 1. Rousseau DL, Thomas CR, Petrelli NJ, Kahlenberg MS. Sqamous cell carcinoma of the anal canal. Surg Oncol 2005; 14: 121-32. 2. But-Hadzic J, Jenko K, Poljak M, Kocjan BJ, Gale N, Strojan P. Sinonasal inverted papilloma associated with squamous cell carcinoma. Radiol Oncol 2011; 45: 267-72. 3. Chapet O, Gerard JP, Riche B, Alessio A, Mornex F, Romestaing P. Prognostic value of tumour regression evaluated after first course of radiotherapy for anal canal cancer. !nt J Radiat Oncol BiolPhys 2005; 63: 1316-24. 4. Cancer incidence in Slovenia 2003. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2006. 5. Cancer incidence in Slovenia 2004. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2007. 6. Cancer incidence in Slovenia 2005. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2008. 7. Cancer incidence in Slovenia 2006. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2009. 8. Cancer incidence in Slovenia 2007. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2010. 9. Nigro ND, Vaitkevicius VK, Considine D. Combined therapy for cancer of the anal canal. Dis Colon Rectum 1974; 27: 763-6. 10. Fraunholz I, Rebeneck D, Weiß C, Rödel C. Combined-modality treatment for anal cancer. Strahlenther Oncol 2010; 186: 361-6. 11. Ferrigno R, Nakamura RA, Dos Santos Novaes PE, Pellizzon AC, Maia MA, Fogarolli RC, et al. Radiochemotherapy in the conservative treatment of anal canal carcinoma: Retrospective analysis of the results and radiation dose effectiveness. Int J Radiat Oncol Biol Phys 2005; 61: 1136-42. 12. Oblak I, Petric P, Anderluh F, Velenik V, Hudej R, Fras AP. Anal cancer chem-oirradiation with curative intent - a single institution experience. Neoplasma 2009; 56: 150-5. 13. Sobin LH, Gospodarowitcz MK, Wittekind C. International Union Against Cancer (UICC): TNM classification of malignant tumours. 7th edition. New York: Wiley-Liss; 2009. 14. Strojan P. Role of radiotherapy in melanoma management. Radiol Oncol 2010; 44: 1-12. 15. Ajani JA, Welch SR, Raber MN, Fiels WS, Krakoff IM. Comprehensive criteria for assessing therapy-induced toxicity. Cancer Invest 1990; 8: 147-59. 16. LENT-SOMA scales for all anatomic sites. Int J Radiat Oncol Biol Phys 1995; 31: 1049-91. 17. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457-81. 18. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 1977; 35: 1-39. 19. Cox DR. Regression models and life-tables. J R StatSoc Bull 1972; 34: 187220. 20. Gerard JP, Ayzac L, Hun D, Romestaing P, Coquard R, Ardiet JM, et al. Treatment of anal canal carcinoma with high dose radiation therapy and concomitant fluorouracil-cisplatinum. Long-term results in 95 patients. Radiother Oncol 1998; 46: 249-56. 21. Flam M, John M, Pajak TF, Petrelli N, Myerson R, Doggett S, et al. Role of mitomycin in combination with florouracil and radiotherapy, and a salvage chemoradiotherapy in the definitive nonsurgical treatment of the epidermoid carcinoma of the anal canal: results of a phase III randomozed intergroup study. J Clin Oncol 1996; 14: 2527-39. 22. Ajani JA, Winter KA, Gunderson LL, Pedersen J, Benson AB 3rd, Thomas CR Jr, et al. Fluorouracil, mitomycin, and radiotherapy vs fluorouracil, cisplatin, and radiotherapy for carcinoma of the anal canal: a randomized controlled trial. JAMA 2008; 299: 1914-21. 23. Peiffert D, Bey P, Pernot M, Guillemin F, Luporsi E, Hoffstetter S, et al. Conservative treatment by irradiation of epidermoid cancers of the anal canal. Prognostic factors of tumoural control and complications. Int J Radiat Oncol Biol Phys 1997; 37: 313-24. 24. Akbari RP, Paty PB, Guillem JG, Weiser MR, Temple LK, Minsky BD, et al. Oncologic outcomes of salvage surgery for epidermoid carcinoma of the anus initially managed with combined modality therapy. Dis Colon Rectum 2004; 47: 1136-44. 25. Cummings BJ, Brierley JD. Anal Cancer. In: Halperin EC, Perez CA, Brady LW, editors. Principles and Practice of Radiation Oncology. 5th edition. Philadelphia: Lippincott Williams& Wilkins, 2008. p. 1383-96. 26. Schiller DE, Cummings BJ, Rai S, Le LW, Last L, Davey P, et al. Outcomes of salvage surgery for squamous cell carcinoma of the anal canal. Ann Surg Oncol 2007; 14: 2780-9. 27. Pocard M, Tiret E, Nugent K, Dehni N, Parc R. Results of salvage abdominoperineal resection for anal cancer after radiotherapy. Dis Colon Rectum 1998; 41: 1488-93. 28. Oblak I, Strojan P, Zakotnik B, Budihna M, Smid L. Hemoglobin as a factor influencing the outcome in inoperable oropharyngeal carcinoma treated by concomitant radiochemotherapy. Neoplasma 2003; 50: 452-8. 29. Horsman MR, Van der Kogel A. Therapeutic approaches to tumour hypoxia. In: Joiner M, Van der Kogel A, editors. Basic Clinical Radiobiology. 4th edition. London: Hodder Arnold, 2009. p. 233-45. 30. Sato H, Koh PK, Bartolo DCC. Management of anal canal cancer. Dis Colon Rectum 2005; 48: 1301-15. 31. Cummings BJ, Keane TJ, O'Sullivan B, Wong CS, Catton CN. Epidermoid anal cancer: treatment by radiation alone or by radiation and 5-fluorouracil with and without mitomycin C. Int J Radiat Oncol Biol Phys 1991; 21: 1115-25. 