vol.54 no.4 december 2020 Skrajšan povzetek glavnih znacilnosti zdravila (kabozantinib) TERAPEVTSKE INDIKACIJE Zdravljenje napredovalega karcinoma ledvicnih celic (KLC) pri predhodno nezdravljenih odraslih bolnikih s srednje ugodnim ali slabim prognosticnim obetom ter pri odraslih bolnikih po predhodnem zdravljenju, usmerjenem v vaskularni endotelijski rastni faktor (VEGF). V monoterapiji zdravljenje hepatocelularnega karcinoma (HCK) pri odraslih bolnikih, ki so se predhodno že zdravili s sorafenibom. ODMERJANJE IN NACIN UPORABE Pri bolnikih s KLC in HCK je priporoceni odmerek 60 mg enkrat na dan. Zdravljenje je treba nadaljevati tako dolgo, dokler bolnik vec nima klinicnih koristi od terapije ali do pojava nesprejemljive toksicnosti. Pri sumu na neželene reakcije bo morda treba zdravljenje zacasno prekiniti in/ali zmanjšati odmerek. Ce je treba odmerek zmanjšati, se priporoca zmanjšanje na 40 mg/dan in nato na 20 mg/dan. Prekinitev odmerka se priporoca pri obravnavi toksicnosti 3. ali višje stopnje po CTCAE (common terminology criteria for adverse events) ali nevzdržni toksicnosti 2. stopnje. Zmanjšanje odmerka se priporoca za dogodke, ki bi lahko cez cas postali resni ali nevzdržni. Za priporocila glede prilagoditve odmerka ob pojavu neželenih ucinkov glejte celoten povzetek glavnih znacilnosti zdravila. Pri blagi ali zmerni ledvicni okvari je treba kabozantinib uporabljati previdno. Uporaba se ne priporoca pri hudi ledvicni okvari. Pri blagi okvari jeter odmerka ni treba prilagajati. Pri zmerni okvari jeter (Child Pugh B) je priporocljivo skrbno spremljanje celokupne varnosti. Pri bolnikih s hudo okvaro jeter (Child Pugh C) uporaba kabozantiniba ni priporocljiva. Nacin uporabe:Tablete je treba pogoltniti cele in jih ni dovoljeno drobiti. Bolnikom je treba narociti, naj vsaj 2 uri pred uporabo zdravila in 1 uro po tem nicesar ne jedo. KONTRAINDIKACIJE Preobcutljivost na ucinkovino ali katero koli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI Vecina dogodkov se pojavi zgodaj v teku zdravljenja, zato mora zdravnik bolnika v prvih 8 tednih zdravljenja skrbno spremljati, da oceni, ali je treba odmerek prilagoditi. Dogodki, ki se obicajno pojavijo zgodaj, vkljucujejo hipokalciemijo, hipokaliemijo, trombocitopenijo, hipertenzijo, sindrom palmarno-plantarne eritrodisestezije (PPES), proteinurijo in GI dogodke (bolecine v trebuhu, vnetje sluznice, zaprtje, driska, bruhanje). Pred uvedbo zdravljenja s kabozantinibom je priporocljivo izvesti preiskave delovanja jeter (ALT, AST in bilirubin), vrednosti skrbno spremljati med zdravljenjem in po potrebi prilagoditi odmerek. Bolnike je treba spremljati glede znakov in simptomov jetrne encefalopatije. Bolnike, ki imajo vnetno bolezen crevesja, ki imajo tumorsko infiltracijo prebavil ali so imeli pred posegom na prebavilih zaplete, je treba pred uvedbo zdravljenja skrbno oceniti, nato pa natancno spremljati za pojav simptomov GI perforacij in fistul, vkljucno z abscesi in sepso. Z uporabo kabozantiniba je treba pri bolnikih, pri katerih se pojavi GI perforacija ali fistula, ki je ni možno ustrezno obravnavati, prenehati. Driska, navzea/bruhanje, zmanjšanje apetita in vnetje ustne sluznice/bolecina v ustni votlini so nekateri od najpogosteje porocanih neželenih ucinkov na prebavila. Nemudoma je treba uvesti ustrezne medicinske ukrepe, vkljucno s podpornim zdravljenjem z antiemetiki, antidiaroiki ali antacidi. Ce pomembni neželeni ucinki na prebavila vztrajajo ali se ponavljajo, je treba presoditi o prekinitvi odmerjanja, zmanjšanju odmerka ali trajni ukinitvi zdravljenja s kabozantinibom. Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da porocajo o katerem koli domnevnem neželenem ucinku zdravila. Kabozantinib je treba uporabljati previdno pri bolnikih, pri katerih obstaja tveganje za pojav venske trombembolije, vkljucno s pljucno embolijo, in arterijske trombembolije ali imajo te dogodke v anamnezi. Z uporabo je treba prenehati pri bolnikih, pri katerih se razvije akutni miokardni infarkt ali drugi klinicno pomembni znaki zapletov trombembolije. Kabozantiniba se ne sme dajati bolnikom, ki hudo krvavijo ali pri katerih obstaja tveganje za hudo krvavitev. Uporaba zaviralcev poti VEGF pri bolnikih s hipertenzijo ali brez nje lahko spodbudi nastanek anevrizem in/ali disekcij arterij. Med zdravljenjem s kabozantinibom je treba spremljati vrednosti trombocitov in odmerek prilagoditi glede na resnost trombocitopenije. Vsaj 28 dni pred nacrtovanim kirurškim posegom je treba zdravljenje ustaviti, ce je mogoce. Kabozantinib je treba ukiniti pri bolnikih z zapleti s celjenjem rane, zaradi katerih je potrebna zdravniška pomoc. Pred uvedbo kabozantiniba je treba dobro obvladati krvni tlak. Med zdravljenjem je treba vse bolnike spremljati za pojav hipertenzije in jih po potrebi zdraviti s standardnimi antihipertenzivi. V primeru trdovratne hipertenzije, kljub uporabi antihipertenzivov, je treba odmerek kabozantiniba zmanjšati oz. prenehati z zdravljenjem. V primeru hipertenzijske krize je treba zdravljenje ukiniti. Pred uvedbo kabozantiniba je treba opraviti pregled ustne votline in le­tega v casu zdravljenja periodicno ponavljati. Ob pojavu osteonekroze celjusti, je treba prenehati z uporabo kabozantiniba. Pri resni PPES je treba razmisliti o prekinitvi zdravljenja. Nadaljevanje zdravljenja naj se zacne z nižjim odmerkom, ko se PPES umiri do 1. stopnje. V casu zdravljenja je treba redno spremljati beljakovine v urinu. Ce se pri bolniku razvije nefroticni sindrom, je treba z uporabo kabozantiniba prenehati. Pri uporabi kabozantiniba so opazili sindrom posteriorne reverzibilne encefalopatije (PRES). Pri bolnikih s PRES je treba zdravljenje ukiniti. Kabozantinib je treba uporabljati previdno pri bolnikih s podaljšanjem intervala QT v anamnezi, pri bolnikih, ki jemljejo antiaritmike, in pri bolnikih z relevantno obstojeco boleznijo srca, bradikardijo ali elektrolitskimi motnjami. Uporaba kabozantiniba je bila povezana z vecjo pojavnostjo elektrolitskih nepravilnosti, zato je priporocljivo spremljati biokemijske parametre in po potrebi uvesti ustrezno nadomestno zdravljenje v skladu s standardno klinicno prakso. Bolniki z redko dedno intoleranco za galaktozo, laponsko obliko zmanjšane aktivnosti laktaze ali malabsorpcijo glukoze/ galaktoze ne smejo jemati tega zdravila. Plodnost, nosecnost in dojenje: Ženskam v rodni dobi je treba svetovati, da v casu zdravljenja s kabozantinibom ne smejo zanositi. Zanositev morajo prepreciti tudi ženske partnerice moških bolnikov, ki uporabljajo kabozantinib. Med zdravljenjem in še vsaj 4 mesece po koncanju terapije je treba uporabljati zanesljiv nacin kontracepcije. Kabozantiniba se ne sme uporabljati med nosecnostjo, razen ce zdravljenje ni nujno potrebno zaradi klinicnega stanja ženske. Matere med zdravljenjem in še 4 mesece po koncanju terapije ne smejo dojiti. Kabozantinib lahko predstavlja tveganje za plodnost pri moških in ženskah. INTERAKCIJE Kabozantinib je substrat za CYP3A4. Pri socasni uporabi mocnih zaviralcev CYP3A4 (npr. ritonavirja, itrakonazola, eritromicina, klaritromicina, soka grenivke) je potrebna previdnost. Kronicni socasni uporabi mocnih induktorjev CYP3A4 (npr. fenitoina, karbamazepina, rifampicina, fenobarbitala ali pripravkov zelišcnega izvora iz šentjanževke) se je treba izogibati. Razmisliti je treba o socasni uporabi alternativnih zdravil, ki CYP3A4 ne inducirajo in ne zavirajo ali pa inducirajo in zavirajo le neznatno. Pri socasni uporabi zaviralcev MRP2 (npr. ciklosporina, efavirenza, emtricitabina) je potrebna previdnost, saj lahko povzrocijo povecanje koncentracij kabozantiniba v plazmi. Ucinka kabozantiniba na farmakokinetiko kontraceptivnih steroidov niso preucili, vendar pa se priporoca dodatna kontracepcijska metoda (pregradna metoda). Zaradi visoke stopnje vezave kabozantiniba na plazemske beljakovine je možna interakcija z varfarinom v obliki izpodrivanja s plazemskih beljakovin, zato je treba spremljati vrednosti INR. Kabozantinib morda lahko poveca koncentracije socasno uporabljenih substratov P-gp v plazmi. Bolnike je treba opozoriti na uporabo substratov P-gp (npr. feksofenadina, aliskirena, ambrisentana, dabigatran eteksilata, digoksina, kolhicina, maraviroka, posakonazola, ranolazina, saksagliptina, sitagliptina, talinolola, tolvaptana) socasno s kabozantinibom. NEŽELENI UCINKI Za popolno informacijo o neželenih ucinkih, prosimo, preberite celoten povzetek glavnih znacilnosti zdravila Cabometyx. Najpogostejši resni neželeni ucinki zdravila v populaciji bolnikov s KLC so bili bolecine v trebuhu, driska, navzea, hipertenzija, embolija, hiponatriemija, pljucna embolija, bruhanje, dehidracija, utrujenost, astenija, zmanjšanje apetita, globoka venska tromboza, omotica, hipomagneziemija in PPES. Najpogostejši resni neželeni ucinki zdravila v populaciji bolnikov s HCK so bili jetrna encefalopatija, astenija, utrujenost, PPES, driska, hiponatriemija, bruhanje, bolecine v trebuhu in trombocitopenija. Zelo pogosti: anemija, trombocitopenija, hipotiroidizem, zmanjšanje apetita, hipomagneziemija, hipokaliemija, hipoalbuminemija, paragevzija, glavobol, omotica, hipertenzija, krvavitev, disfonija, dispneja, kašelj, driska, navzea, bruhanje, stomatitis, obstipacija, bolecine v trebuhu, dispepsija, bolecina v zgornjem predelu trebuha, PPES, izpušcaj, bolecine v okoncinah, utrujenost, vnetje sluznice, astenija, periferni edem, zmanjšanje telesne mase, zvišanje ALT v serumu, zvišanje AST. Pogosti:absces, nevtropenija, limfopenija, dehidracija, hipofosfatemija, hiponatriemija, hipokalciemija, hiperkaliemija, hiperbilirubinemija, hiperglikemija, hipoglikemija, periferna nevropatija (vkljucno s senzoricno), tinitus, globoka venska tromboza, venska tromboza, arterijska tromboza, pljucna embolija, GI perforacija, fistula, GERB, hemoroidi, bolecina v ustni votlini, suha usta, disfagija, glosodinija, jetrna encefalopatija, pruritus, alopecija, suha koža, akneiformni dermatitis, sprememba barve las oz. dlak, hiperkeratoza, mišicni krci, artralgija, proteinurija, zvišanje ALP v krvi, GGT, kreatinina v krvi, amilaze, lipaze, holesterola v krvi, trigliceridov v krvi. Obcasni: konvulzije, pankreatitis, holestaticni hepatitis, osteonekroza celjusti, zapleti z ranami. Neznana pogostnost: možganska kap, miokardni infarkt, anevrizme in disekcije arterij. Vrsta ovojnine in vsebina: Plastenka vsebuje 30 filmsko obloženih tablet. Režim izdaje: Rp/Spec Imetnik dovoljenja za promet z zdravilom: Ipsen Pharma, 65 quai Georges Gorse, 92100 Boulogne-Billancourt, Francija Pred predpisovanjem, prosimo, preberite celoten povzetek glavnih znacilnosti zdravila! CAB-300420 SAMO ZA STROKOVNO JAVNOST PharmaSwiss d.o.o., Brodišce 32, 1236 Trzin CAB0720-03, julij 2020 telefon: +386 1 236 47 00, faks: +386 1 236 47 05 Publisher Association of Radiology and Oncology Aims and Scope Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers and review articles, pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, medical physics and radiation protection. Therefore, the scope of the journal is to cover beside radiology the diagnostic and therapeutic aspects in oncology, which distinguishes it from other journals in the field. Editor-in-Chief Gregor Serša, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Executive Editor Viljem Kovac, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Clinical Oncology, Radiotherapy) Editorial Board Subject Areas: Radiology and Nuclear Medicine Sotirios Bisdas, University College London, Department of Neuroradiology, London, UK Boris Brkljacic, University Hospital “Dubrava”, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Maria Godény, National Institute of Oncology, Budapest, Hungary Gordana Ivanac, University Hospital Dubrava, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luka Ležaic, University Medical Centre Ljubljana, Department for Nuclear Medicine, Ljubljana, Slovenia Katarina Šurlan Popovic, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Jernej Vidmar, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psyhology, Department of Life Sciences, Trieste, Italy Vassil Hadjidekov, Medical University, Department of Diagnostic Imaging, Sofia, Bulgaria Marko Hocevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Deputy Editors Andrej C, University of Primorska, Faculty of Health Science, Izola, Slovenia (Subject Areas: Clinical Oncology, Experimental Oncology) Božidar Casar, Institute of Oncology Ljubljana, Department for Dosimetry and Quality of Radiological Procedures, Ljubljana (Subject Area: Medical Physics) Maja Cemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Subject Areas: Clinical Oncology and Radiotherapy Serena Bonin, University of Trieste, Department of Medical Sciences, Cattinara Hospital, Surgical Pathology Blg, Molecular Biology Lab, Trieste, Italy Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Blaž Grošelj, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Luka Milas, UT M. D. Anderson Cancer Center, Houston, USA Miha Oražem, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Gaber Plavc, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Center Ljubljana, Division of Internal Medicine, Department of Gastroenterology, Ljubljana, Slovenia Andrea Veronesi, Centro di Riferimento Oncologico- Aviano, Division of Medical Oncology, Aviano, Italy Branko Zakotnik, Institute of Oncology Ljubljana, Department of Medical Oncology, Ljubljana, Slovenia Mikl Kásler, National Institute of Oncology, Budapest, Hungary Maja Osmak, Ruder Boškovic Institute, Department of Molecular Biology, Zagreb, Croatia December 2020 Vol. 54 No. 4 Pages 371-512 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Igor Kocijancic, University Medical Center Ljubljana, Institute of Radiology, Ljubljana, Slovenia (Subject Areas: Radiology, Nuclear Medicine) Karmen Stanic, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy; Clinical Oncology) Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy, Clinical Oncology) Subject Area: Experimental Oncology Metka Filipic, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Janko Kos, University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Tamara Lah Turnšek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavcic, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Justin Teissié, CNRS, IPBS, Toulouse, France Gillian M. Tozer, University of Sheffield, Academic Unit of Surgical Oncology, Royal Hallamshire Hospital, Sheffield, UK Subject Area: Medical Physics Robert Jeraj, University of Wisconsin, Carbone Cancer Center, Madison, Wisconsin, USA Mirjana Josipovic, Rigshospitalet, Department of Oncology, Section of Radiotherapy, Copenhagen, Denmark Häkan Nystr, Skandionkliniken, Uppsala, Sweden Ervin B. Podgoršak, McGill University, Medical Physics Unit, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. Reader for English Vida Kološa Secretary Mira KlemencicZvezdana Vukmirovic Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanovic Layout Matjaž Lužar Printed by Tiskarna Ozimek, Slovenia Published quarterly in 400 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJE Zaloška cesta 2 1000 Ljubljana Slovenia Beneficiary bank account number: SI56 02010-0090006751 IBAN: SI56 0201 0009 0006 751 Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2, 1520 Ljubljana; Slovenia SWIFT: LJBASI2X Subscription fee for institutions EUR 100, individuals EUR 50 The publication of this journal is subsidized by the Slovenian Research Agency. 