32. Marshall DT, Thomas CR. Carcinoma of the anal canal. Oncol Rev 2009; 3: 27-40. 33. Mitchell SE, Mendenhal WM, Zlotecki RA, Carroll RR. Squamous cell carcinoma of the anal canal. Int J Radiat Oncol Biol Phys 2001; 45: 1007-13. 34. Hwang JM, Rao AR, Cosmatos HA, Wang R, Kaptein JS, Kagan RA, et al. Treatment of T3 and T4 anal carcinoma with combined chemotharapy and interstitial 192-Ir implantation: a 10-year experience. Brachytherapy 2004; 3: 95-100. 35. 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. The diffuse large B-cell lymphoma - where do we stand now in everyday clinical practice Brigita Gregoric1, Vesna Zadnik2, Barbara Jezersek Novakovic1 1 Division of Medical Oncology and 2 Department of Epidemiology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2012; 46(2): 153-159. Received 3 September 2011 Accepted 2 November 2011 Correspondence to: Assist. Prof. Barbara Jezeršek Novaković, MD, PhD, Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška2, SI-1000 Ljubljana, Slovenia. Phone: +386 15879280; Fax: +386 15879305; E-mail: bjezersek@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Due to superior results observed with the addition of rituximab into treatment of patients with the diffuse large B-cell lymphoma (DLBCL),the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) regimen and its variants became the standard initial treatment of these patients. However, the treatment recommendations are based on results of clinical studies while the conditions of routine treatment are far different from the ones in clinical studies. The aim of this retrospective study was therefore to compare the treatment results of routinely treated patients with the DLBCL to results reported by some larger studies. Patients and methods. Two hundred and ninety five patients with the DLBCL were treated between 2004 and 2008 according to the then protocol with R-CHOP or R-CHOP-like regimens at the Institute of Oncology Ljubljana. Treatment response was evaluated according to Cheson's criteria and the disease-free and overall survival by means of Kaplan Meier survival curves. Results. Response to treatment in our evaluation diverged from the reported one predominately in the low risk group (international prognostic index [IPI] categorisation) and in the very good prognosis group (revised international prognostic index (R-IPI) categorisation). The determined complete response (CR) rates in other IPI and R-IPI groups were generally within expectations. Also in the disease-free survival the largest discrepancy occurred in the low-risk patient group (3 year disease-free survival rate of 75%) and in the very good prognosis group (4 year disease-free survival rate of 59%). In all other IPI risk groups, the disease-free survival at 3 years (low intermediate risk 76%, high intermediate risk group 57%, and high risk group 53%) agreed very well with the quoted ones. Slightly worse was the compliance of the 4 year disease-free survival rates (72% in the good prognosis and 51 % in the poor prognosis group) with the results from the literature. The 3 year overall survival rates (low risk patients 87%, high intermediate risk 61% and high risk patients 51%) were somewhat worse than the reported ones in all IPI subgroups except in the low intermediate risk group (82%). On the other hand, the 4 year overall survival rates of the R-IPI categories (94% in the very good prognosis group, 80% in the good prognosis group, 56% in the poor prognosis group) were much better correlated with the data from the literature. Conclusions. In total, the treatment outcomes of routinely treated patient with the DLBCL at our institute are quite encouraging when compared to results of some larger studies. There are probably no dilemmas about how to treat young good prognosis patients and patients aged over 60 years at present. However, the 5 year overall survival rate of 76% for the young poor prognosis group is unsatisfying and needs to be improved. At present, quite a few studies are underway to clarify which of the regimens will perform best in this population. Key words: diffuse large B-cell lymphoma; R-CHOP; treatment result; routine treatments Introduction The diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodg-kin's lymphomas.12 However, the disease is quite heterogeneous in terms of morphology, genetics, biologic behaviour, and consequently response to treatment and prognosis.1 Beside histopathol-ogy and genetics, similar like in other malignancies, clinical parameters have been identified that gm ■a FIGURE 1. Percentage of patients achieving complete and other responses according to various IPI categories. IPI = international prognostic index. CR = complete response influence the prognosis of patients with DLBCL.3-5 Namely, the age above 60 years, serum lactate dehydrogenase concentration above normal, ECOG performance status of 2 or more, Ann Arbor stage III or IV and number of involved extranodal sites above 1 have been shown to