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Sio, Bilgehan Sahin, Gorkem Gungor, Gokhan Aydin, Bulent Yapici, Enis Ozyar 488 Does regular quality control improve the quality of surgery in Slovenian breast cancer screening program? Andraz Perhavec, Sara Milicevic, Barbara Peric, Janez Zgajnar radiophysics 495 Experimental validation of Monte Carlo based treatment planning system in bone density equivalent media Djeni Smilovic Radojcic, Bozidar Casar, David Rajlic, Manda Svabic Kolacio, Ignasi Mendez, Nevena Obajdin, Dea Dundara Debeljuh, Slaven Jurkovic 505 Comparison of three film analysis softwares using EBT2 and EBT3 films in radiotherapy Tamás Pza, Zsuzsánna Zongor, Barbara Melles-Bencsik, Da Zita Tatai-Szab Tibor Major, Csilla Pesznyák I slovenian abstracts 371 review Modern treatment of vulvar cancer Sebastjan Merlo1,2 1 Gynaecological Oncology Unit, Department of Surgery, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(4): 371-376. Received 28 June 2020 Accepted 21 July 2020 Correspondence to: Assist. Sebastjan Merlo, M.D., Ph.D., Gynaecological Oncology Unit, Department of Surgery, Institute of Oncology Ljubljana, Zaloška, SI-1000 Ljubljana, Slovenia. E-mail: smerlo@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Vulvar cancer accounts for 3–5% of malignant diseases of the female genital tract. The Slovenian inci­dence rate is 5.5/100,000, which means 57 new cases per year. The most common histological type (90%) is squamous cell carcinoma. Based on etiology, it can be classified into the first type which correlates with human papillomavirus (HPV) infection and the second type which is not associated with HPV. The most common and long-lasting symptom of vulvar cancer is pruritus. The preferred diagnostic procedure to confirm the diagnosis is a punch or incision biopsy. Surgery in combination with radiotherapy is the standard treatment for vulvar cancer. Sentinel lymph node biopsy with lymphoscintigraphy is now a standard part of surgical treatment. Chemotherapy is a palliative treatment option. Conclusions. Vulvar cancer is a rare disease. Because of the pathogenesis, surgery and radiotherapy are the main treatment modalities. The sentinel node biopsy (SNB) represents a contemporary approach to the vulvar cancer treat­ment and significantly reduces morbidity. Improvements in treatment of vulvar cancer contributed to the decrease of mortality among Slovenian women. Key words: vulvar cancer; surgical treatment; sentinel lymph node biopsy; lymphoscintigraphy; radiotherapy Introduction Vulvar cancer is the fourth most common gynae­cological malignancy.1 The basic treatment for vul-var cancer is still surgery, but the radical nature of the procedure has changed or decreased over the last twenty years. Historically treatment included radical vulvectomy and a radical inguino-femoral lymphadenectomy. The procedure was associated with a high rate of postoperative complications. For this reason, a minimally invasive surgical tech­nique was developed. This is the sentinel lymph node biopsy, which is now a standard procedure in the treatment of patients with early-stage vulvar cancer. This procedure significantly reduced mor­bidity and improved quality of life.2 Epidemiology Vulvar cancer accounts for 3–5% of all gynaeco-logical cancers in the world. This puts it in fourth place among gynaecological malignancies. The first three places are occupied by cancer of the uter­us, ovaries and cervix. Every year 27,000 women worldwide are diagnosed with vulvar cancer. The highest incidence is in Europe, North and South America, Oceania, and the lowest in Asia.1 In 2016, 57 women were diagnosed with vul-var cancer in Slovenia with an incidence of 5.5 / 100,000. An analysis of the time trends over the last 15 years shows an increase in incidence since 2003, while mortality has remained constant. It should be noted that this coincides with the introduction of the national program for the early detection of precancerous changes in the cervix. This has led to an increased number of gynaecological examina­tions, including the older population. Vulvar can­cer occurs most frequently in women over 80 years of age. In 2016 there were no cases of women under 30 years of age in Slovenia. According to the 2016 data, 59.6% of patients had a limited stage of the disease at diagnosis, 29.8% an advanced stage and 7% of patients had metastatic disease.3 372 The survival of patients with vulvar cancer improved slightly over time. According to the Slovenian Cancer Registry, the 5-year survival rate of patients with vulvar cancer was 43% between 2004 and 2009, while between 2010 and 2016 5-year survival rate was 48%. Patients diagnosed with lo­calized disease have significantly longer overall sur­vival than patients with locally advanced disease.4,5 The international EUROCARE-5 study showed a relative 5-year survival rate for different cancers. In this study patients with vulvar and vaginal can­cer were pooled. The average European relative 5-year survival rate in this study was 56% for pa­tients between 2000 and 2007.6 Etiopathogenesis More than 90% of cases of vulvar cancer are de­fined as squamous cell carcinoma. It can develop in two different ways. In younger women (aged 35– 65 years), HPV infection plays a key role in the de­velopment of squamous cell carcinoma, especially strains 16 and 18. Risk factors include a history of genital warts and other sexually transmitted dis­eases, low socioeconomic status, smoking and im­munodeficiency. The second type of development is independent of HPV and occurs more frequently in older patients (aged 55–85 years). It is a gradual process of development of cellular atypia leading to vulvar intraepithelial neoplasia (VIN) and then squamous cell carcinoma. The risk factor is the li­chen sclerosus. The crossing of the two pathogenetic pathways is also possible.7-9 Clinical manifestation and diagnostic procedures The most common symptom of vulvar cancer is persistent itching. Less common symptoms are bleeding from the vulvar skin, bleeding or dis­charge from the vagina, dysuria and pain. In ad­vanced cases, a tumour can be seen on the vulva. The tumour may be ulcerated, leukoplactic or warty.1 Treatment of a woman with suspected malignant disease of the vulva starts with a thorough medical history, followed by a clinical examination. It is im­portant to accurately describe suspicious changes, their size, number, position, mobility, assessment of infiltration of deeper structures and safety margins in case of excision. A bimanual vaginal and rectal examination should always be performed to assess vaginal and rectal involvement. Since HPV occurs in 86% of precancerous changes in the vulva and in 28.6% of vulvar cancers, the examination should also include a colposcopic examination of the vagi­na and cervix. Assessment of the size, mobility and consistency of the inguinal lymph nodes is manda­tory. The condition of the skin above the inguinal lymph nodes should also be noted. Palpation of the supraclavicular lymph nodes is important as well. If there is already a pathology of the vulva (atroph­ic lichen sclerosus, pathological cytological smear of the vulva), vulvoscopy is also advisable.2 A targeted biopsy with histological examination of the tissue taken is necessary to make a definitive diagnosis. It is important that the sample is taken at the site of the vital tissue, so it is advisable to take a tissue sample near the edge of the alteration. Necrosis, granulation tissue, fibrin or inflammation are often found in the middle of the changes in the form of ulcers, blisters, atrophy and scarring. Such a hysto-pathological pattern is neither appropri­ate nor representative. The size of the biopsy taken should be at least 4 mm3.2 The preferred method of sampling is the punch biopsy. Excisional biopsy is not recommended as it may prevent proper further treatment. In patients with multiple vulvar lesions, a separate biopsy of all lesions should be per­formed and the sampling site should be indicated.2 Treatment The treatment of vulvar cancer often involves a combination of surgery and radiotherapy. Systemic treatment is rarely used. Treatment can be long lasting and have a major impact on quality of life.2,9 Surgical treatment Primary vulvar lesion.The basic criterion for the treatment of a tumour lesion is the depth of the stromal invasion into the biopsy tissue taken. If an early stage disease is defined as T1a (= 1 mm of stromal invasion), a wide local excision is per­formed. If the disease is defined as T1b (> 1 mm stromal invasion) or T2 = 4 cm and the lesion is 1 cm from the median line, wide local excision or a modified radical vulvectomy and ipsilateral senti­nel node biopsy (SNB) is performed. However, in the case of a lesion in the median line, a wide local excision and a bilateral SNB is required. If the disease is locally advanced (T2 > 4 cm and T3) and the lesion is =1 cm from the median line, a radical vulvectomy and ipsilateral dissection of Merlo S / Treatment of vulvar cancer 373 the inguino-femoral lymph nodes is performed. If the lesion is in the median line, a radical vulvecto-my and bilateral dissection of the inguino-femoral lymph nodes are performed. If the lymph nodes are positive, we opt for external beam radiotherapy (EBRT) of the primary tumour, lymph nodes and pelvis. In case of negative lymph nodes, we choose EBRT of the primary tumour and/or selected in-guino-femoral lymph nodes. In all cases, adjuvant treatment follows. If the patient has metastatic disease outside the pelvis (any T, any N, M1 outside the pelvis), we do not opt for surgical treatment, but for palliative EBRT and/or symptomatic treatment.2,9 Lymph nodes. The most basic method for deter­mining the status of inguino-femoral lymph nodes is palpation, but its accuracy is only 9% preopera­tively and 55% intraoperatively. The status can also be determined by ultrasound examination of the inguinal regions. The sensitivity and specificity of lymph node ultrasound examination for vulvar cancer is 76.3% and 91.3%, with positive and nega­tive predictive values of 82.9% and 87.5%, respec­tively. Fine needle biopsy and cytological verifica­tion follow if lymph node involvement is suspect­ed.10,11 Other imaging methods have proven to be less reliable compared to ultrasound.12,13 In the absence of a reliable method for detect­ing inguino-femoral lymph node involvement, in-guino-femoral lymphadenectomy was part of the standard treatment of vulvar cancer.1 Metastases in the inguino-femoral lymph nodes in early stages of the disease are found in only 20–30% of patients, which means that all other patients do not benefit from a complete lymphadenectomy. The possible postoperative complications following a complete lymphadenectomy are lymphedema of the lower extremity (14–49%), lymphocyst formation (11– 40%) and wound infections with dehiscence.14,15 Due to the lack of non-invasive techniques to determine the status of inguino-femoral lymph nodes, the absence of lymph node metastases in most patients with early stage disease and the fre­quent morbidity following inguino-femoral lym­phadenectomy, the minimally invasive surgical technique, SNB biopsy was developed. SNB is now part of the standard treatment of early-stage vul-var cancer. Vulvar cancer has a predictable course of the lymphatic vessels and lymphatic drainage is predictable. Therefore, SNB of inguino-femoral lymph nodes is a safe replacement for inguino-femoral lymphadenectomy. The sentinel lymph node is defined as the first lymph node in the lym­phatic basin into which the lymph of the primary tumour drains. Histological examination of the sentinel lymph node is representative for all other lymph nodes in the area, and histologically, a nega­tive sentinel lymph node means the absence of me­tastases in subsequent lymph nodes.16-19 The sentinel lymph node is marked in two ways, with a nanocolloid bound to 99mTc (Technetium) and with a patent blue. This method is the most re­liable, as the sentinel lymph node is found in 97.7% of cases. Only by injecting patent blue the sentinel lymph node is identified in 68.7%, and only by the nanocolloid bound to technetium in 94%.20,21 At the Institute of Oncology Ljubljana, the tech­nique has changed over the years. It is crucial that patent blue is injected intradermally and not subcutaneously. The volume of the injected, un­diluted dye is 2 ml. On the day of surgery, 0.5 ml of technetium-labeled nanocolloid is injected in-tradermally with a thin needle at four points near the outer edge of the tumour. Lymphoscintigraphy with a gamma camera follows. The first active ac­cumulation point of the radiopharmaceutical is the sentinel lymph node, and its position is marked on the skin. Sometimes several points of high activity appear, in this case we mark them all. Immediately before the beginning of the surgical procedure, 2 ml of patent blue is injected intradermally in the same place as radiopharmaceutical. Then a 3 to 4 cm long skin incision is made at the marked site. The tissue is carefully dissected until a blue stained lymph node is found. Its activity is checked with a hand-held gamma detector and then removed.2 Women with histologically confirmed unifo-cal vulvar carcinoma, less than 4 cm in diameter, with an invasion depth of more than 1 mm, and in whom there are clinically no metastases in the inguino-femoral lymph nodes, are candidates for sentinel lymph node biopsy.2,22 A tumour located 1 cm or more from the midline of the vulva is usually drained into the unilateral lymphatic system, so a sentinel lymph node biopsy is performed on the same side. Bilateral drainage is present in tumours that are central or less than 1 cm from the median line. In this case a biopsy of the sentinel lymph node should be performed bi­laterally. If the lymph node is detected in the lym­phoscintigraphy on one side only, inguino-femoral lymphadenectomy on the opposite side is recom­mended to avoid a false negative result.2,9,22 Patients with a multifocal tumour are not suit­able candidates for sentinel lymph node biopsy because they have a higher incidence of disease recurrence (10.5%) compared to patients with a unifocal tumour (2.3%).23 Previous surgery and 374 excisions of the vulva that may interfere with lymphatic flow in the inguino-femoral region are relative contraindications for sentinel lymph node biopsy, but the decision in these cases is made on a patient-specific basis. Lymphadenectomy is rec­ommended in patients with recurrent disease or in patients who have already had an inguino-femoral sentinel lymph node biopsy.2,20 Radiotherapy The purpose of postoperative radiotherapy is to reduce the likelihood of local and/or regional re­currence, prolong disease-free survival and overall survival.2 Due to the low incidence of vulvar can­cer, the number of randomized clinical trials and evidence-based treatment outcomes is also low. As a result, there are no standard indications and recommendations for adjuvant treatment of vulvar cancer. The data collected suggest that adjuvant treatment is not necessary in patients with early-stage cancer, negative inguino-femoral lymph node status and a favourable prognosis.24 However, treatment of locally advanced disease sometimes requires adjuvant treatment following surgery. Lymph node metastases, large primary tumours, deep stromal invasion, lymphovascular invasion and close surgical margins are associated with a higher incidence of disease recurrence. The role of adjuvant therapy in these patients is still not fully understood. Radiation or radiation combined with lymph node dissection is very effective in pre­venting disease recurrence in the inguino-femoral lymph nodes in patients with squamous cell carci­noma of the vulva. According to the recommenda­tions of the Gynecologic Oncology Group (GOG), adjuvant radiation is considered the standard treat­ment for squamous cell carcinoma of the vulva in patients with 2 or more positive lymph nodes with extracapsular spread or inguino-femoral dissection is not feasible. The benefit of adjuvant radiother­apy has been demonstrated in patients with two or more positive inguino-femoral lymph nodes, while the role of irradiation of patients with only one positive inguino-femoral lymph node remains undetermined.2,24,25 Systemic treatment Data on the role of systemic therapy in the treat­ment of vulvar cancer are very sparse, as they are based on small, non-randomized phase II clinical trials involving fewer than 50 patients treated with various regimens of chemotherapy. Currently, chemotherapy is not recommended as a stand­alone preoperative (neoadjuvant) or postoperative (adjuvant) systemic treatment. Chemotherapy can only be considered as a palliative treatment for metastatic disease if other treatments are not fea­sible. Various cytostatic drugs (cisplatin, paclitaxel, bleomycin, navelbin, 5-fluorouracil) were used in the trials in combination or monotherapy. The re­sponse rate was 0–40%, progression-free survival 1–10 months and overall survival up to 19 months. Due to the toxicity of cisplatin, the less toxic carbo­platin has been increasingly used in recent years to treat metastatic vulvar cancer. In analogy to meta­static cervical cancer, the combination of carbopl­atin and paclitaxel has been increasingly used in recent years for the treatment of metastatic vulvar cancer because the combination is similarly effec­tive and less toxic than the combination of cisplatin and paclitaxel.26 Chemotherapy can be used in combination with concomitant radiation (chemoradiotherapy), either as a stand-alone treatment or as preopera­tive (neoadjuvant) treatment in patients with lo­cally advanced disease. Various cytostatic drugs (cisplatin, 5-fluorouracil, mitomycin-C) are used in chemoradiotherapy to improve the local effect of radiation (chemosensitization). Since concomitant treatment with chemotherapy and radiation is as­sociated with higher toxicity, lower doses of cyto-static drugs are used during radiation, so in this case it is actually a topical rather than a systemic treatment.26,27 The role of targeted therapeutics in the treat­ment of advanced vulvar cancer is still unknown. We have data from a small clinical trial with the tar­geted therapeutic erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, which included 41 patients with advanced disease. Partial response was achieved in 27%, progression-free survival was short (median treatment time 3 months), and treat­ment was associated with many adverse events.28,29 Follow-up There is currently insufficient evidence to support a uniform follow-up pattern after radical treatment of vulvar cancer. Experts and professional asso­ciations therefore disagree on follow up schedule. Local recurrence can occur at any time, so lifelong follow-up is recommended. Depending on the type of treatment, the European Society of Gynaecological Oncology (ESGO) suggests the following follow-up scheme. After primary surgical treatment, the first exam­ination is performed 6–8 weeks after the surgical procedure, then clinical examinations of the vulva and groin region are performed every 3–4 months for a period of two years. In the following three years, follow-up examinations are scheduled twice a year. After this period, it is advisable to perform annual clinical examinations. This is particularly important for patients at increased risk, such as pa­tients diagnosed with lichen sclerosus / planus. 10–12 weeks after chemotherapy or radiothera­py, a computed tomography or positron emission tomography-computed tomography (PET-CT) ex­amination is recommended to confirm remission. Later, clinical examinations of the vulva and groin region are recommended every 3–4 months for the first two years, followed by examinations twice a year for 3 years and then annual examinations. If a local recurrence is suspected, a biopsy should be performed, and if there is a suspicion of groin region relapse of the disease or extended dis­ease, appropriate imaging diagnostics should fol­low. The early detection of malignant recurrences that can still be treated surgically can significantly improve quality of life, but there is currently no firm evidence of the effects on morbidity and mor­tality.2,6,30 Conclusions Surgical treatment is still standard treatment of vulvar cancer. The greatest progress in this field in recent years has been the development of a mini­mally invasive surgical technique, sentinel lymph node biopsy, which is now standard treatment in selected cases. The replacement of inguino-femoral lymphadenectomy with this procedure significant­ly reduced morbidity and improved quality of life. Due to the rarity of vulvar cancer, patients should be treated in specialized centres where appropriate equipment, knowledge and experience are avail­able. References 1. Alkatout I, Schubert M, Garbrecht N, Weigel MT, Jonat W, Mundhenke C, et al. Vulvar cancer: epidemiology, clinical presentation, and management options. Int J Womens Health 2015; 7: 305-13. doi: 10.2147/IJWH.S68979 2. Oonk MHM, Planchamp F, Baldwin P, Bidzinski M, Brännstr M, Landoni F, et al. European Society of Gynaecological Oncology Guidelines for the management of patients with vulvar cancer. Int J Gynecol Cancer 2017; 27: 832-7. doi: 10.1097/IGC.0000000000000975 3. Cancer Registry of Republic of Slovenia [Internet] 2016 [cited 2020 May 6]. Available from: https://www.onko-i.si/rrs/ Merlo S / Treatment of vulvar cancer 375 4. Zadnik V, Primic Zakelj M, Lokar K, Jarm K, Ivanus U, Zagar T. Cancer burden in Slovenia with the time trends analysis. Radiol Oncol 2017; 51: 47-55. doi:1 0.1515/raon-2017-0008 5. Cancer in Slovenia 2016. Ljubljana: Cancer in Slovenia 2016. Ljubljana: Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Slovenian Cancer Registry; [internet] 2019 [cited 2020 May 12]. Available from: https://www.onko-i.si/fileadmin/onko/datoteke/dokumenti/RRS/ LP_2016.pdf 6. De Angelis R, Sant M, Coleman MP, Francisci S, Baili P, Pierannunzio D, et al. Cancer survival in Europe 1999-2007 by country and age: results of EUROCARE-5-a population-based study. Lancet Oncol 2014; 15: 23-34. doi: 10.1016/S1470-2045(13)70546-1 7. Del Pino M, Rodriguez-Carunchio L, Ordi J. Pathways of vulvar intraepithelial neoplasia and squamous cell carcinoma. Histopathology 2013; 62: 161-75. doi: 10.1111/his.12034 8. Pils S, Gensthaler L, Alemany L, Horvat R, de Sanjosé S, Joura EA. HPV preva­lence in vulvar cancer in Austria. Wien Klin Wochenschr 2017; 129: 805-9. doi: 10.1007/s00508-017-1255-2 9. Wohlmuth C, Wohlmuth-Wieser I. Vulvar malignancies: an interdiscipli­nary perspective. J Dtsch Dermatol Ges 2019; 17: 1257-76. doi: 10.1111/ ddg.13995 10. Angelico G, Santoro A, Inzani F, Spadola S, Fiorentino V, Cianfrini F, et al. Ultrasound-guided FNA cytology of groin lymph nodes improves the management of squamous cell carcinoma of the vulva: results from a com­parative cytohistological study. Cancer Cytopathol 2019; 127: 514-20. doi: 10.1002/cncy.22154 11. de Gregorio N, Ebner F, Schwentner L, Friedl TWP, Deniz M, LátK, et al. The role of preoperative ultrasound evaluation of inguinal lymph nodes in patients with vulvar malignancy. Gynecol Oncol 2013; 131: 113-7. doi: 10.1016/j.ygyno.2013.07.103 12. Kataoka MY, Sala E, Baldwin P, Reinhold C, Farhadi A, Hudolin T, et al. The accuracy of magnetic resonance imaging in staging of vulvar cancer: a retro­spective multi-centre study. Gynecol Oncol 2010; 117: 82-7. doi: 10.1016/j. ygyno.2009.12.017 13. Andersen K, Zobbe V, Thranov IR, Pedersen KD. Relevance of computerized tomography in the preoperative evaluation of patients with vulvar cancer: a prospective study. Cancer Imaging 2015; 15: 8. Available from: https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC4470090/. doi: 10.1186/s40644­015-0044-2 14. Cham S, Chen L, Burke WM, Hou JY, Tergas AI, Hu JC, et al. Utilization and outcomes of sentinel lymph node Biopsy for vulvar cancer. Obstet Gynecol 2016; 128: 754-60. doi: 10.1097/AOG.0000000000001648 15. Huang J, Yu N, Wang X, Long X. Incidence of lower limb lymphedema after vulvar cancer: a systematic review and meta-analysis. Medicine 2017; 96: 8722. doi: 10.1097/MD.0000000000008722 16. Slomovitz BM, Coleman RL, Oonk MHM, van der Zee A, Levenback C. Update on sentinel lymph node biopsy for early-stage vulvar cancer. Gynecol Oncol 2015; 138: 472-7. doi: 10.1016/j.ygyno.2015.05.017 17. Oonk MHM, Hollema H, van der Zee AGJ. Sentinel node biopsy in vulvar cancer: implications for staging. Best Pract Res Clin Obstet Gynaecol 2015; 29: 812-21. doi: 10.1016/j.bpobgyn.2015.03.007 18. Zigras T, Kupets R, Barbera L, Covens A, Liu Y, Gien LT. Uptake of sentinel lymph node procedures in women with vulvar cancer over time in a population based study. Gynecol Oncol 2019; 153: 574-9. doi: 10.1016/j. ygyno.2019.03.010 19. Brincat MR, Muscat Baron Y. Sentinel lymph node biopsy in the manage­ment of vulvar carcinoma: an evidence-based insight. Int J Gynecol Cancer 2017; 27: 1769-73. doi: 10.1097/IGC.0000000000001075 20. van Doorn HC, van Beekhuizen HJ, Gaarenstroom KN, van der Velden J, van der Zee AGJ, Oonk MHM, et al. Repeat sentinel lymph node procedure in patients with recurrent vulvar squamous cell carcinoma is feasible. Gynecol Oncol 2016; 140: 415-9. doi: 10.1016/j.ygyno.2016.01.013 21. Verbeek FPR, Tummers QRJG, Rietbergen DDD, Peters AAW, Schaafsma BE, van de Velde CJH, et al. Sentinel lymph node biopsy in vulvar cancer using combined radioactive and fluorescence guidance. Int J Gynecol Cancer 2015; 25: 1086-93. doi: 10.1097/IGC.0000000000000419 22. Ghoniem K, Shazly SA, Dinoi G, Zanfagnin V, Glaser GE, Mariani A. Sentinel lymph nodes and precision surgery in gynecologic cancer. Clin Obstet Gynecol 2020; 63: 12-23. doi: 10.1097/GRF.0000000000000517 376 23. Van der Zee AGJ, Oonk MH, De Hullu JA, Ansink AC, Vergote I, Verheijen RH, et al. Sentinel node dissection is safe in the treatment of early-stage vulvar cancer. J Clin Oncol 2008; 26: 884-9. doi: 10.1200/JCO.2007.14.0566 24. Mitra S, Sharma MK, Kaur I, Khurana R, Modi KB, Narang R, et al. Vulvar carcinoma: dilemma, debates, and decisions. Cancer Manag Res 2018; 10: 61-8. doi: 10.2147/CMAR.S143316 25. Swanick CW, Eifel PJ, Huo J, Meyer LA, Smith GL. Challenges to delivery and effectiveness of adjuvant radiation therapy in elderly patients with node-positive vulvar cancer. Gynecol Oncol 2017; 146: 87-93. doi: 10.1016/j. ygyno.2017.05.004 26. Deppe G, Mert I, Belotte J, Winer IS. Chemotherapy of vulvar cancer: a review. Wien Klin Wochenschr 2013; 125: 119-28. doi: 10.1007/s00508-013­0338-y 27. Domingues AP, Mota F, Durăo M, Frutuoso C, Amaral N, de Oliveira CF. Neoadjuvant chemotherapy in advanced vulvar cancer. Int J Gynecol Cancer 2010; 20: 294-8. doi: 10.1111/igc.0b013e3181c93adc 28. Inrhaoun H, Elghissassi I, Gutierrez M, Brain E, Errihani H. Long term re­sponse to erlotinib in a patient with recurrent vulvar carcinoma: case report and review of literature. Gynecol Oncol Case Rep 2012; 2: 119-20. doi: 10.1016/j.gynor.2012.07.002 29. Horowitz NS, Olawaiye AB, Borger DR, Growdon WB, Krasner CN, Matulonis UA, et al. Phase II trial of erlotinib in women with squamous cell carci­noma of the vulva. Gynecologic Oncology 2012; 127: 141-6. doi: 10.1016/j. ygyno.2012.06.028 30. Schnch HG, Ackermann S, Alt-Radtke CD, Angleitner L, Barinoff J, Beckmann MW, et al. Diagnosis, therapy and follow-up of vaginal cancer and its precursors. Guideline of the DGGG and the DKG (S2k-Level, AWMF Registry No. 032/042, October 2018). Geburtshilfe Frauenheilk 2019; 79: 1060-78. doi: 10.1055/a-0919-4959 377 review Combining radiotherapy and immunotherapy in definitive treatment of head and neck squamous cell carcinoma: review of current clinical trials Gaber Plavc1,2, Primoz Strojan1,2 1 Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(4): 394-408.; 54(4): 377-393. Received 18 August 2020 Accepted 22 September 2020 Correspondence to: Assist. Gaber Plavc, M.D., Institute of Oncology Ljubljana, Department of Radiation Oncology, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: gplavc@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Head and neck squamous cell carcinoma (HNSCC) presents as locally advanced disease in a major­ity of patients and is prone to relapse despite aggressive treatment. Since immune checkpoint inhibitors (ICI) have shown clinically significant efficacy in patients with recurrent/metastatic HNSCC (R/M HNSCC), a plethora of trials are investigating their role in earlier stages of disease. At the same time, preclinical data showed the synergistic role of concurrently administered radiotherapy and ICIs (immunoradiotherapy) and explained several mechanisms be­hind it. Therefore, this approach is prospectively tested in a neoadjuvant, definitive, or adjuvant setting in non-R/M HNSCC patients. Due to the intricate relationship between host, immunotherapy, chemotherapy, and radiotherapy, each of these approaches has its advantages and disadvantages. In this narrative review we present the biological background of immunoradiotherapy, as well as a rationale for, and possible flaws of, each treatment approach, and provide readers with a critical summary of completed and ongoing trials. Conclusions. While immunotherapy with ICIs has already become a standard part of treatment in patients with R/M HNSCC, its efficacy in a non-R/M HNSCC setting is still the subject of extensive clinical testing. Irradiation can overcome some of the cancer’s immune evasive manoeuvres and can lead to a synergistic effect with ICIs, with possible ad­ditional benefits of concurrent platinum-based chemotherapy. However, the efficacy of this combination is not robust and details in trial design and treatment delivery seem to be of unprecedented importance. Key words: head and neck neoplasms; immunoradiotherapy; radiotherapy; immunotherapy Introduction Head and neck squamous cell carcinoma (HNSCC) accounts for more than 800,000 new cancer cases and over 400,000 deaths each year worldwide.1 Despite aggressive therapeutic approaches the outcomes are still highly dependent on disease burden. Five-year disease control ranges from al­most 100% in patients with T1a glottic carcinoma to below 30% in patients with locally-advanced hy­popharyngeal cancer.2,3 More than 60% of all cases are locally-advanced at diagnosis with a 50% rate of relapse in the first two years, despite the use of multimodal state-of-the-art treatment.4 Therefore, while treatment-related toxicity is now of primary concern in early stage HNSCC and low-risk human papilloma virus (HPV) mediated oropharyngeal carcinomas, with 3-year overall survival rates in excess of 90%5,6, in other patients the focus of re­search is on treatment intensification and/or modi­fication. After intrinsic tumour suppressor mechanisms fail, further tumour progression is the result of an inefficient elimination phase or equilibrium phase 378 of the extrinsic tumour suppression by the im­mune system.7 Genetically unstable cancer cells under constant immune selection pressure evade immune recognition and destruction. Thus, they become invisible to immune cells by reducing the presentation of tumour antigens, decreasing their sensitivity to the cytotoxic effects of immune cells, and rendering their microenvironment im­munosuppressive.7 In the fight against the latter, immune checkpoint inhibitors (ICI) targeting im­mune checkpoint programmed cell death protein 1 (anti-PD-1) are now considered standard care in recurrent and metastatic HNSCC (R/M HNSCC).8,9 Because of their proven efficacy and significantly improved toxicity profile as well as positive effect on quality of life as compared to standard chemo­therapy regimens, an increasing number of trials are testing ICIs in the earlier stages of HNSCC.10–12 Besides a well-known immunosuppressive ef­fect of radiotherapy (RT), it can also lead to posi­tive alterations in innate and adaptive immunity.13 The same is true for the positive effects of the im­mune system on radiation efficacy, as a tumoricid­al effect of RT is dependent on functional T cells, even at ablative doses.14 Furthermore, RT induces programmed death-ligand 1 (PD-L1) expression in dendritic cells (DCs) and cancer cells which con­tributes to acquired cancer radioresistance, which could be overcome by concurrent anti-PD-1/L1.15 These intricate interactions form the basis for com­bined treatment with RT and ICIs (immunoradio-therapy). This combination was shown to cause similar toxicity compared to either RT or ICI alone across different cancer types.16 Encouraging effi­cacy of this treatment combination has also been shown in early prospective trials in metastatic malignant melanoma and non-small cell lung cancer.17–21 The first results of trials using immu­noradiotherapy in non-R/M HNSCC are now also available and many are underway. In this review we presented a biological rationale for the combi­nation of RT and anti-PD-1/L1 and performed a systematic search for, and critical assessment of, completed and ongoing trials using a combination in non-R/M HNSCC. Role of anti-PD-1 and radiotherapy in immune rejection of HNSCC The efficacy of anti-PD1 therapy in HNSCC is poor with less than 20% of responding patients.8,22,23 These high rates of primary or acquired resistance in R/M HNSCC to anti-PD1 agents are a result of absent antigenic proteins, defective antigen pres­entation, T cell exhaustion/absence, insensibility of tumours to T cells, presence of immunosuppres­sive cells, and/or presence of other inhibitory im­mune checkpoints.24 For the immune system to exert its cytotoxic function, mutant peptides, also known as tumour neoantigens (TNA) or ectopically expressed anti­gens, must be presented to antigen-presenting cells by cancer cells on major histocompatibility com­plex I (MHC I).25 Even though the tumour muta­tion burden in HNSCC is rather high with 5 muta­tions per million base pairs, a proper presentation is needed for them to elicit an immune response.26,27 A vital role of antigen processing machinery in this step is evident by the absence of CD8+ T cell recog­nition of HNSCC in the case of defective antigen processing machinery (defect present in 20–80% of HNSCCs).28–30 The next step is presentation of the TNA by MHC I. The complete loss of MHC I re­sults in natural killer (NK) cells’ activation, while aberrant expression is beneficial for cancer cells