correlate significantly with a shorter disease-free and overall survival of patients treated with anthracycline containing regimen. These five factors have been included in the original international prognostic index (IPI).3 The addition of rituximab to standard chemotherapy (cyclophosphamide, doxorubicin, vincris-tine, and prednisolone [CHOP] and CHOP-like) in patients with DLBCL has resulted in significant improvements of the disease-free and overall survival rates.6-8 Beside the original IPI that has been later on validated also in patients receiving rituximab containing treatment9 similarly the revised international prognostic index (R-IPI)10has been proposed to predict outcome in patients with the DLBCL receiving R-CHOP or R-CHOP-like regimens. It is still unclear which of the two indexes is more appropriate for presentation of study results in this population. Due to superior results with rituximab, the R-CHOP and variants have become the standard initial treatment of patients with the DLBCL.11 However, the conditions of routine treatment are far different from the ones in clinical studies where the study population is highly selected, the histopathology and staging procedures are thoroughly revised and treatment and side effects are strictly controlled. The aim of our retrospective study was therefore to analyse and to compare the treatment results of routinely treated patients with the DLBCL at the Institute of Oncology Ljubljana to results reported by some larger studies. TABLE 1. Distribution of patients according to the selected regimens Number % R-ACVBP 19 6.4 R-CHOP 253 85.8 R-CHOP+MTX 12 4.1 R-CHOP/R-other 11 3.7 Total 295 100.0 R-ACVBP = rituximab, doxorubicin, cyclophosphamide, vincristine, bleomycin, prednisolone; R-CHOP = rituximab, doxorubicin, cyclophosphamide, vincristine, prednisolone; R-CHOP+MTX = rituximab, doxorubicin, cyclophosphamide, vincristine, prednisolone, middle-dose methotrexate; R-CHOP/R-other = R-CHOP with reduced doses of doxorubicin + etoposide Patients and methods Two hundred and ninety five patients with the DLBCL were treated between 2004 and 2008 according to the then protocol with R-CHOP or R-CHOP-like regimens at the Institute of Oncology Ljubljana. The patients' characteristics, patohistological diagnosis, disease stage, response to treatment and survival data were taken from patients' records. Treatment response was evaluated according to Cheson' criteria1213and the disease-free and overall survival by means of Kaplan Meier survival curves. For the determination of statistical differences the log rank test and Chi-square test were applied. Results Patients' characteristics and treatment Among 295 patients, there were 132 males (44.7%) and 163 females (55.3%). Their median age was 64 years (range from 19 to 86 years). One hundred and sixteen patients (39.3%) were aged below 60 years and 179 patients (60.7%) were aged 60 or more years. Ninety three (31.5%) patients had limited disease (stage I or II) and 198 (67.1%) patients had advanced disease (stages III and IV) at presentation. The stage of disease could not be defined in 4 patients. According to the IPI categories, there were 34 (11.5%) patients with IPI 0, 63 (21.4%) patients with IPI 1, 66 (22.4%) patients with IPI 2, 69 (23.4%) patients with IPI 3, 46 (15.6%) patients with IPI 4 and 17 (5.7%) patients with IPI 5, respectively. All patients were treated with R-CHOP or R-CHOP-like regimens (Table 1). The selection of regimen was influenced only by adverse prognostic factors (e.g. massive infiltration of bone marrow and/or bones where middle dose MTX was added to R-CHOP). In just few young poor-prognosis TABLE 2. Response to treatment according to different IPI categories ...... l i i., „, Low intermediate High intermediate ... . . , ,„, . .. All patients Low risk IPI=0,1 risk |p|=2 ^ |p|=3 High risk IPI=4-5 N % N % N % N % N % CR 226 76,6 83 85,6 54 81,8 47 68,1 42 66,7 CRu 4 1,4 1 1,0 0 0,0 0 0,0 3 4,8 PR 36 12,2 10 10,3 4 6,1 14 20,3 8 12,7 SD 2 0,7 0 0,0 0 0,0 1 1,4 1 1,6 PD 13 4,4 1 1,0 4 6,1 4 5,8 4 6,3 Unclassified 14 4,7 2 2,1 4 6,1 3 4,3 5 7,9 Total 295 100,0 97 100,0 66 100,0 69 100,0 63 100,0 CR = complete response; CRu= complete response unconfirmed; PR = partial response; SD = stable disease; PD = progressive disease; unclassified -unclassified response to treatment; IPI = international prognostic index; N = number of patients rï^— ■ \_ os- H s .......___. >o.i- iPijnuu '—--- 3 -nHiTir»Uniil ri-; ^ 0 Hl II ej- Lo j i «-t Irl- M AO] 00-t-r-1-1-r e » » H Tim» [monthsl FIGURE 2. Disease-free survival according to different IPI risk groups. IPI = international prognostic index. patients, the more dose-intensive R-ACVBP21 regimen was used instead of the R-CHOP21 regimen. The R-CHOP14 regimen has never been applied. In patients with compromised cardiac function, reduced doses of anthracyclines were applied and were sometimes compensated for with the addition of etoposide (reduced intensity R-CHOEP). Patients with stage I or II of the disease received 6 cycles while patients with stage I.X, II.X, III and IV received 8 cycles of rituximab containing treatment. Patients treated with R-ACVBP received 6 cycles at maximum. Response to treatment The response to treatment for all patients and for distinct IPI categories is presented in Table 2. The difference in response between the low risk group and low intermediate risk group was statistically insignificant as was the difference between the high intermediate and high risk groups. But the significant Chi-square for the entire table (p=0.045) indicates the significant difference between both low risk and both high risk groups. The response to treatment is given also for distinct R-IPI categories (Table 3). In this case, the difference between the very good and good prognosis groups was statistically insignificant, as well as the entire table Chi-square p value (p=0,088). A statistically significant difference in the response was observed between the IPI 2 and IPI 3 categories which was detected by both indexes - namely by the IPI and the R-IPI and is also clearly presented in Figure 1. The disease-free survival according to IPI and R-IPI categories With the median observation period of 22 months, the estimated 3 year disease-free survival rates were 75.3% for low risk, 75.6% for low intermediate risk, 57.2% for high intermediate risk, and 53.1% for high risk group, respectively (Figure 2). The difference between the groups was statistically significant (log rank, p = 0.001). The progression-free survival was plotted also according to the R-IPI categories - the estimated 4 year disease-free survival rates were 59.4% for very good prognosis, 71.6% for good prognosis, and 51.1% for bad prognosis group, respectively (Figure 3). Again, the difference between the groups was statistically significant (log rank, p= 0.000). The overall survival according to IPI and R-IPI categories With the median observation period of 31 months, the estimated 3 year overall survival rates were TABLE 3. Response to treatment according to different R-IPI categories All patients Very good prognosis IPI=0 Good prognosis IPI=1,2 Bad prognosis IPI=3 N % N % N % N % CR 226 76,6 29 85,3 108 83,7 89 67,4 CRu 4 1,4 0 0,0 1 0,8 3 2,3 PR 36 12,2 5 14,7 9 7,0 22 16,7 SD 2 0,7 0 0,0 0 0,0 2 1,5 PD 13 4,4 0 0,0 5 3,9 8 6,1 Unclassifed 14 4,7 0 0,0 6 4,6 8 6,1 Total 295 100,0 34 100,0 129 100,0 132 100,0 CR = complete response; CRu= complete response unconfirmed; PR = partial response; SD = stable disease; PD = progressive disease; unclassified : unclassified response to treatment; R-IPI = revised international prognostic index; IPI = international prognostic index; N = number of patients FIGURE 3. Disease-free survival according to different R-IPI risk groups. R-IPI = revised international prognostic index. FIGURE 4. Overall survival according to different IPI risk groups. IPI = international prognostic index. 86.9% for low risk, 81.6% for low intermediate risk, 60.9% for high intermediate risk, and 50.9% for high risk group, respectively (Figure 4). The difference between the groups was statistically significant (log rank, p = 0.000). The overall survival was plotted also according to the R-IPI categories - the estimated 4 year overall survival rates were 93.7% for very good prognosis, 79.5% for good prognosis, and 55.9% for bad prognosis group, respectively (Figure 5). Again, the difference between the groups was statistically significant (log rank, p= 0.000). Treatment outcomes according to clinical categories of patients Treatment outcomes were evaluated also according to clinical categories - namely, response to treatment, disease-free survival and overall survival were followed separately for young good prognosis patients (younger than 60 years with IPI 0 or 1), young poor prognosis patients (younger than 60 years with IPI of 2 or more), and older patients (aged over 60 years regardless of IPI), respectively. The response to treatment is given in Table 4, while the disease-free and overall survivals are plotted in Figures 6 and 7, respectively. The difference in the disease-free survival between all three groups was statistically significant (log rank, p = 0.005), but it was insignificant when only young good prognosis and young poor prognosis groups were compared (p = 0.365). Also the difference in the overall survival between all there groups was significant (p = 0.000) as was the difference between young good prognosis and young poor prognosis group (p = 0.005). TABLE 4. Response to treatment according to different clinical categories. The difference among groups was insignificant (p=0.150) All patients <60years, IPI=0,1 <60years, IPI>1 >A0years N % N % N % N % CR 226 76,6 58 85,3 34 70,8 134 74,9 CRu 4 1,4 1 1,5 0 0,0 3 1,7 PR 36 12,2 8 11,8 7 14,6 21 11,7 SD 2 0,7 0 0,0 1 2,1 1 0,6 PD 13 4,4 0 0,0 5 10,4 8 4,5 Unclassified 14 4,7 1 1,5 1 2,1 12 6,7 Total 295 100,0 68 100,0 48 100,0 179 100,0 CR = complete response; CRu= complete response unconfirmed; PR = partial response; SD = stable disease; PD = progressive disease; unclassified : unclassified response to treatment; IPI = international prognostic index; N = number of patients t n-i-1-1-1-rt JO M 60 Tim* [tnûmhî] FIGURE 5. Overall survival according to different R-IPI risk groups. R-IPI = revised international prognostic index. Discussion The treatment outcomes in patients with the DLBCL have been significantly improved with the addition of rituximab to standard anthracycline containing chemotherapies both in terms of the disease-free as well as the overall survival. This has been demonstrated by various researchers during the last decade14-21, which resulted in the introduction of rituximab into standard first-line treatment of these patients. However, it is somewhat difficult to compare the results of different studies due to variable study designs and regimens applied and therefore we are still uncertain about the optimal therapy for a given patient or for a given group of patients.22 Consequently, quite problematic is also the evaluation of treatment outcomes in patients t-r-r-1 a ai 40 BD H Tim» [m emh i] FIGURE 6. Disease-free survival of different clinical categories of patients. DLBCL = diffuse large B-cell lymphoma. IPI = international prognostic index. treated in everyday clinical practice. The introduction of the standard IPI by Shipp et al.3, its validation in patients receiving rituximab containing treatments by Ziepert et al.9 and the proposal of R-IPI by Sehn et al.10, beside determining the patients' prognosis at least partially facilitated the comparison of study results as well as the comparison of routine treatment outcomes with the study results. Response to treatment in our evaluation diverged from the reported one predominately in the low risk group (CR rate of 85.6%) where it was even lower than the reported 87% CR rate in the original IPI study where patients received chemotherapy without rituximab.3 The same observation holds true for the very good prognosis group in the R-IPI categorisation in which a higher CR FIGURE 7. Overall survival of different clinical categories of patients. DLBCL = diffuse large B-cell lymphoma. IPI = international prognostic index rate from the observed 85.3% could have been expected. The determined CR rates in other IPI and R-IPI groups were generally within expectations. The CR rates observed in the group of young good prognosis patients (86.8% of patients achieving CR or CRu) are completely in agreement with the results reported by Pfreundschuh et al. in the MInT study.19 However, the overall response rate of 88.3% achieved in our patients aged over 60 years was much better than the 77% overall response rate reported by Habermann et al.18 Regarding the duration of response given by the disease-free survival, again the largest discrepancy occurred in the low-risk patient group where the 3 year disease-free survival rate was 75% compared to 87% reported by Ziepert et al.9 In all other risk groups the disease-free survival at 3 years (low intermediate risk 76%, high intermediate risk group 57%, high risk group 53%, respectively) agreed very well with the reported ones (75%, 59% and 50%, respectively).9 An even larger discrepancy was noted in case of the R-IPI categories - namely, the 4 year disease-free survival rate was 59% in the very good prognosis, 72% in the good prognosis and 51% in the poor prognosis group, respectively, as compared to the reported 94%, 80%, and 53%, respectively.10 The 3 year disease-free survival of young good prognosis patients in our evaluation was 78% while Pfreundshuh et al.19 reported of 85% rate in equivalent population. On the other hand, Habermann et al.18 achieved a 53% 3 year disease-free survival rate in older patients as compared to the 61% rate in our study. The 3 year overall survival rate of the low risk patients (87%) in our analysis was somewhat worse than the 91% reported by Ziepert et al.9 Equivalent or slightly worse were also the 3 year overall survival rates of low intermediate risk, high intermediate risk and high risk patients (82%, 61%, 51%, respectively) as compared to the reported rates (81%, 65%, 59%, respectively).9 On the other hand, the 4 year overall survival rates of the R-IPI categories (94% in the very good prognosis group, 80% in the good prognosis group, and 56% in the poor prognosis group, respectively) were much better correlated with the reported ones of 94%, 79%, and 55%, respectively.10 The 3 year overall survival of young good prognosis patients in our evaluation was 93% which completely corresponds to the rate reported by Pfreundshuh et al.19 in equivalent population. Then again, Habermann et al.18 achieved a 67% 3 year overall survival rate in older patients (aged over 60 years) as compared to the 63% rate in our study. The repeating pattern of worst results achieved in our low risk and/or the very good prognosis group raises the question of whether those patients have been in some way "understaged". Another possible explanation is the existence of