and is present in up to 60% of HNSCCs.31–33 Up to 80% of HNSCC patients overexpress the epidermal growth factor receptor (EGFR), which also down-regulates MHC I.34 Treatment with anti-PD-1 was shown to be less efficient in cancers with aberrant MHC I.35,36 Yet tumour antigenicity is not enough to elicit immune response by itself. TNA presentation must be put in context by accompanying adjuvants in the form of danger-associated molecular patterns (DAMP) which are recognised by pattern recog­nition receptors on the cells of innate immunity. Different types of DAMPs are exposed by differ­ent modes of cell death and even by stressed can­cer cells.37 These include membrane-bound calreti­culin, emitted ATP, and passively released nuclear high-mobility group box protein 1 (HMGB1). This leads to the recruitment and activation of dendritic (DCs) and other mononuclear cells.38,39 DCs cross-present antigens to naďve CD8+ T and by co-stim­ulatory signals (ligands and cytokines provided by DCs upon stimulation by DAMPs and type I interferons [IFNs]) prime these cytotoxic T lym­phocytes in regional lymph nodes.40 Type I IFN is produced by cancer cells as a result of a stimulator of interferon genes (STING) responding to DNA in the cytosol of cancer cell, which is a consequence of cancer’s unstable genome.41,42 To prevent unnecessary damage to surrounding tissue in their fight against viruses, CD8+ T lym­phocytes also express inhibitory receptors, such 379 as PD-1, with its ligand PD-L1 on host tissue and immune cells.43 The same PD-L1 expression is ex­ploited by cancer cells to escape immune surveil­lance.44 An active PD-1/PD-L1 pathway in tumour microenvironment (TME) also promotes T cell ex­haustion and differentiation of regulatory T cells (Treg).45 Primed tumour-infiltrating lymphocytes (TILs) that are suppressed due to PD-1/PD-L1 in­teraction are vital for anti-PD-1 efficacy, which also tips the balance from differentiation of exhausted T cells and Tregs towards generation of effector T cells.45,46 Immunostimulatory effect of RT depends a great deal on inducing the above-described im­munogenic cell death, with dose-dependent (from 2 to 20 Gy) increase in concentrations of DAMPs calreticulin, HMGB1, and ATP.47 RT also produces free cytosolic DNA which is more pronounced in cancers with a loss of p53 function, as is the case in the majority of HNSCC.48,49 Cytosolic DNA is sensed by various pattern recognition receptors with STING being a central connecting protein. Activation of the cyclic GMP–AMP synthase-STING (cGAS-STING) pathway by free cytosolic DNA leads to type I IFN production in cancer and DCs.41,50 Regarding antigenicity, RT increases MHC I expression and diversifies the tumour-infiltrating T cell receptor repertoire which is a positive pre­dictor of response to anti-PD-1/L1.51–53 Previously silent mutated genes can be expressed by RT, thus leading to presentation of these TNAs by MHC I.54,55 RT also induces some constituents of antigen processing machinery by enhancing degradation of proteins into peptides.51 The positive effects of RT are also apparent in TME. By reducing tumour hypoxia and consequently reducing the expression of vascular endothelial growth factor , SBRT can in­hibit mobilisation of myeloid-derived suppressor cells (MDSC).56 Some authors also observed an en­hanced recruitment of T cells into TME after RT.57 RT-enhanced death receptor Fas expression fur­ther promotes the antitumour activity of recruited T cells.58,59 Furthermore, RT promotes the function and differentiation of cytotoxic T cells by inducing interleukin-1B, tumour necrosis factor-a, and inter­leukin-6.13 Considering vasculature, low dose RT increases the ratio of antitumoural macrophages type 1 and tumour-promoting macrophages type 2, which leads to vascular normalisation and T cell recruitment.60 Besides, low dose RT also appears to decrease TME’s immunosuppressive cells such as Tregs and MDSCs.61 Another beneficial vascu-lature-related effect of RT is induction of cell adhe­sion molecules, for example Intercellular Adhesion Molecule 1 and E-selectin, that help leukocytes ex-travasate to TME.62 Importantly, as a part of standard treatment in HNSCC, concurrent platin-based chemoradiother­apy (CRT) was also shown to induce immunogenic cell death.47 In the in vitro model, antigen presen­tation and T cell cytotoxicity were enhanced by moderate doses of cisplatin. In the in vivo mouse model synergism of cisplatin and anti-PD-1 was observed.63 However, cisplatin also resulted in PD-L1 upregulation on cancer cells and higher dos­es were immunosuppressive. Nevertheless, Luo et al. showed on murine cancer models that cisplatin combined with anti-PD-1 treatment enhances RT-induced abscopal effect in non-irradiated nodes.64 It should be noted that all the above-mentioned effects of RT were observed in preclinical studies and are not universally beneficial, as was shown in clinical setting. Release of DAMPs HMGB1 and ATP, which is degraded into extracellular adenosine, can have many immunosuppressive effects.65–70 Activation of cGAS-STING can lead to increased concentrations of MDSC in TME and even increase cancer aggressiveness.71,72 STING ac­tivation can also lead to depletion of tryptophan in TME via upregulation of Indoleamine 2,3-di­oxygenase, resulting in reduced T cell cytotoxic-ity and increased tumour-associated macrophages and MDSCs.73,74 Even sustained type I IFN signal-ling is detrimental as it results in increased Treg and MDSC concentrations in TME and enhanced expression of PD-1.75 Besides, RT increases tumour growth factor beta concentration which was shown to promote tumour-promoting macrophages type 2 differentiation and inhibit DCs and cytotoxic T cells.13 In addition, RT was shown to even upregu-late hypoxia inducible factor-1a, leading to even­tual Treg and MDSC accumulation and DC and T cell inhibition via vascular endothelial growth fac­ tor.76–80 Methods We searched PubMed and Clinicaltrials.gov da­tabases with search terms ((immunoradiotherapy OR radioimmunotherapy) OR ((head and neck) OR (oral cavity) OR (oropharyngeal) OR (oropharynx) OR (larynx) OR (laryngeal) OR (hypopharynx) OR (hypopharyngeal)) AND (immunotherapy OR checkpoint OR pembrolizumab OR avelumab OR atezolizumab OR camrelizumab OR durvalumab OR avelumab OR nivolumab OR toripalimab OR PD-1 OR PD-L1 OR tremelimumab OR CTLA-4) 380 TABLE 1. Neoadjuvant immunoradiotherapy trials NIRT-HNC, NCT03247712,89 2018 I 10 HPV+ resectable HNSCC stage I-III or CUP with clinical indications for adj. RT or TORS ineligible NIVO+SBRT 5 weeks before surgery, followed by NIVO 3x NIVO neoadj. and 3x adj. NIVO starting 4 weeks postop. SBRT to GTV+3mm; 5pts: 5x8Gy daily (A), and 5 pts: 3x8Gy (B) every other day; delivered between 1st and no surgical delays; G3 postop. toxicity higher in cohort A; pCR: 100% in cohort A, and 80% in cohort B. 2nd NIVO cycle II 11, ongoing cohort C: same as phase I, cohort D: stage III-IV HPV- resectable HNSCC cohort C: SBRT alone 5 weeks before surgery, followed by NIVO, cohort D: same as phase I cohort C: only adj. NIVO, same as in phase I cohort D: same as phase I cohort C (6pts): SBRT 3 x 8 Gy cohort D (5 pts): SBRT 3 x 8 Gy no G3-4 toxicity; major pathologic response in majority of pts NCT03635164,91 2018 I 18 HPV- resectable LAHNSCC DURVA+SBRT 3–6 weeks before surgery, followed by DURVA DURVA neoadj. with the first SBRT fraction and up to 6x DURVA postop. SBRT to gross disease only, starting dose of 2x6Gy (planned increase to NA 3x6Gy, cohort size of 3 patients) every other day, starting concurrently with DURVA NCT03618134,92 2018 I/II 82 TORS eligible HPV+ oropharyngeal HNSCC DURVA+SBRT+/­tremelimumab 5–7 weeks before TORS, followed by DURVA DURVA+/­tremelimumab neoadj. with the first SBRT fraction and on day 27, followed by up to 4x adj. DURVA SBRT in 5fx, starting concurrently with DURVA+/­tremelimumab NA adj. = adjuvant; CUP = cancer of unknown primary; DURVA = durvalumab; fx = fraction; GTV = gross tumour volume; G3 = grade 3; HNSCC = head and neck squamous cell carcinoma; HPV- = human papilloma virus negative cancer; HPV+ = human papilloma virus associated cancer; LAHNSCC = locally advanced HNSCC; N = planned number of enrolled patients, NA = not available; neoadj. = neoadjuvantly, NIVO = nivolumab; pCR = pathological complete response; postop. = postoperatively; pts = patients; RT = radiotherapy, SBRT = stereotactic body RT; TORS = transoral robotic surgery AND (radiotherapy OR SBRT OR RT OR SABR OR irradiation) and with the start date of the stud­ies from 15th July 2013 to 15th July 2020. In total, 39 completed or ongoing trials were found, using con­current (chemo)radiotherapy and ICIs in primary definitive treatment of non-R/M HNSCC (non-na­sopharyngeal). Trials using anti-PD-1/L1 and radiotherapy combination in HNSCC: different approaches In completed and ongoing trials, concurrent anti­PD-1/L1 and RT was delivered either before or af­ter surgery, or as a sole definitive treatment. Few delivered anti-PD-1/L1 also as an extended con-solidative treatment. Taking the intricate relation­ship between the immune system and therapy into account, attention to the below-described caveats should help shed light on the pros and cons of these research approaches. Neoadjuvant immunoradiotherapy Except for the earliest stages of HNSCC, elec­tive neck treatment either by lymphadenectomy or irradiation is part of the standard treatment.81 Lymph nodes are also one of the places where DCs cross-prime CD8+ T lymphocytes.82 Even though the immediate treatment effect of concurrent anti­PD-1 and RT depends primarily on TILs already present in the primary tumour, T cells from lymph nodes are responsible for long-lasting tumour con­trol.83,84 Preclinical studies in murine cancer models clearly showed the vital role of functioning drain­ing lymph nodes for RT efficacy with or without concurrent ICI.85,86 Removal of draining lymph nodes or elective nodal irradiation led to reduced tumour-specific TILs.85,86 Furthermore, clinical data show reduced efficacy of anti-PD-1 in previ­ously treated patients with HNSCC.87 This speaks strongly in favour of using an immunoradiothera­py combination before surgery as compared to its postoperative application. 381 Neoadjuvant RT is not considered a standard of care in HNSCC, therefore these “window of oppor­tunity trials” serve mostly to advance our under­standing of the underlying mechanisms and to lay the ground for further studies.88 Special attention must be therefore given to patient safety. In the, so far only, immunoradiotherapy “window of oppor­tunity” trial that reported results, no surgical de­lays were noted.89 The possibility of anti-PD-1 in­duced hyperprogression must nevertheless be kept in mind as it was reported in up to 29% of patients with R/M HNSCC.90 The ongoing trials are presented in detail in Table 1. Leidner et al. completed phase I of their phase I/II trial and already provided intriguing results.89 In the first phase, 10 patients with stage I-III HPV associated HNSCC or cancer of unknown primary with clinical indications for adjuvant RT or who were ineligible for transoral robotic sur­gery were accrued. Two cohorts were formed of which five patients received neoadjuvant SBRT with 5x8 Gy (A cohort), and another five patients had SBRT with 3x8 Gy (B cohort), both with con­current nivolumab. No grade 4 toxicity was ob­served, with somewhat higher grade 3 toxicity in the A cohort. Notably, grade 2 renal insufficiency was observed in 50% of patients. Both fractionation regimens were shown to be effective with 100% and 80% complete pathological responses in the A and B cohort, respectively. However, on presurgi-cal imaging evaluated by RECIST criteria, no com­plete responses were found. Recently, preliminary results of their phase II cohort expansion were also presented.91 Only the SBRT fractionation of the B cohort was further pursued. In cohort C inclusion criteria were the same as in cohorts A and B, while these six patients were treated with only neoad­juvant SBRT, followed by surgery and adjuvant nivolumab. Cohort D included only patients with HPV-negative HNSCC, and these five patients were treated the same as those in cohort B (SBRT with 3x8 Gy concurrently with nivolumab). Results were so far only vaguely described: there was no limiting toxicity, but the complete pathological re­sponse rate was somewhat lower than in cohorts A and B. In-detail results are awaited. The approach to treatment was similar in HPV-negative HNSCC patients in the NCT03635164 trial, with the difference that anti-PD-L1 agent durvalumab was used instead of nivolumab.91 The third ongoing trial (NCT03618134) with a similar approach is testing whether the addition of tremelimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4), to durvalumab can improve the outcome in HPV-positive HNSCC patients.92 T hese two ICIs provide complementary effects, albeit at the expense of increased toxic­ ity.93,94 Definitive immunoradiotherapy Considering only non-cancer/TME-related factors, synergism between anti-PD-1 and RT is probably most pronounced when these two treatment mo­dalities are delivered concurrently in previously untreated patients with intact draining lymph nodes and no lymphopenia.85–87,95–98 Definitive im­munoradiotherapy as a sole treatment fulfils these criteria, except for nodal irradiation. If, in a neo­adjuvant setting, elective nodal irradiation is not mandatory, its omission would be ill-advised in a definitive (chemo)radiotherapy setting based on our current knowledge.81 However, advancement in diagnostic imaging and treatment (e.g. sentinel lymph node biopsy) provides the basis for ongoing trials testing reduced dose and/or volume of elec­tive nodal irradiation which would be welcomed in immunoradiotherapy as well.99 Preclinical studies also provide rather strong support for greater efficacy of hypofractionated RT compared to conventionally fractionated RT. 56,100,101 In contrast to all the above-listed trials with immu­notherapy in the neoadjuvant setting, however, the definitive setting immunoradiotherapy trials most­ly utilise conventionally fractionated RT courses as compared to hypofractionated SBRT. This could be an important outcome-defining factor. Concurrent chemoradiotherapy with cisplatin causes severe radiomucositis (grade 3–4) in around 40% of HNSCC patients.102,103 Even though anti­PD-1/L1 induced oral mucositis or stomatitis occurs in less than 3% of patients and is usually mild, it can nevertheless occasionally be severe.104 Special atten­tion should be paid to this when using an approach with combined CRT and anti-PD-1/L1, despite the fact that pertinent trials have so far not reported exacerbated toxicity in oral mucosa (see below). Another important aspect of concurrent CRT and immunotherapy is the effect of chemotherapy on immunotherapy’s efficacy which seems to be ben­eficial in low doses, whereas high-dose chemother­apy is known to cause myelosuppression and could be detrimental to the efficacy of immunotherapy.63,64 Several trials use ICI combined with cetuximab, an anti- EGFR agent. Cetuximab is a mouse/human chimeric monoclonal IgG1 antibody.105 Besides act­ing through targeting EGFR and dysregulating its signaling pathway, it also stimulates NK cells anti- 382 tumour activity, activates DCs, and recruits cyto­toxic CD8+ T cells.105 Cetuximab’s ability to prime adaptive and innate immunity is met with regula­tory immunosuppressive mechanisms. Targeting these immunosuppressive mechanisms (induction of Tregs, MDSC, PD-1, PD-L1, CTLA-4) by immu­notherapy such as ICI has great potential and is still being tested in several trials.106 A prospective trial using anti-PD-1 combined with cetuximab in 33 patients with platinum-refractory/ineligible R/M HNSCC showed a 41% response rate. About a third of patients experienced treatment-related grade 3 toxicity.107 Furthermore, retrospectively gathered data on a triple combination of cetuximab, chemo­therapy and anti-PD-1 used in 15 patients with R/M HNSCC was presented in 2018 by Lin et al.108 The combination seemed effective with 58% partial re­sponses and acceptable toxicity. Completed and ongoing trials treating patients with non-R/M HNSCC with a definitive immuno-radiotherapy combination are presented in Table 2, while important details are presented below. JAVELIN Head and Neck 100 (NCT02952586) is the first randomised phase III trial combining CRT with concomitant ICI in patients with LAHNSCC to be terminated due to inefficiency.109 Concurrent administration of a PD-L1 inhibitor avelumab and standard CRT (70 Gy and high-dose cisplatin) fol­lowed by maintenance avelumab for 12 months was compared to a placebo arm receiving the same CRT but with placebo instead of avelumab in 697 high-risk LAHNSCC patients.110 A pre-planned interim analysis showed that this combination is unlikely to show a significant improvement in pro-gression-free survival and the trial was therefore terminated. Detailed study findings are awaited. In a GORTEC 2017-01 REACH trial (NCT02999087), two standard arms (CRT with a three-weekly high-dose cisplatin in a cohort of pa­tients fit for high-dose cisplatin, and RT with con­current cetuximab in a cohort of patients unfit for high-dose cisplatin) were compared to experimen­tal arms with the same RT regimen and concur­rent avelumab and cetuximab (preliminary results, Table 2).111,112 All patients completed RT except for one cisplatin-ineligible patient receiving RT concurrently with avelumab and cetuximab. 