some not yet determined aspect that negatively influenced response to treatment, disease-free survival and to some extent also the overall survival of these patients. This aspect could be of patohistological origin - e.g. the inclusion of patients with immuno-blastic variants of the DLBCL which are no longer recognized as a separate entity in the WHO classification but have been associated with a worse outcome even after treatment with R-CHOP23 or of genetic origin - namely patients with the activated B-cell type gene expression profile have a much worse 5 year overall survival compared to patients with the germinal centre type gene expression profile.2425 It is, however, quite unlikely that patients with immunoblastic lymphomas or activated B-cell type lymphomas would have been gathered prevailingly in the low risk and/or very good prognosis groups. In total, the treatment outcomes of routinely treated patient with the DLBCL at our institute are quite encouraging when compared to results of some larger studies. There are probably no dilemmas about how to treat young good prognosis patients at present - it is with 6 cycles of R-CHOP21.19 On the other hand, for patients aged over 60 years the Ricover-60 study reported the best results with 6 cycles of R-CHOP14 (and total 8 applications of rituximab).26 This regimen is unfortunately associated with serious toxicity and therefore not applicable in the routine setting. Regarding our results also the treatment with 6 or 8 cycles (considering the stage of the disease) of R-CHOP21 will be appropriate for everyday management of the DLBCL in this fragile population. As for the young poor prognosis group - the 5 year overall survival rate of 76% is unsatisfying and needs to be improved. At present, quite a few studies are underway to clarify which of the regimens will perform best in this population. Most probably this will have to include routine determination of the gene expression profile in each patient in order to tailor his individual treatment. Acknowledgement This research was partially supported by Slovenian Ministry of Science (research program J3-0321). References 1. Swerdlow SH, Campo E, Harris NL, Pileri SA, Stein H, Thiele J, et al, editors. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th edition. Lyon, France: International Agency for Research on Cancer; 2008. 2. Žakelj Primic M, Bračko M, Pompe-Kirn V, Zakotnik B, editors. Rak v Sloveniji 2007. Ljubljana: Onkološki inštitut Ljubljana, Epidemiologija in register raka, Register raka Republike Slovenije, 2010. 3. Shipp M, Harrington D, Anderson J, Armitage JO, Bonadonna G, Brittinger G, et al. A predictive model for aggressive non-HodgkinCs lymphomas. N Engl J Med 1993; 329: 987-94. 4. Strojan-Flezar M, Lavrencak J, Zganec M, Strojan P. Image cytometric nuclear texture features in inoperable head and neck cancer: a pilot study. Radiol Oncol 2011; 45: 40-50. 5. Velnar T, Smrdel U, Popovic M, Bunc G. Genetic markers in oligodendroglial tumours. Radiol Oncol 2010; 44: 13-8. 6. Cvetkovic RS, Perry CM. Rituximab: a review of its use in non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Drugs 2006; 66: 791-820. 7. Molina A. A decade of rituximab: improving survival outcomes in non-Hodgkin's lymphoma. Annu Rev Med 2008; 59: 237-50. 8. Jezeršek Novaković B, Benigar A. Treatment of non-Hodgkin's lymphomas with rituximab in Slovene patients. Med Oncol 2010; 27: 167-76. 9. Ziepert M, Hasenclever D, Kuhnt E, Glass B, Schmitz N, Pfreundschuh M, et al. Standard International prognostic index remains a valid predictor of outcome for patients with aggressive CD20+ B-cell lymphoma in the rituximab era. J Clin Oncol 2010; 28: 2373-80. 10. Sehn LH, Berry B, Chhanabhai M, Fitzgerald C, Gill K, Hoskins P, et al. The revised International Prognostic Index is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Blood 2007; 109: 1857-61. 11. Tilly H, Dreyling M. Diffuse large B-cell non-Hodgkin's lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010; 21(Suppl 5): 172-4. 12. Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, et al. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. J Clin Oncol 1999: 17: 1244-53. 13. Cheson BD. New staging and response criteria for non-Hodgkin lymphoma and Hodgkin lymphoma. Radiol Clin North Am 2008; 46: 213-23. 14. Pettengell R, Linch D. Position paper on the therapeutic use of rituximab in CD20-positive diffuse large B-cell non-Hodgkin's lymphoma. Br J Haematol 2003; 121: 44-8. 15. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346: 235-42. 16. Sehn LH, Donaldson J, Chhanabhai M, Fitzgerald C, Gill K, Klasa R, et al. Introduction of combined CHOP plus rituximab therapy dramatically improved outcome of diffuse large B-cell lymphoma in British Columbia. J Clin Oncol 2005; 23: 5027-33. 17. Feugier P, Van Hoof A, Sebban C, Solal-Celigny P, Bouabdallah R, Fermé C, et al. Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d'Etude des Lymphomes de l'Adulte. J Clin Oncol 2005; 23: 4117-26. 18. Habermann TM, Weller EA, Morrison VA, Gascoyne RD, Cassileth PA, Cohn JB, et al. Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J Clin Oncol 2006; 24: 3121-7. 19. Pfreundschuh M, Trümper L, Osterborg A, Pettengell R, Trneny M, Imrie K, et al. CHOP-like chemotherapy plus rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis diffuse large-B-cell lymphoma: a randomised controlled trial by the MabThera International Trial (MInT) Group. Lancet Oncol 2006; 7: 379-91. 20. Coiffier B, Thieblemont C, Van Den Neste E, Lepeu G, Plantier I, Castaigne S, et al. Long-term outcome of patients in the LNH-98.5 trial, the first randomized study comparing rituximab-CHOP to standard CHOP chemotherapy in DLBCL patients: a study by the Groupe d'Etudes des Lymphomes de l'Adulte. Blood 2010; 116: 2040-5. 21. Horvat M, Jezeršek Novaković B. Effect of response quality and line of treatment with rituximab on overall and disease-free survival of patients with B-cell lymphoma. Radiol Oncol 2010, 44: 232-8. 22. Kovač V, Smrdel U. Meta-analyses of clinical trials in patients with non-small cell lung cancer. Neoplasma 2004; 51: 334-40. 23. Ott G, Ziepert M, Klapper W, Horn H, Szczepanowski M, Bernd HW, et al. Immunoblastic morphology but not the immunohistochemical GCB/ nonGCB classifier predicts outcome in diffuse large B-cell lymphoma in the RICOVER-60 trial of the DSHNHL. Blood 2010; 116: 4916-25. 24. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403: 503-11. 25. Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 2008; 359: 2313-23. 26. Pfreundschuh M, Schubert J, Ziepert M, Schmits R, Mohren M, Lengfelder E, et al. Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60). Lancet Oncol 2008; 9: 105-16. Effectiveness of L-thyroxine treatment on TSH suppression during pregnancy in patients with a history of thyroid carcinoma after total thyroidectomy and radioiodine ablation Blaz Krhin1 and Nikola Besic2 1 Department of Laboratory Diagnostics, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2012; 46(2): 160-165. Received 29 September 2011 Accepted 24 November 2011 Correspondence to: Prof. Nikola Bešič MD, PhD, Dept. of Surgical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 953; Fax:+386 1 5879400; E-mail: nbesic@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Introduction. There are scarce data about the optimal increase of L-thyroxine dose during pregnancy in patients with a history of thyroid carcinoma. The first aim of the study was to find out if routine therapeutic measures enable adequate TSH suppression in pregnancy. The other aim was to find out the optimal dose of L-thyroxine for TSH suppression in pregnant women. Patients and methods. In this retrospective observational study, we analysed 36 pregnancies of 32 women with a history of thyroid carcinoma. Before pregnancy, all of them underwent total thyroidectomy and radioiodine ablation of thyroid remnant, and they were on suppressive doses of L-thyroxine. Thyroid function tests were obtained before, during and after pregnancy. Results. Mean L-thyroxine dose before pregnancy, in the first, second and, third trimester and after delivery was 149, 147, 155, 165 and 158 micrograms daily, respectively. TSH concentration remained suppressed in 9 pregnancies, it was within normal range in 22 and elevated in 5 pregnancies. The mean dose of L-thyroxine in patients with suppressed TSH before pregnancy, in the first, second and, third trimester and after delivery was 154, 154, 164, 160 and 161 micrograms daily, respectively. When the dose had to be changed, the mean increase of the dose was 31.5 micrograms daily. Conclusions. The range of changes in TSH concentration during pregnancy in the patients who have been on suppressive L-thyroxine therapy before conception is quite wide. TSH was adequately suppressed in only 25% of pregnancies. The dose of L-thyroxine in patients with suppressed TSH in the first, second and third trimester was 154, 164 and 160 micrograms daily, respectively. Key words: pregnancy; TSH suppression; L-thyroxine; thyroid carcinoma. Introduction Thyroid hormones are important for normal pregnancy and the foetal development.12 During pregnancy, maternal thyroid hormones requirements increase.34 It is well known that the reference values of TSH, free T3 and free T4 for healthy the nonpregnant population are not the same as during pregnancy. Dashe et al.5 published the data about the TSH concentration in 13,599 pregnancies. They found out that the normal physiological concen- tration of TSH during the first trimester of pregnancy was as low as 0.1 mU/L. According to the trimester-specific reference ranges of serum, TSH concentrations above 2.3 mU/L in the first trimester and 3.1-3.5 mU/L in the second and third trimester may already be indicative of subclinical hypothyroidism.16 Safety of pregnancy during subclinical hyperthyroidism was reported by Casey et al.7 They found out that the subclinical hyperthyroidism was not associated with the adverse outcome of pregnancy.7 The patients having undergone thyroidectomy