88% and 76% of patients received all planned doses of avelumab and cetuximab, respectively. A grade =4 adverse effect occurred in 5/41 (12%) patients in experimental arms (all in the cohort of patients in­eligible for high-dose cisplatin), and in 5/41 (12%) patients in standard arms (14% in high-dose cispl­atin eligible and 10% in high-dose cisplatin ineli­gible patients) where one grade 5 toxicity was also observed. The trial continues. In 2019, results of the lead-in phase of randomised phase II/III trial NRG-HN004 (NCT03258554) were presented. Ten out of a planned 523 cisplatin-ineli­gible patients received durvalumab concomitantly with RT and all completed RT as planned, while 8/10 patients received all the planned durvalumab cycles. Randomisation will continue to either RT with durvalumab or RT with cetuximab.113 The GORTEC 2015-01 PembroRad randomised phase II trial’s safety-related results were present­ed in 2018.114 In 133 cisplatin ineligible patients with LAHNSCC cetuximab or pembrolizumab were added to conventional RT, which resulted in a similar completion rate of RT (86 vs. 88%) and dys­phagia (34 vs. 39%). However, mucositis was more prevalent in the cetuximab arm and the same goes for dermatitis (49 vs. 17%) (Table 2). Final results are still awaited. The results of the first 16 randomised patients of the planned 120 patients with HPV- LAHNSCC in a DURTRE-RAD trial (NCT03624231) were re­cently presented.115 Among the first six patients treated with a combination of RT, durvalumab and tremelimumab (arm A), five patients (83%) stopped treatment due to immune-related adverse effects (irAE), of which one was grade 5. This arm was terminated due to excessive toxicity. Arm B with only durvalumab added to RT, which resulted in only 1/10 patients stopping treatment due to irAE, is continuing to enrol. Weiss et al. (NCT02609503) presented the results of their phase II trial after a median follow-up of 21 months.116 In 29 cisplatin ineligible patients with LAHNSCC pembrolizumab was given concurrent­ly with definitive RT and for an additional three adjuvant cycles (Table 2). The estimated two-year overall and progression-free survival was 75% and 71% respectively. RT was delivered in full in 28/29 patients, and 25/29 patients received all pembroli­zumab doses. Toxicities were mild with a major exception being grade 3–4 lymphopenia, which oc­curred in 59% of patients, however, absolute lym­phopenia did not predict for progression. Further characterisation of this unexpected lymphopenia showed declines in blood concentrations of B cells and CD4+ T cells, whereas CD8+ T cells were rela­tively preserved.116 Powel et al. presented results from their phase I trial (NCT02586207), testing pembrolizumab with chemoradiotherapy in 59 patients with LAHNSCC.117 Pembrolizumab was discontinued due to irAE in 9% during CRT and for non-irAE 383 TABLE 2. Definitive immunoradiotherapy trials NCT02586207,117 2015 I 59 LAHNSCC eligible for CRT (34 pts HPV + and 23 pts HPV-) PEMBRO + CRT, followed by PEMBRO PEMBRO on days -7 (before CRT), 15 and 36 (conc. with CRT), and adj. for 5 cycles starting on day 1: CRT with IMRT 70 Gy (2Gy/fx) and LD-CDDP for 6 cycles HPV + : 85% CR 12 weeks after CRT; HPV-: 78% CR 12 weeks after CRT; HPV + : 2-year OS 97% and PFS 93%; HPV-: 1-year OS 87% and PFS 73% GORTEC 2015-01 “PembroRad” (NCT02707588),114 2016 II, rand. 133 LAHNSCC ineligible for CDDP arm A: CETUX + RT; arm B: PEMBRO + RT arm A: CETUX during RT; arm B: PEMBRO during RT IMRT (69.99Gy/33fx) arm A: 94% grade 3 toxicity, 57% grade 3 mucositis, 86% received full RT; arm B: 78% grade 3 toxicity, 24% grade 3 mucositis, 88% received full RT KEYNOTE-412 (NCT03040999),124 2017 III, rand. 780 LAHNSCC eligible for CRT arm A: PEMBRO + CRT, followed by PEMBRO; arm B: placebo + CRT, followed by placebo arm A: priming dose of PEMBRO followed by 2x PEMBRO + CRT, followed by 14x maint. PEMBRO; CRT (70Gy/35fx) and HD-CDDP NA arm B: placebo instead of PEMBRO NCT02759575,131 2016 I/II 47 LAHNSCC of larynx PEMBRO + CRT PEMBRO starting 3 weeks before CRT, maximum 4x CRT (70Gy/35fx) and HD-CDDP NA NCT02609503,116 2016 II 29 LAHNSCC ineligible for CDDP PEMBRO + RT, followed by PEMBRO PEMBRO conc. with RT and 3 adj. cycles IMRT (70Gy/35fx) 2-year OS 75% and PFS 71%; 59% grade 3–4 lymphopenia NCT02777385,130 II, rand. 90 LAHNSCC arm A: PEMBRO + arm A: 8x CRT with IMRT NA 2016 CRT; arm B: CRT followed by PEMBRO PEMBRO 1 week prior to RT; arm B: 8x PEMBRO (70Gy/35fx) and LD-CDDP beginning in week 10 NCT03532737,132 2018 II 50 LAHNSCC PEMBRO + CRT or PEMBRO + CETUX + RT PEMBRO starting 3 weeks before (C)RT and during CRT or during RT + CETUX CRT with IMRT (66–70Gy/30–35fx) and HD-CDDP or conc. CETUX NA KEYCHAIN (NCT03383094),133 2018 II, rand. 114 HPV + LAHNSCC arm A: PEMBRO + RT; arm B: CRT arm A: conc. and adj. PEMBRO for 20 cycles; arm B: CDDP-based CRT IMRT (70Gy/33– 35fx) (arm A) and HD-CDDP in arm B NA PEACH (NCT02819752),134 2017 I 36 LAHNSCC PEMBRO + CRT, followed by PEMBRO pre-loading dose of PEMBRO (dose-escalation trial, standard CRT NA 100–200mg) and conc. CRT and PEMBRO and 4x adj. PEMBRO NCT04369937,127 II 50 IR HPV + HPV-16 vaccination 3x ISA101b CRT with IMRT NA 2020 HNSCC (ISA101b) + PEMBRO + CRT starting 1 week prior to PEMBRO and two weeks (70Gy/35fx) and HD-CDDP prior to CRT RTOG 3504 (NCT02764593),120 2016 I 40 IR-HR LAHNSCC conc. and adj. NIVO added to each of 4 (C)RT cohorts conc. NIVO starting 2 weeks before (C)RT and adj. NIVO starting 3 months after CRT all cohorts: IMRT (70Gy/35fx); cohort 1: CRT with LD-CDDP; cohort 2: CRT with HD­CDDP; cohort 3: adj. NIVO infeasible after HD-CDDP or in CDDP-ineligible pts; low rates of NIVO DLT RT + CETUX; cohort 4: RT NCT03162731,121 2017 I 24 HR LAHNSCC NIVO + ipilimumab + RT 17x NIVO and 6x ipilimumab, both starting 2 weeks before RT IMRT (70Gy/35fx) first 12 pts: grade 3 in-RT-field toxicity in 50% of pts, 3 pts discontinued therapy >3 months post-RT, 1 grade 3 colitis, 1 grade 5 bleeding, irAE in 50% of pts NCT03894891,135 II 70 LAHNSCC of induction docetaxel standard standard NA 2019 larynx and hypopharynx + CDDP + NIVO, followed by NIVO + RT institutional dosing institutional dosing NCT03829722,136 2019 II 40 HR HPV + OP cancer NIVO + CRT, followed by adj. NIVO 4x NIVO before and conc. with CRT, followed by 4x NIVO CRT (70Gy/35fx) and carboplatin + paclitaxel combination once NA (temporarily suspended due to COVID-19) per week NRG-HN005 (NCT03952585),126 2019 II/III, rand. 711 early-stage HPV + OP cancer arm A: NIVO + deescalated RT; arm B: CRT arm C: 6x NIVO, starting 1 week prior to RT IMRT, CRT with HD­CDDP NA deescalated CRT NCT03799445,137 2019 II 180 low-intermediate volume HPV + OP cancer NIVO + ipilimumab + RT NIVO on days 1, 15, 29, and ipilimumab on day 1; for 2 cycles IMRT 50–66Gy starting on day 1 of 2. cycle of NIVO + ipilimumab NA GORTEC 2017­01 “REACH” (NCT02999087),138 2017 III, rand. 688 LAHNSCC Cohort 1 (fit for CDDP): CRT with CDDP (arm 1A), RT + AVEL + CETUX (arm 1B); Cohort 2 (unfit for CDDP); RT + CETUX (arm 2A), RT + AVEL + CETUX AVEL and CETUX starting 1 week prior to RT, followed by AVEL maint. for 12 months IMRT 69.96Gy with either HD-CDDP or CETUX first 82 pts: thresholds of the safety monitoring rule not crossed; trial continues (arm 2B) JAVELIN HEAD AND NECK 100 (NCT02952586),110 2016 III, rand. 697 LAHNSCC arm A: AVEL + CRT; arm B: placebo + CRT AVEL starting 1 week prior to CRT, followed by maint. AVEL for 12 months CRT with IMRT (70Gy/35fx) and HD-CDDP preplanned interim analysis: unlikely to show improvement, terminated NCT02938273,122 2017 I 10 LAHNSCC ineligible for CDDP AVEL + CETUX + RT AVEL starting 1 week prior to RT, followed by maint. AVEL for VMAT (70Gy/35fx) tumour recurrence in 50% after a median follow 4 months; CETUX conc. up of 12months; transient and manageable irAE DUCRO-HN (NCT03051906),139 2018 I/II 69 LAHNSCC DURVA + CETUX + RT DURVA and CETUX, both conc. with RT, IMRT (69.9Gy/33fx) NA followed by adj. DURVA for 6 months DURTRE-RAD (NCT03624231),115 2018 II, rand. 120 HPV- LAHNSCC arm A: DURVA + TREM + RT; arm B: DURVA + RT DURVA started 2 weeks prior to RT and TREM started with RT, followed RT (70Gy/35fx) first 16 patients: in arm A 5/6 stopped treatment due by DURVA for up to 9 cycles to toxicity -> terminated; in arm B 1/10 patients stopped treatment CheckRad-CD8 (NCT03426657),123 2018 II 120 LAHNSCC induction DURVA + TREM + CDDP + docetaxel and in case of increased CD8 + TILs compared to pre-treatment Bx -> DURVA + TREM after induction: DURVA with RT and TREM with RT, followed by DURVA for up to 12 cycles RT (70Gy/35fx) first 10pts after induction (re-biopsies): pCR in 8/10pts, 2 grade 3 + toxicities + RT 385 CITHARE (NCT03623646),140 2019 II, rand. 66 early-stage HPV + OP cancer arm A: DURVA + RT; arm B: CRT DURVA conc. with RT RT 70Gy with CDDP in arm B NA REWRITe (NCT03726775),129 2018 II 73 HNSCC T1-2 or HNSCC T3-4 and not eligible for CRT/CETUX + RT DURVA + RT, followed by additional 6 months of DURVA DURVA conc. with RT, followed by 6 months of DURVA RT to only primary tumour and immediately adjacent nodal level without extended neck NA irradiation NCT04405154,141 II 32 LAHNSCC CRT + camrelizumab camrelizumab CRT with NA 2020 conc. with CRT and after for total of 8 cycles IMRT/VMAT (66–70Gy/33–35fx) and HD-CDDP adj. = adjuvantly; AVEL = avelumab; CETUX = cetuximab; ,; CDDP = cisplatin; conc. = concurrently; CR = complete response; CRT = chemoradiotherapy; DLT = dose-limiting toxicity; DURVA = durvalumab; , fx = fractions; HD-CDDP = high dose cisplatin 100 mg/m2 every three weeks during RT; HR = high-risk; HPV+ = human papilloma virus associated cancer, HPV- = human papilloma virus negative cancer; IMRT = intensity modulated RT; IR = intermediate-risk; irAE – immune-related adverse effects; LAHNSCC = locally advanced head and neck squamous cell carcinoma; LD-CDDP = low dose cisplatin 40 mg/m2 every week during RT; maint. = maintenance; N = planned enrolment; NA = not available; NIVO = nivolumab; OP = oropharyngeal; OS = overall survival; PEMBRO = pembrolizumab; PFS = progression-free survival; RT = radiotherapy, TILs = tumour infiltrating lymphocytes; TREM – tremelimumab; VMAT = volumetric modulated arc therapy related causes in 12% after CRT. The goal cisplatin dose of 200 mg/m2 or more was received by 88% of patients and 98% of patients received all 70 Gy of RT. 76% of patients received all eight planned pembrolizumab cycles. Grade 4 toxicities were solely hematologic and electrolyte abnormalities. Outcomes are described in Table 2. In the RTOG 3504, a phase I trial enrolling 40 pa­tients with intermediate risk (HPV-associated oro-pharyngeal HNSCC, T1-2N2b-N3/T3-4N0-3, >10 pack-years or T4N0-N3, T1-3N3 =10 pack-years) or high-risk LAHNSCC (oral cavity, laryngeal, hypopharyngeal, or HPV-negative oropharyngeal HNSCC, T1-2N2a-N3 or T3-4N0-3), nivolumab was added to each of four (C)RT cohorts in a concurrent and adjuvant setting.118–120 RT was delivered with either a weekly low-dose or three-weekly high-dose cisplatin, with cetuximab, or as monotherapy (Table 2). T he addition of nivolumab concurrently to all four (C)RT regimens was found safe. Levels of dose-limiting toxicity were acceptable and af­ter 17, 16, 10, and 6 months of median follow-up in each of the four RT cohorts there were 0/10 (RT plus weekly cisplatin), 1/9 (RT plus three-weekly cisplatin), 1/10 (RT plus cetuximab), and 3/10 (RT only) events (i.e. death or disease progression), re­spectively. However, adjuvant administration of nivolumab was infeasible after (C)RT in cisplatin-ineligible patients or in those who received high-dose three-weekly concurrent cisplatin. Data from the first 12 patients (planning to enrol 24 patients) from the NCT03162731 phase I trial, adding nivolumab and ipilimumab to standard RT in high-risk LAHNSCC, were also presented.121 After a follow-up of 7.2–18.4 months, 10 of the 12 patients are alive with no evidence of disease. Major toxicities are presented in Table 2. Elbers et al. recently reported results from their phase I trial (NCT02938273) in 10 cisplatin ineligi­ble patients with LAHNSCC that received avelum-ab and cetuximab in conjunction with RT, followed by avelumab as a maintenance therapy for an addi­tional four months (Table 2).122 After a median fol­low-up of 12 months disease recurred in 50% of the patients. The majority of adverse effects were re­lated to RT and cetuximab; grade 3 irAE occurred in four patients and were successfully managed. An innovative approach is used in the CheckRad-CD8 phase II trial (NCT03426657) in which 120 patients with LAHNSCC have a second biopsy after induction durvalumab, tremelimum-ab, cisplatin, and docetaxel therapy. In the case of increased CD8+ TILs compared to pre-treatment biopsy, patients receive concurrent durvalumab, tremelimumab, and RT. Non-responders continue with standard therapy outside of the trial. The interim analysis for the first 10 patients was pre­sented in 2019. After induction therapy re-biopsies showed a complete pathological response in 8/10 patients with another two patients showing an in- 386 crease in CD8+ TILs. There were two cases of grade III-IV toxicity: hepatitis and infectious diarrhoea.123 Further results are awaited. There are an additional 16 ongoing trials em­ploying a combination of RT and ICIs that have not presented their results yet. Two of these are randomized phase III studies. The first one, KEYNOTE-412, will hopefully provide robust data to clarify the role of anti-PD-1 agent pembrolizum-ab given concomitantly with CRT and as a main­tenance therapy in patients with locally advanced HNSCC.124 The interpretation of the results could be hindered by the inability to discern the distinct effects of the priming, concurrent, and maintenance applications of pembrolizumab. Notably, a similar international phase III trial has previously been terminated due to slow accrual, and another simi­lar trial, JAVELIN Head and Neck 100, testing the addition of anti-PD-L1 agent to CRT in LAHNSCC was terminated due to inefficiency.109,125 An addi­tional phase III trial, NRG-HN005, is a non-inferior­ity trial, testing treatment de-escalation in patients with early stage HPV-positive oropharyngeal car­cinoma.126 A reduced dose RT, concurrently with either cisplatin or nivolumab, will be compared to standard CRT with cisplatin. The results will add valuable information to expanding pool of knowl­edge from the de-escalation trials in patients with HPV-positive HNSCC. A somewhat different approach will be exam­ined in the NCT04369937.127 HPV-16 E6/E7-specific therapeutic vaccination (ISA101b) will be adminis­tered to 50 patients with intermediate risk of HPV+ HNSCC one week prior to the start of pembroli­zumab and two weeks prior to the start of CRT with cisplatin (Table 2). The combination of ISA101 and nivolumab was already examined in a single-arm phase II trial where 24 patients with incurable HPV-positive cancers (22 oropharyngeal and one cervical and one anal cancer) were enrolled. An overall response rate of 33% with a median dura­tion of response of 10.3 months and a median over­all survival of 17.5 months seemed promising.128 REWRITe (NCT03726775), a phase II trial that started in 2018, follows the recommendations from preclinical studies about omitting extended elec­tive nodal irradiation when combining RT with im­munotherapy. In this trial, patients with early stage T1–2 HNSCC or those with T3–4 disease and who are ineligible for cisplatin or cetuximab concurrent­ly with RT will simultaneously receive durvalum-ab and RT to the primary tumour and immediately adjacent lymph nodes