and radioiodine therapy are dependent on exogenous L-thyroxine.8 Some authors advocate that the increase of dose of L-thyroxine during pregnancy should be determined from the results of thyroid function tests8-11, while others propose to increase L-thyroxine dose as soon as pregnancy is confirmed.1213 There are very limited data in the literature about the changes of TSH and thyroid hormones during pregnancy in patients with a history of thyroid carcinoma. To our knowledge, these studies included only a small number of patients8, 12-15 with the largest study group of 18 such cases reported by Loh et al.8 There are scarce data about the optimal increase of L-thyroxine dose during pregnancy in the patients with thyroid carcinoma after total thyroidectomy and radioiodine ablation of thyroid remnant. The first aim of the study was to find out if routine therapeutic measures enable adequate TSH suppression in pregnancy. The other aim was to find out the optimal dose of L-thyroxine for TSH suppression in pregnant women. Material and methods In this retrospective observational study we analysed 36 pregnancies of 32 women (mean age at conception 29.9 ± 0.6 years) with a history of thyroid carcinoma during the period from 2000 to 2009. T1, T2 and T3 tumour was diagnosed in 15, 7 and 10 patients, respectively. In 23 and 9 patients, the tumour stage was assessed as N0 and N1, respectively. None of the patients had distant metastases. All of them had no evidence of disease at conception and after delivery. Histopathology of bioptic specimen revealed the presence of Hashimoto's thyroiditis in 11/36 (=31%) of patients. Before pregnancy, all of them underwent total thyroidectomy and radioio-dine ablation of the thyroid remnant and they were all on suppressive doses of L-thyroxine. All patients were advised to take L-thyroxine four hours before vitamins, iron or calcium drugs. At conception, the age of patients was 22 to 37 years (mean age 29.9 ± 0.6 years). Among our patients, 28 women were pregnant once and four twice. Pregnancy passed without any events in 30 cases and with complications in six cases (premature delivery in two cases, preeclampsia in one, increased blood pressure in one, high serum glucose concentration in two, vaginal bleeding in the seventh month of gestation in one case). Thyroid function tests were performed before, during and after pregnancy. The last test of thyroid function before pregnancy was done 1-12 (median 3) months before conception. During pregnancy, a clinical exam and thyroid function tests were performed every 6-8 weeks. When more than one set of tests was performed in any one trimester, the highest TSH concentration with the corresponding free T3 and free T4 concentrations were used in the statistical analysis in order to minimize any bias towards avoidance of dose change. The median number of thyroid function tests performed during pregnancy in each woman was four (range3-6). L-thyroxine dose was modified to maintain serum TSH below 0.3 mU/L. Suppression dose was adequate if TSH was 0.01-0.29 mU/L and free T3 was within normal range. From 2000-2006, TSH was measured by the two-site immunoluminometric assay (sandwich principle) LIASON TSH (Byk-Sangtec Diagnostica, Dietzenbach, Germany). Free T3 and free T4 were measured by commercially available kits (LIAISON FT3, LIAISON FT4) with "LIAISON" Immunoassay System (Byk-Sangtec, Germany later DiaSorin, Italy). Reference values for TSH, free T3 and free T4 were 0.27-4.2 mU/L, 2.93-6.8 pmol/L and 7.7-23.2 pmol/L, respectively. From 2007 onwards, TSH, free T3 and free T4 were measured by commercially available kits (TSH, FT3, FT4) with "Modular Analytics E170" Immunoassay System (Roche Diagnostics, Mannheim, Germany). Reference values for TSH, free T3 and free T4 were 0.27-4.20 mIU/L, 3.1-6.8 pmol/L a3nd 12-22 pm4 ol/L, respectively. The study was reviewed by the appropriate medical ethics committee. The Institute's Protocol Review Board approved the study, which was performed in accordance with the medical ethics standards laid down in an appropriate version of the 1964 Declaration of Helsinki. The participants gave informed consent. Statistical analysis Changes in the results of thyroid-function tests, and L-thyroxine doses throughout pregnancy were analysed by repeated measures ANOVA or Friedman's test, followed by Wilcoxon signed rank test in case of non-normal data distribution. P-values of less than 0.05 were considered to indicate statistical significance. The software package SPSS 16.0 for Windows (SPSS Inc., Chicago, IL USA) was used. Daily dose of L-Thiyroxire (ng) before lsl trimester 2nd ifinittstf Jrd trimester alter delivery FIGURE 1. Changes of TSH during 36 pregnancies. The bottom and top of the box are the 25th and 75th percentile (the lower and upper quartiles, respectively), and the band near the middle of the box is the 50th percentile (the median). The ends of the whiskers represent the minimum and maximum of all the data. TSH (mU/l) Iwfore 1st trimesief 2nnf jr