only. This will be followed by six months of maintenance durvalumab.129 NCT02777385 is a phase II trial, planning to ran-domise 90 patients with LAHNSCC to either con­current CRT with cisplatin and pembrolizumab or to CRT followed by pembrolizumab (Table 2).130 It will hopefully help to answer if concurrent appli­cation is better than sequential or vice versa. Adjuvant (postoperative) immunoradiotherapy Testing novel treatments in an adjuvant setting offers a unique opportunity to stratify operated patients by risk of recurrence based on a detailed histopathological report, and therefore to avoid overtreatment. However, one should be aware of the above-described disadvantages when using immunotherapy with or without concurrent radio­therapy in patients with resected draining lymph nodes or after intensive treatment. Two trials testing the potentials of adjuvant im­munoradiotherapy reported early results. Wise-Draper et al. presented results of the lead-in stage of their phase II trial (NCT02641093). One to three weeks before planned surgery, patients who were clinically at high risk (cT3/4 stage and/or =2 +LNs) had one priming application of pembrolizumab followed by risk adjusted administration of adju­vant pembrolizumab in combination with RT or CRT. The pathological response to priming appli­cation of pembrolizumab was seen in 47% and was correlated with increased TILs. Adjuvant combina­tion treatment with pembrolizumab and RT/CRT has an acceptable safety profile (Table 3).142 The other trial is a phase I NRG-HN003 trial that was conducted with the aim of determining a sched­ule for a phase II study. The tested regimen con­sisted of pembrolizumab added to adjuvant RT in patients with previously resected HPV-negative HNSCC with microscopically positive margins or an extracapsular extension of nodal metastases.143 Pembrolizumab administered every three weeks in a dose of 200 mg for eight doses, starting the week before adjuvant CRT, was declared as worth pur­suing. irAE were rare and non-significant (Table 3). Beside these, there are six more ongoing trials registered in the international databases delivering different concurrent immunoradiotherapy combi­nations in an adjuvant setting and three of them are randomised phase 3 trials. The experimental arm in KEYNOTE-689 (NCT03765918) is similar to the one in trial by Wise-Draper et al., except that two cycles of neoadjuvant pembrolizumab will be administered and longer maintenance therapy with pembrolizumab is planned. This will be com- 387 TABLE 3. Trials utilizing adjuvant immunoradiotherapy NCT02641093,142 2016 II 80 LAHNSCC neoadj. PEMBRO followed by resection, followed by PEMBRO + (C)RT PEMBRO 1 week prior to surgery and conc. with RT for total of 7 doses IMRT (60–66Gy/30fx) + /- LD-CDDP (if ECE + /R1) first 23 pts (lead-in phase): 47% pathological response, no DLT, 2 pts recurred NRG-HN003 (NCT02775812),143 2016 I 34 resected R1/ECE + HPV- HNSCC adj. PEMBRO + CRT 3 different schedules aimed to determine phase II schedule CRT with IMRT (60Gy/30fx) and LD-CDDP No irAE unacceptably delayed RT, 50% got all 8 doses of PEMBRO KEYNOTE-689 (NCT03765918),144,145 2018 III, rand. 600 resected LAHNSCC arm A: neoadj. PEMBRO followed by resection then PEMBRO + (C)RT; arm B: resection then (C)RT arm A: 2x neoadj. PEMBRO and PEMBRO conc. with adj. (C) RT, followed by PEMBRO for up to 15 cycles (C)RT 60–70Gy/30–35fx + /- HD-CDDP depending on risk factors NA GORTEC 2018-01 “NIVOPOSTOP” (NCT03576417),146 2018 III, rand. 680 resected R1/ECE + LAHNSCC arm A: adj. NIVO + CRT; arm B: adj. CRT NIVO starting 3 weeks before CRT for total of 4 doses CRT with IMRT (66Gy/33fx) and HD-CDDP NA ADHERE (NCT03673735),147 2019 III, rand. 650 resected HR HPV- HNSCC arm A: adj. DURVA + CRT; arm B: adj. CRT 1 dose of DURVA 1 week prior to CRT and maint. DURVA for 6 CRT 66Gy/33fx and HD-CDDP NA doses ADRISK (NCT03480672),149 2018 II, rand. 240 resected LAHNSCC with >1LN/ECE + /R1 arm A: adj. PEMBRO + CRT; arm B: adj. CRT PEMBRO conc. with RT and for up to 12 months CRT with CDDP NA NCT03715946,150 2018 II 135 resected IR­HR HPV + adj. NIVO + deescalated RT NIVO conc. with RT and for RT (45–50Gy/25fx) NA oropharyngeal cancer additional 6 doses after RT adj. = adjuvant; CDDP = cisplatin; conc. = concurrent; CRT = chemoradiotherapy; DLT = dose-limiting toxicity; DURVA = durvalumab; ECE+ = extracapsular extension of metastasis in lymph node; fx = fractions; HD-CDDP = high dose cisplatin 100 mg/m2 every three weeks during RT; HPV+ = human papilloma virus associated cancer; HPV- = human papilloma virus negative cancer; HR = high-risk; IMRT = intensity modulated RT; IR = intermediate-risk; irAE = immune-related adverse effects; LAHNSCC = locally advanced head and neck squamous cell carcinoma; LD-CDDP = low dose cisplatin 40 mg/m2 every week during RT; N = planned enrolment; neoadj. = neoadjuvant; NIVO = nivolumab; PEMBRO = pembrolizumab; RT = radiotherapy; R1 = microscopically positive resection margin, LN = lymph node; NA = not available pared to standard adjuvant CRT in LAHNSCC patients with either more than one pathological lymph node, microscopically positive margins or an extracapsular extension of nodal metasta­ses.144,145 The two other randomised phase III trials, GORTEC 2018-01 (NCT03576417, also known as NIVOPOSTOP)146 and ADHERE (NCT03673735)147, will both enrol patients with resected high-risk HNSCC and randomise them to either adjuvant CRT with concurrent nivolumab (NIVOPOSTOP)/ durvalumab (ADHERE), or to standard of care ad-juvant CRT. Th ese three phase III trials could set ground for the new era in the setting of adjuvant treatment of a high-risk HNSCC based on patho­logical data (microscopically positive margins or extracapsular extension of nodal metastases). Currently, with adjuvant CRT locoregional relapse rates as well as distant metastases rates at five years are around 20% in these patients.102,148 Based on the preclinical data described above, it would be reasonable to expect a synergistic locoregional ac­tivity of radioimmunotherapy. A major drawback of adding immunotherapeutics to RT in postopera­tive setting could be the absence of regional lymph nodes that could hinder the efficacy of this com­bination. Nevertheless, ICIs will be delivered in doses that were shown to be effective systemically, therefore, it is justified to expect improved distant control of the disease.8,10 The other three phase I and phase II trials are presented in Table 3. A djuvant/maintenance therapy with immune checkpoint inhibitor In several of the above-described trials anti-PD-1/ L1 therapy is also applied as a prolonged adjuvant or maintenance therapy. Support for this approach 388 comes from two other tumour types. In patients with unresectable locally-advanced non-squamous cell carcinoma lung cancer (NSCLC) without pro­gression after definitive CRT, consolidation dur­valumab was shown to prolong survival.152 Also, after a complete resection of stage III melanoma, adjuvant ipilimumab prolonged overall survival compared to placebo, while adjuvant nivolumab compared head-to-head to adjuvant ipilimumab showed better relapse-free survival and less toxic­ity. Long-term data of the latter study are not yet available.153,154 Besides differences in tumour-in­trinsic factors and the composition of their TME, another important aspect to consider is the dif­ferent recurrence pattern of these tumours. While melanoma and NSCLC are prone to dissemination, HNSCC tends to recur more often locoregionally in previously treated tissue. After resection alone, stage III melanoma spreads to distant sites in more than 60% of cases, and stage III NSCLC relapses distantly after CRT alone in up to 50% of cases.154,155 On the other hand, the risk of distant metastases is around 15% in HNSCC, whereas isolated lo-coregional relapses are much more common.4,156 Whether consolidation anti-PD-1/L1 agents can de­crease rates of distant metastases as well as locore­gional relapses in HNSCC is still to be determined. Another important consideration in prolonged treatment with anti-PD-1/L1 agents is toxicity. Even though the overall effect on the quality of life with anti-PD-1 agents in R/M HNSCC was found to be positive and there were fewer adverse effects compared to standard chemotherapy, irAE never­theless occurred in around 60% of patients with 17% of them experiencing a grade 3 or higher toxic event.22,157 Prolonged treatment with anti-PD-1/L1 agents should therefore be approached carefully and weighted against its toxicity. It should not be ignored that there is also financial toxicity associ­ated with these treatments. It was estimated that in CheckMate 141 the incremental cost-effectiveness ratio per quality-adjusted life year for nivolumab was around 90,000 euros.158 Even if the methods used in such calculations had some flaws, the fi­nancial burden of these new drugs is obvious and therefore special attention should already be paid in trial design.158 Importantly, with the above-de­scribed trials it will be hard to discern the benefit of concurrent immunoradiotherapy from the benefit of maintenance immunotherapy as none of these trials compares this extended adjuvant treatment to a comparator arm without it. In either case, care­ful patient selection for immunotherapy, probably biomarker driven, will help to prevent unneces­sary additional toxicity and the financial burden of this treatment. Potential biomarkers for immuno-therapy in HNSCC have recently been extensively reviewed by Gavrielatou et al.159 Conclusions Researchers pursue different strategies in using a RT-ICI combination in a non-R/M HNSCC setting and the first results are already available. Window of opportunity trials are most welcomed since bio­logical mechanisms behind the synergistic effect of combined immunoradiotherapy are not fully un­derstood and reliable criteria for patient selection are lacking. The first results of these trials that use immunoradiotherapy neoadjuvantly are encourag­ing. In a definitive setting results are more varied. A large phase III trial employing concurrent and maintenance avelumab for 12 months post-chemo-radiotherapy was terminated because of inefficacy. Prolonged RT courses with large treatment fields and high doses of concomitant chemotherapy agents could be detrimental to the success of im­munotherapy. In an adjuvant setting it is hard to overlook factors such as a changed anatomy of lym­phatics and a changed microenvironment of possi­ble remaining cancer cells due to previous surgery, which could both adversely affect the effective­ness of immunoradiotherapy. Additionally, many of these trials administer anti-PD-1/L1 agents not only concurrently with RT but also as prolonged adjuvant treatment, without a comparator arm for proper evaluation of this approach. However, im­munoradiotherapy is evolving rapidly in HNSCC and final results of the herein presented ongoing trials are eagerly awaited. Acknowledgments This study was funded by the Slovenian Research Agency (program no. P3-0307). References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality world­wide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394-424. doi: 10.3322/caac.21492 2. 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Adjuvant ipilimumab versus placebo after complete re­section of stage III melanoma: long-term follow-up results of the European Organisation for Research and Treatment of Cancer 18071 double-blind phase 3 randomised trial. Eur J Cancer 2019; 119: 1-10. doi: 10.1016/j. ejca.2019.07.001 155. Steuer CE, Behera M, Ernani V, Higgins KA, Saba NF, Shin DM, et al. Comparison of concurrent use of thoracic radiation with either carbo­platin-paclitaxel or cisplatin-etoposide for patients with stage III non– small-cell lung cancer. JAMA Oncol 2017; 3: 1120. doi: 10.1001/jamaon­col.2016.4280 156. Duprez F, Berwouts D, De Neve W, Bonte K, Boterberg T, Deron P, et al. Distant metastases in head and neck cancer. Head Neck 2017; 39: 1733-43. doi: 10.1002/hed.24687 157. Wang H, Mustafa A, Liu S, Liu J, Lv D, Yang H, et al. Immune checkpoint inhibitor toxicity in head and neck cancer: from identification to manage­ment. Front Pharmacol 2019; 10: doi: 10.3389/fphar.2019.01254 158. 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Cancer Treat Rev 2020; 84: 101977. doi: 10.1016/j.ctrv.2020.101977 394 research article Correlations between DTI-derived metrics and MRS metabolites in tumour regions of glioblastoma: a pilot study Eduardo Flores-Alvarez1 , Edgar-Anselmo Rios-Piedra2, Griselda-Adriana Cruz-Priego3, Coral Durand-Muz4, Sergio Moreno-Jimenez5, Ernesto Roldan-Valadez6,7 1 Department of Neurosurgery, General Hospital of Mexico, Secretariat of Health, Mexico City, Mexico 2 Department of Radiology, Stanford University, CA, USA; Department of Electrical Engineering, Stanford University, CA, USA 3 Postgraduate Unit, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico 4 Department of Internal Medicine, Medica Sur Clinic and Foundation, Mexico City, Mexico 5 Radioneurosurgery Unit, The National Institute of Neurology and Neurosurgery, Mexico City, Mexico 6 Department of Radiology, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia 7 Directorate of Research, General Hospital of Mexico, Secretariat of Health, Mexico City, Mexico Radiol Oncol 2020; 54(4): 394-408. Received 9 July 2020 Accepted 31 July 2020 Correspondence to: Ernesto Roldan-Valadez, M.D., M.Sc., D.Sc., Department of Radiology, I.M. Sechenov First Moscow State Medical University (Sechenov University), Trubetskaya str., 8, b. 2, 119992, Moscow, Russia. Email: ernest.roldan@usa.net Disclosure: No potential conflicts of interest were disclosed. Introduction. Specific correlations among diffusion tensor imaging (DTI)-derived metrics and magnetic resonance spectroscopy (MRS) metabolite ratios in brains with glioblastoma are still not completely understood. Patients and methods. We made retrospective cohort study. MRS ratios (choline-to-N-acetyl aspartate [Cho/NAA], lipids and lactate to creatine [LL/Cr], and myo-inositol/creatine [mI/Cr]) were correlated with eleven DTI biomarkers: mean diffusivity (MD), fractional anisotropy (FA), pure isotropic diffusion (p), pure anisotropic diffusion (q), the total magnitude of the diffusion tensor (L), linear tensor (Cl), planar tensor (Cp), spherical tensor (Cs), relative anisotropy (RA), axial diffusivity (AD) and radial diffusivity (RD) at the same regions: enhanced rim, peritumoral oedema and nor-mal-appearing white matter. Correlational analyses of 546 MRS and DTI measurements used Spearman coefficient. Results. At the enhancing rim we found four significant correlations: FA . LL/Cr, Rs = -.364, p = .034; Cp . LL/Cr, Rs = .362, p = .035; q . LL/Cr, Rs = -.349, p = .035; RA . LL/Cr, Rs = -.357, p = .038. Another ten pairs of significant correla­tions were found in the peritumoral edema: AD . LL/Cr, AD . mI/Cr, MD . LL/Cr, MD . mI/Cr, p . LL/Cr, p . mI/ Cr, RD . mI/Cr, RD . mI/Cr, L . LL/Cr, L . mI/Cr. Conclusions. DTI and MRS biomarkers answer different questions; peritumoral oedema represents the biggest chal­lenge with at least ten significant correlations between DTI and MRS that need additional studies. The fact that DTI and MRS measures are not specific of one histologic type of tumour broadens their application to a wider variety of intracranial pathologies. Key words: brain neoplasms; diffusion tensor imaging; magnetic resonance spectroscopy; statistics as topic; soft­ware tools Introduction Since the last decade, a particular interest prevails for the identification of clinical prognostic mark­ers for glioblastoma.1 During this time, medical imaging research has focused its attention in the conventional magnetic resonance imaging (MRI) diagnosis of gliomas, identifying regional tu­mour infiltration and oedema boundaries in those qualitative patterns observed in the T2-weighted imaging (T2-w), fluid-attenuated inversion recov­ery (FLAIR), pre-contrast T1-w weighted imaging Flores-Alvarez E et al. / Correlations between diffusion tensor imaging -derived metrics and MRS metabolites 395 (T1-w), and post-contrast T1-w.2 Other MRI-based quantitative morphological features that have been reported include the contrast-enhancing (CE) rim width and surface regularity3, residual tumour volume (RTV) and extent of resection (EOR).4 A recent meta-analysis highlighted the limitations of the current conventional MRI-based Response Assessment in Neuro-Oncology (RANO) criteria for treatment evaluation in glioblastoma.5 Some volumetric features of the oedema region might have a role as predictors of progression-free survival (PFS) in patients with glioblastoma.6 Diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) biomarkers are currently reported in glioblastoma research as a consequence of their higher diagnostic accuracy than conventional MRI for the detection of tumour progression.7,8 A recent meta-analysis found the sensitivity and specificity of MRS were 91% and 95%, respectively.9 MRS found that the choline-to-N-acetyl aspartate (Cho/Naa) ratio is the most substantial survival predictor in glioblastoma with a log-hazard function of 2.672 (each unit of increase in the Cho/Naa ratio represents a 267% increase in the risk of death in glioblastoma).10 The usefulness of DTI-derived biomarkers has been proved in the differentiation of glioblastoma from brain abscess­es and metastatic brain tumours11 and between glioblastoma and healthy brains.12 Up to 11 DTI-derived biomarkers have calculated in brain MRI, each one with different diagnostic performance de­pending on the selected tumour region.13 However, despite the above technological ad­vances in glioblastoma imaging, there is a low cor­relation between the conventional MR images and the gross pathologic margin of the tumour with the actual margins of the areas of neoplastic infiltra­tion.14 Most of the advanced MRI techniques have been reported as separated diagnostic methods without a correlational assessment.5 For exam­ple, some studies have been published about the whole brain MRS correlations with Sox2-positive cell density8, but no with other advanced MRI tech­niques. We found only one article in the literature that studied the correlations between DTI and MRS in schizophrenic patients and healthy controls.15 Although it is known that MRS and DTI use dif­ferent mechanisms to visualizer abnormal patholo­gies, they can provide complementary imaging data on white matter changes in brain.15 The assessment of MRS and DTI biomarkers in glioblastoma is one of the leading research lines for our group. To the best of our knowledge, no previous studies have evinced a correlation among these variables; we aimed to analyse the correla­tions between the three most commonly reported MRS metabolites ratios and the eleven-known DTI-derived metrics in glioblastoma. Our null hy­pothesis considered no correlations between MRS metabolite ratios and DTI metrics; our alternative hypothesis expects that at least one pair of signifi­cant correlations were found at each tumour region in glioblastoma. Patients and methods Patients Retrospective cohort of patients with at first (sus­pected) diagnosis and later pathology confirmation of glioblastoma according to the WHO; inclusion criteria considered examinations between January 2010 and December 2014. Exclusion criteria applied to corticosteroid or antibiotic treatment, lesions with areas related to calcification and haemorrhage and previous brain surgery. MR examinations with other structural abnormalities were excluded. The local Institutional Review Board approved the study and the study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Brain image acquisition MR was performed by using a 3T unit (Signa HDxt, GE Healthcare, Waukesha, WI, USA) with a high-resolution eight-channel head coil (Invivo, Gainesville, FL, USA). MR sequences included conventional axial T2-w, axial Fluid-Attenuated Inversion Recovery (Flair), and pre-contrast axial T1-w. Post-contrast axial T1-w used 0.1 mmol/kg of body weight of gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany). Pre-contrast axial Spoiled Gradient Echo (SPGR) that exploited the T1 shortening effects of methemo­globin allowed direct visualization of lesions with haemorrhage. Diffusion-weighted imaging (DWI) was performed using a single-shot SE EPI sequence with b-values of 1000 s/mm2 and an image without diffusion weighting with b-value of 0 s/mm2. DTI was performed using a single-shot SE EPI sequence. Diffusion gradients were applied in 25 directions with b-values of 1000 s/mm2 and an im­age without diffusion weighting with b-value of 0 s/mm2. DTI sequences were acquired in the axial plane with 44 contiguous sections, 2.4 mm section thickness, no intersection gap, TR/TE of 17,000/80 ms, with parallel imaging to reduce off-resonance 396 artefacts (PI factor was 2); 25 x 25 cm FOV, and 128 x 128 matrix size. Selected tumour regions A board-certified radiologist (ERV) blinded to the clinical history of each patient, manually traced the boundaries of the tumour regions. For all param­eters derived from MRS and DTI, measurements were acquired in three areas: normal-appearing white matter (NAWM), drawn in the patient’s con-tralateral hemisphere; viable tumour region (area of the enhanced rim at T1-w post-contrast); and peritumoral oedema (arbitrarily chosen as an ad­jacent immediate zone with a 10-mm-wide band). Metabolites measurements using MRS Multi-voxel spectroscopic imaging (MV-MRS) was performed using a point-resolved spectroscopic sequence technique (PRESS). The volume of inter­est (VOI) size was individually adjusted position­ing the voxel over the lesion and trying to mini-mise partial-volume effects resulting from other neighbouring tissues including bones and cerebral spinal fluid (CSF) of the ventricles. Proton spectra were recorded in the axial plane with T1-w post-contrast images via TR; 1500 ms, TE; 26 and 144 ms, FOV; 24 × 24 cm, 1–1.5 cm section thickness, 256 × 256 matrix and 24 × 24 phase encoding. Knowing that cerebral metabolites have different inherent T1 and T2 relaxation times, a TE of 24 ms allowed us to quantify metabolites that are identified only at short TE (Lipids and Myo-inositol). The interme­diate TE of 144 ms let us identified the Cho and Lactate peaks, which are the primary metabolites altered in neoplasms. Because fewer metabolites were observed with longer TE values, the spectrum obtained is easier to interpret (we could quickly identify the rest of selected metabolites (NAA and Cr). Additionally, a TE of 144 ms identified the Lactate peak invert below baseline.16 The MRS data were transferred to a clini­cal workstation, with FDA-cleared software (GE Advantage). A short echo time allowed the acqui­sition of four brain spectra with metabolite signal peaks centred within a range of 0–4.35 ppm as fol­lows: methyl protons of N-acetylaspartate (NAA) at 2.0 ppm, N-trimethyl protons of choline-con­taining metabolites at 3.2 ppm (Cho), creatine (Cr) at 3–3.1 ppm, a compound peak containing lipids and lactate (LL) at 0.8–1.4 ppm, and a compound peak of the protons of myo-inositol (mI) at 3.56 and 4.06 ppm.16 Automatic shimming of the linear x, y, z channels was used to optimise field homogene­ity, water resonance and water suppression pulses were optimised. Relative quantification of metabo­lites was performed after Gaussian curve fitting using standard spectroscopic analysis software FuncTool 9.4.04b, (GE Healthcare, Milwaukee, WI, USA). Three metabolite ratios were calculated: Cho/NAA, lipids and lactate to creatine (LL/Cr), and and myo-inositol/creatine (mI/Cr). Figure 1 A–F show examples of the MRS measurements at the enhancing rim and peritumoral oedema. DTI-derived metrics We used the FA maps, and T1-post gadolinium orientation maps to draw three regions of inter­est (ROI) from each selected region (NAWM, en­hancing rim and peritumoral oedema). For each ROI, we obtained the major (.1), intermediate (.2), and minor (.3) eigenvalues at the selected re­gions using a GE Advantage Workstation with the software FuncTool 9.4.04b (GE Medical Systems, Milwaukee, WI, USA). The three eigenvalues were applied to the eleven formulas previously pub­lished for the calculation of DTI-derived metrics: mean diffusivity (MD), fractional anisotropy (FA), pure isotropic diffusion (p), pure anisotropic diffu­sion (q), the total magnitude of the diffusion tensor (L), linear tensor (Cl), planar tensor (Cp), spherical tensor (Cs), relative anisotropy (RA), axial diffusiv­ity (AD) and radial diffusivity (RD)13; Figure 1 G–I presents an example of FA map used to locate the ROI at the selected regions: enhancing rim, peritu-moral oedema, and NAWM. Statistical analysis Sample size We used the sample-size formula published by Browner et al. for determining whether a correla­tion coefficient differs from zero.17 N = [(Z. + Z.) C]2 + 3, for this formula: N = Total number of measurements required Z. = the standard normal deviate for . (If the alternative hypothesis is two-sided, Z. = 1.96 when . = 0.05) Z. = the standard normal deviate for . (Z. = 0.84 when . = 0.20) C = 0.5 × ln [(l + r)/( l – r)] r = expected correlation coefficient Considering that Tang et al. reported a correla­tion coefficient between DTI and MRS biomarkers up to 33.2% in schizophrenic patients15, our alter­native hypothesis was that correlation coefficients Flores-Alvarez E et al. / Correlations between diffusion tensor imaging -derived metrics and MRS metabolites 397 FIGURE 1. (A-F) magnetic resonance spectroscopy (MRS) measurements at the enhancing rim and peritumoral edema. (G-I) example of a FA map used to locate the ROI at the selected regions: enhancing rim, peritumoral oedema, and normal-appearing white matter (NAWM). between DTI and MRS biomarkers would be above 50%. With this expected correlation coefficient, a two-sided alternative hypothesis, . = 0.05, . = 0.20, and statistical power = 80%; N = 29. We had 33 dif­ferent measurements per each DTI biomarkers. Correlation analyses Bivariate correlations were performed using the Spearman correlation coefficient (Rs)18 to describe the degree of the linear relationship between three metabolites ratios (Cho/Naa, LL/Cr, and mI/Cr) and the eleven DTI-derived biomarkers (MD, FA, p, q, L, Cl, Cp, Cs, RA, AD and RD). We chose the Rs because it is a non-parametric test that can be used with variables that have a non-normal distri­bution.19 Each correlation coefficient was interpret­ed as Very strong (at least of 0.8), Moderately strong (0.6 up to 0.8), Fair (0.3 up to 0.6) and Poor (less than 0.3). Squaring R-values represented the coefficient of determination, the proportion of variance that each two compared variables had in common.18 We ad­ditionally tested the statistical significance of the difference between R coefficients between groups 398 Scatterplot Matrix Mean 5 di usivity 3 (MD) 1 -1 Pure 2.5 isotropic di usion (p) 1.0 -0.5 Pure 0.5 anisotropic di usion (q) 0.2 Total -0.1 magnitude 6 of the 4 di usion 2 tensor (L) 0 Fractional 0.35 anisotropy 0.30 (FA) 0.25 0.20 -0.010 Linear -0.020 tensor (Cl) -0.030 -0.040 -0.04 Planar -0.06 tensor (Cp) -0.08 -0.10 -0.12 1.12 Spherical tensor (Cs) 1.10 1.08 1.06 1.04 Relative 0.20 anisotropy 0.15 (RA) 0.10 0.05 Axial 4 3 di usivity 2 (AD) 1 0 Radial 5 di usivity 3 (RD) 1 -1 0.2 0.4 0.6 0.8 1 02468 10 12 05 10 Cho/Naa LL/Cr mI/Cr FIGURE 2. Scatter plots showing the correlation between magnetic resonance spectroscopy (MRS) metabolites and diffusion tensor imaging (DTI) metric at the normal-appearing white matter (NAWM). by converting each pair of R values into standard z scores, then using the formula proposed by Pallant and colleagues20: Observed Z value (Zobs) . -1.96 or . 1.96 were considered statistically significantly different. Software All analyses were carried out using the IBM. SPSS. Statistics software (version 26.0.0.1 IBM Corporation; Armonk, NY, USA) and JMP® Pro software (version 14.3, SAS Institute Inc., Cary, NC, USA). Statistical significance was indicated by p < 0.05 (two-tailed). Results DTI and MRS measurements For each patient, we recorded MRS and DTI measurements at three selected regions: NAWM, enhancing rim and oedema. The three MRS measures for each metabolite ratio (Cho/Naa, LL/ Cr, and mI/Cr) were recorded at all tumour re­gion, adding 9 MRS measurements per patient. Similarly, 11 DTI-derived metrics (MD, FA, p, q, L, Cl, Cp, Cs, RA, AD and RD) were calculated at each tumour region for each patient, with a to­tal of 33 DTI measurements. Then, for each pa­tient, we got 42 measurements (9 from MRS and 33 from DTI), this amount multiplied by 13 pa­tients added 546 measurements that integrated 33 MRS-DTI parameter pairs per region. A total of 99 bivariate pairs were obtained in our correlation analyses. DTI.MRS correlation at the NAWM We found five pairs of bivariate correlations showing statistical significance all of them with the same metabolite LL/Cr. Only one correlation was positive, Cp . LL/Cr, Rs = .468, p = .014. The other four depicted negative Rs coefficients: FA . LL/Cr, Rs = -.475, p = .012; q . LL/Cr, Rs = -.495, p = .009; RA . LL/Cr, Rs = -.490, p = .010; Cs . LL/ Cr, Rs = -.488, p = .010. Table 1 shows the correla­tions between DTI metrics and MRS metabolites at the NAWM region. Figure 2 depicts a scatter-plot matrix of the DTI and MRS correlations at the NAWM region. DTI.MRS correlation at the gadolinium-enhanced tumour region Similar to the findings in the NAWM, we found only four significant correlations between only one MRS metabolite and 4 DTI-derived metrics: FA . LL/Cr, Rs = -.364, p = .034; Cp . LL/Cr, Rs = .362, p = .035; q . LL/Cr, Rs = -.349, p = .035; RA . LL/Cr, Rs = -.357, p = .038. Table 2 depicts the correlations between DTI metrics and MRS metabolites at the tumor region. Figure 3 show a scatterplot matrix of the DTI and MRS correla­tions at the enhancing rim region. Flores-Alvarez E et al. / Correlations between diffusion tensor imaging -derived metrics and MRS metabolites 399 TABLE 1. Correlations between diffusion tensor imaging (DTI) metrics and magnetic resonance spectroscopy (MRS) metabolites for the normal-appearing white matter (NAWM) region Cho/Naa -0.2862 0.1479 Axial diffusivity (AD) LL/Cr 0.1900 0.3426 mI/Cr -0.1777 0.3751 Cho/Naa 0.2300 0.2485 Fractional anisotropy (FA) LL/Cr -0.4749 0.0123* mI/Cr -0.2110 0.2907 Cho/Naa -0.2827 0.1530 Linear tensor (Cl) LL/Cr 0.2061 0.3024 mI/Cr -0.2147 0.2822 Cho/Naa -0.1441 0.4732 Planar tensor (Cp) LL/Cr 0.4680 0.0138* mI/Cr 0.3139 0.1108 Cho/Naa -0.0961 0.6336 Pure isotropic diffusion (p) LL/Cr -0.1020 0.6126 mI/Cr -0.2683 0.1761 Cho/Naa 0.2217 0.2665 Relative anisotropy (RA) LL/Cr -0.4898 0.0095* mI/Cr -0.2290 0.2506 Cho/Naa -0.0680 0.7363 LL/Cr -0.1408 0.4836 mI/Cr -0.2781 0.1602 Cho/Naa = choline-to-N-acetyl aspartate; LL/Cr = lipids and lactate to creatine; mI/Cr = and myo-inositol/creatine [mI/Cr] 400 DTI.MRS correlation at the oedema region At the edema region we found that besides the LL/ Cr metabolite, the concentrations of mI/Cr also de­picted statistical significance with five DTI metrics different than the observed correlations in the tu­mor and NAWM regions. It meant we found ten significand correlations: AD . LL/Cr, Rs = .658, p < .001; AD . mI/Cr, Rs = .493, p = .006; MD . LL/Cr, Rs = .685, p < .001; MD . mI/Cr, Rs = .513, p = .004; p . LL/Cr, Rs = .685, p < .001; p . mI/Cr, Rs = .513, p = .004; RD . mI/Cr, Rs = .693, p < .001; RD . mI/Cr, Rs = .508, p = .004; L . LL/Cr, Rs = .685, p < .001; L . mI/Cr, Rs = .513, p = .004. Table 3 presents the corre­lations between DTI metrics and MRS metabolites at the edema region. Figure 4 show a scatterplot matrix of the DTI and MRS correlations at the peri­tumoral edema. Figure 5 depicts a diagram show­ing the significant correlations observed between DTI-MRS bivariate correlations at the NAWN, tu­mor and edema regions. Statistical significance between identical DTI-MRS bivariate pairs in different regions The assessment of the statistical significance of the difference between R coefficients found only four pairs of DTI-MRS correlations that were coinciden­tally significant at NAWM and tumor enhanced regions (Figure 4). We did not find statistical sig­nificances between their R coefficients: Cp . LL/ Cr, Z = .54, p = .589; FA . LL/Cr, Z = .57, p = .568; q . LL/Cr, Z = .76, p = .447; RA . LL/Cr, Z = .69, p = .490. Discussion Between 1998 and 2009, quantitative biomark­ers from MRS (NAA, Cho, LL, and mI) were ac­cepted to be measured with sufficient sensitivity in the millimoles per litre range to be used in clinical diagnosis.21 Recent studies have shown the impor­tance of Cho/NAA and LL/Cr ratios in assembling significant survival models in glioblastoma.10 The use of DTI allows diffusion directionality to be quan­tified as different DTI-derived metrics21; it yields ultrastructural information on cellular density and properties of the extracellular matrix.22 In 2006, Pena et al. expressed that it was not completely un­derstood the magnitudes and associations among DTI measurements observed in the evaluation of brain tumours.23 Cortez-Conradis et al. in 2015, evaluated correlations among DTI-derived metrics in glioblastoma24, but without exploring the asso­ciations with MRS metabolites in the same tumour regions. In this study, we were able to probe the alter­native hypothesis posed at the introduction and methods sections: bivariate correlations among DTI-metrics and MRS metabolite ratios are sig­nificant at selected tumour regions and above 50% of Rs value in glioblastoma (NAWM, enhancing rim and peritumoral oedema). To the best of our knowledge, there are no similar studies in the lit­erature with whom compare our findings. Flores-Alvarez E et al. / Correlations between diffusion tensor imaging -derived metrics and MRS metabolites 401 TABLE 2. Correlations between diffusion tensor imaging (DTI) metrics and magnetic resonance spectroscopy (MRS) metabolites for the tumour region Cho/Naa -0.0961 0.5886 Axial diffusivity (AD) LL/Cr 0.2044 0.2463 mI/Cr -0.0824 0.6432 Cho/Naa 0.0165 0.9262 Fractional anisotropy (FA) LL/Cr -0.3643 0.0342* mI/Cr -0.1238 0.4855 Cho/Naa 0.0017 0.9924 Linear tensor (Cl) LL/Cr 0.0674 0.7048 mI/Cr 0.0395 0.8246 Cho/Naa -0.1699 0.3369 Planar tensor (Cp) LL/Cr 0.3629 0.0349* mI/Cr 0.0604 0.7342 Cho/Naa -0.1152 0.5167 Pure isotropic diffusion (p) LL/Cr 0.0790 0.6569 mI/Cr -0.1713 0.3327 Cho/Naa 0.0200 0.9105 Relative anisotropy (RA) LL/Cr -0.3569 0.0382* mI/Cr -0.1241 0.4843 Cho/Naa -0.1232 0.4877 LL/Cr 0.0799 0.6532 mI/Cr -0.1606 0.3643 Cho/Naa = choline-to-N-acetyl aspartate; LL/Cr = lipids and lactate to creatine; mI/Cr = and myo-inositol/creatine Scatterplot Matrix ent mechanisms of damage by glioblastoma, to­gether they provide complementary imaging data 5 Mean 3 on white matter integrity in brain. The supplemen­ di usivity (MD) 1 tary information provided by DTI and MRS is what -1 we consider the rationale of our study, both tech- Pure 3 isotropic 2 niques should complement the information from di usion (p) 1 conventional MRI in day-to-day practice. The clini­ 0 cal implications will allow researchers to combine Pure 0.5 DTI and MRS metrics to test several prediction anisotropic 0.2 di usion (q) models for tumour progression or the presence of -0.1 Total tumour cells in peritumoral oedema and decrease 8 magnitude 6 the patient-to-patient prognostic variability. For of the 4 di usion 2 example, you could combine the variables of two tensor (L) 0 significant bivariate pairs with Rs > 65% in our Fractional 0.30 study (for example AD . LL/Cr and RD . mI/Cr anisotropy 0.25 (FA) 0.20 measured in peritumoral oedema) together with 0.15 0.10 age, in a Cox’s proportional-hazards regression -0.005 Linear -0.015 model for prediction of survival. The results might tensor (Cl) -0.025 be compared with previously published models.10 -0.035 To simplify the discussion of our findings, we -0.02 Planar grouped them into four sections: -0.04 tensor (Cp) -0.06 -0.08 -0.10 Lack of significant correlations between 1.08 1.06 Spherical Cho/NAA and any of the 11 DTI tensor (Cs) 1.04 1.02 biomarkers in the three selected regions 1.00 Relative 0.15 This was the first finding that caught our atten- anisotropy 0.10 (RA) 0.05 tion. To explain this fact, we should remember that 0.00 Cho peak is the most complex, receiving contri­ 5 Axial butions from a range of choline-containing com­ 3 di usivity (AD) 1 pounds (acetylcholine, glycerophosphocholine, -1 phosphocholine, free choline, phosphatidylcho­ 5 Radial line and choline-plasmalogen); its concentration 3 di usivity (RD) 1 is frequently taken as an empirical marker of the -1 density and turnover of cell membranes.26 Because 01 234 0246810 0 0.51 1.52 Cho/Naa LL/Cr mI/Cr increased Cho may be seen in diverse pathologies like infarction (from gliosis or ischemic damage to FIGURE 4. Scatter plots showing the correlation between magnetic resonance myelin) or inflammation (glial proliferation); it is spectroscopy (MRS) metabolites and diffusion tensor imaging (DTI) metric at the peritumoral edema. considered to be nonspecific.26 NAA is present in the soma of neurons, in dendrites and axons, its regional variability is likely related to differences The clinical relevance of our findings is the sta-in neural architecture, population and density. A tistical evidence that DTI and MRS depict signifi-simple linear relationship of NAA with the mass of cant associations in glioblastoma. MRS measure-neurons has been considered unlikely given that it ments represent a biochemical profile of brains also reflects reversible metabolic changes.27 A high with glioblastoma: decreased N-acetylaspartate concentration of Cho has been observed in brain (NAA) is a putative indicator of persistent axonal tumours and in vitro tumour proliferation markers damage; increases of choline and myo-inositol with Cho/NAA ratio significantly more elevated correspond to glial proliferation, and elevated lac-in high-grade gliomas than in low-grade gliomas. tate has been associated with in.ammation.25 DTI However, threshold values are not well estab-metrics measure the amount of coherence of water lished.28 glioblastoma exhibit high choline-con-diffusion, which putatively reflects the amount of taining compound levels, especially in the tumour myelination in axonal bundles or the coherence of regions, Cho/NAA quantifies those lipid compo-fibre tracts.15 Although DTI and MRS reflect differ-nents, and the DTI-derived metrics evaluates ultra- Flores-Alvarez E et al. / Correlations between diffusion tensor imaging -derived metrics and MRS metabolites 403 TABLE 3. Correlations between diffusion tensor imaging (DTI) metrics and magnetic resonance spectroscopy (MRS) metabolites for the oedema region Cho/Naa 0.0913 0.6315 Axial diffusivity (AD) LL/Cr 0.6575 <.0001* mI/Cr 0.4926 0.0057* Cho/Naa 0.0939 0.6217 Fractional anisotropy (FA) LL/Cr -0.2817 0.1316 mI/Cr -0.1444 0.4465 Cho/Naa 0.0571 0.7645 Linear tensor (Cl) LL/Cr 0.1461 0.4412 mI/Cr -0.0161 0.9329 Cho/Naa -0.1556 0.4115 Planar tensor (Cp) LL/Cr 0.3295 0.0754 mI/Cr 0.2033 0.2813 Cho/Naa 0.1155 0.5435 Pure isotropic diffusion (p) LL/Cr 0.6845 <.0001* mI/Cr 0.5132 0.0037* Cho/Naa 0.1197 0.5286 Relative anisotropy (RA) LL/Cr -0.2294 0.2226 mI/Cr -0.1104 0.5615 Cho/Naa 0.1155 0.5435 LL/Cr 0.6845 <.0001* mI/Cr 0.5132 0.0037* Cho/Naa = choline-to-N-acetyl aspartate; LL/Cr = lipids and lactate to creatine; mI/Cr = and myo-inositol/creatine 404 FIGURE 5. Diagram representation of the significant correlations between diffusion tensor imaging (DTI)- magnetic resonance spectroscopy (MRS) biomarkers at the selected regions: normal-appearing white matter (NAWM), enhancing rim and peritumoral edema. Notice that NAWM and the enhancing rim share four pairs of biomarkers correlations; while in peritumoral oedema ten pairs of correlations were exclusive of that region. structural properties of water molecules and their movements, then the non-significant correlation. Significant correlations between four DTI metrics and LL/Cr at NAWM and enhancing tumour regions In our second group of findings, four significant correlations pairs (Cp . LL/Cr, FA . LL/Cr, q . LL/Cr, RA . LL/Cr) coincidentally appeared in the NAWM and the enhancing tumour regions. They showed some direction of correlation on both region: Three were negative (the more LL/Cr, the less concentration of FA, q and RA); and one posi­tive (LL/Cr and Cp increase or decrease in the same direction). To understand these relationships, we begin mentioning that creatine, Cr, is a marker of en­ergetic systems and intracellular metabolism; it is considered a stable metabolite for its relatively constant concentration and is used as an internal reference for calculating metabolite ratios.29 In the combined ratio, LL/Cr, lipid resonances frequently dominate, and lactate (that can be seen in all tu­mour grades) is mainly present at high levels in glioblastoma.30 About the four selected DTI metrics (Cp, FA, q, and RA) that assembled significant bivariate cor­relations with LL/Cr; FA measures the directional­ity of water diffusion (shape of the diffusion tensor in each voxel). FA values vary between 0 (isotropic diffusion) and 1 (infinite anisotropy).31 FA is de­creased in glioblastoma.11 Diffusion is anisotropic in white matter fibre tracts, as axonal membranes and myelin sheaths present barriers to the mo­tion of water molecules, in directions not parallel to their orientation. Reduced FA (water diffusion parallel to axonal tracts) is indicative of axonal de­generation.32 We found two articles in the last 15 years men­tioning the q biomarker: q is the anisotropic com­ponent of the diffusion tensor, with a marked decrease of q in disrupted tracts; q-value in the low-grade tumours is slightly higher than in high-grade tumours, although this is not significantly different.33 In 2006 Price et al. conclude that q may provide a complete picture of the diffusion profile of a brain tumour.34 Cp is the planar, geometric representation of the diffusion tensor, and since one decade has been used in the differential diagnosis among abscess­es, glioblastomas, and metastases.11 Mean values of Cp have been quantified at the enhancing rim, peritumoral oedema and NAWM regions.13 RA is a ratio of the normalised standard devia­tions between the anisotropic part of the diffusion coefficient and its isotropic part35; it is a function of the variance of the eigenvalues of the diffusion tensor, which is not equal to the variance of the dif­fusivities along with all directions.36 It was not sur­prising to find significant correlations of RA and LL/Cr in NAWM, as it has been reported as one of the best biomarkers to characterise NAWM.13 Cs . LL/Cr, the only significant correlations exclusive of NAWM Cs and LL/Cr depicted a negative correlation, meaning the increase or decrease in opposite direc­tions. Cs describes the spherical, geometric prop­erties of the diffusion tensor11; after RA, Cs is the second DTI metric with the best diagnostic perfor­mance to characterise the NAWM.13 It is not clear for us why Cs . LL/Cr, was the only significant correlation observed at the NAWM, but not ob­served in peritumoral oedema and enhancing rim. Significant bivariate correlations exclusive of the peritumoral region In our fourth and last group of observations, we found ten significant bivariate correlations only observed in that region (AD . LL/Cr, MD . LL/ Flores-Alvarez E et al. / Correlations between diffusion tensor imaging -derived metrics and MRS metabolites 405 Cr, p . LL/Cr, RD . LL/Cr, L . LL/Cr, AD . mI/ Cr, MD . mI/Cr, p . mI/Cr, RD . mI/Cr, L . mI/Cr). All correlations had a positive sign, mean­ing that any increase in LL/Cr or mI/Cr, will coin­cide with increases in AD, MD, p, RD and L. Although scarce, there are independent pub­lications on MRS and DTI metrics that helped us understand better these observations. Firstly, we briefly mention basic concepts of the mI/Cr metab­olite ratio, after the five DTI metrics observed for this region (AD, MD, p, RD and L). mI/Cr includes a range of compounds: phos­phatidylinositol, inositol polyphosphate, inositol monophosphate, myo-inositol and, to a smaller extent, glycine; because inositol is elevated within astrocytes, it increased peak is taken as an empiri­cal marker of glial density and proliferation.37 The exact biological significance of mI/Cr, measurable only at short echo time, had been considered un­certain in gliomas.21 MD measures the average motion of water mol­ecules, independent of tissue directionality31; it is considered a synonym of the coefficient of dif­fusion in different space guidelines.38 Increased MD has been observed in the peritumoral region of high-grade gliomas.39 The best diagnostic per­formance by MD in the peritumoral region13 is explained because it measures the magnitude of molecular motion of water. However, MD does not depend directly on the integrity of myelinated fi­bre tracts.35 p is the isotropic component of the diffusion tensor; p values are significantly higher in the low-grade tumours, possibly reflecting the increased cellularity and restriction of water diffusion in high-grade gliomas; disrupted tracts, however, show a marked increase in p.33 p showed one of the three best diagnostic performance to characterise peritumoral oedema.13 AD and RD describes micro­scopic water movement parallel and perpendicular to the axon tract, respectively; inconsistent changes of RD and AD appeared in axonal injury.40-42 L rep­resents the total magnitude of the diffusion tensor; it shows an increased mean in peritumoral oedema in glioblastoma.43 DTI and MRS features of peritumoral oedema in glioblastoma Characterisation of peritumoral oedema is one of the most challenging topics in glioblastoma. Discrimination of tumour-in.ltrated oedema from vasogenic oedema using DTI metrics has demon­strated con.icting results.44 Since last ten years, authors coincide that there is no threshold value at which a clear distinction could be made between tumour infiltration and purely vasogenic oedema; no DTI metric can, by itself, definitively distinguish between these re­gions.43 Tumour infiltration may occur in brains that appear normal on T2-weighted images in 40% of cases.34 Gliosis (measured by mI/Cr), is an as-trocytic response to any central nervous system injury, which can occur in perifocal oedema. In the relatively long-standing oedema surrounding glioblastoma, glial fibres assume a more regular ar­rangement, resulting in more organised water dif­fusion detected with DTI.11 Limitations of the study Some limitations need to be acknowledged: we did not use the single-voxel technique that it is favour­ite in clinical practice (widely available, usually good field homogeneity, can be readily performed at short echo times, and is relatively easy to process and interpret). However, its highest single limita­tion is the lack of ability to determine the spatial heterogeneity of spectral patterns and the fact that only a small number of brain regions can be cov­ered within the time constraints of a routine clini­cal MR exam.45 We did not measure metabolite re­laxation rates due to scan-time limitations related to a large number of voxels under investigation. We were not able to calculate concentrations of additional metabolites such as glutamine, gluta­mate, alanine, amino acids, separation of lipids and lactate; they required special software pack­ages ready to fit short-echo and long-echo spectra, such as LCModel 46 and jMRU I47; these were not available at our institution when the MRS data for this project were acquired. We would have liked to obtain a higher number of directional motion-probing gradients (MPG) like other studies report­ing up to 40- and 81- for the DTI acquisition.48 It is known that the minimal mathematic requirement for DTI-parameters calculation is 6 independent directional MPG settings.48 Because the amount of imaging time is limited in most clinical situa­tions, we followed the recommendations of the MRI scanner vendor. Our choice of 25 MPG set­tings thus involved a trade-off between minimiz­ing directional bias and minimizing scanning time, it also complied with the minimum of 20 unique sampling orientations necessary for a robust esti­mation of anisotropy.49 Our statement that tumour in.ltration coexist with vasogenic oedema in a heterogeneous pattern 406 in the peritumoral region was not con.rmed with histopathology. The limited explanations to our findings might support the statement by Pena et al. “it is still not known a priori which tensor measure is the most appropriate to quantify pathological changes in brain tissue”.23 Future directions We acknowledge the unmet need of generalising the MRI studies in glioblastoma acquiring ad­vanced imaging techniques, including perfusion-weighted imaging, MR spectroscopy, and DTI, to assess tumour infiltration.50 Because the MRS and DTI biomarkers have been measured in other types of tumours11,16, we believe that the results of this study also apply to those tumours. However, fu­ture studies should address if similar correlations are also observed for them. To achieve a deeper understanding of the DTI and MRS interactions; multivariate analysis of DTI metrics and MRS metabolites, controlling the effect of confounders (gender, age, regional location of the tumour, infil­tration patterns using MRS and DTI) might unveil unknown interactions of these biomarkers at the ultrastructural level in glioblastoma to support the speculation in our explanations. We believe MRS and DTI will be incorpo­rated soon in the context of the World Health Organization (WHO) updated the central nerv­ous system (CNS) tumour classification. In the updated 2016 WHO CNS tumour classification version, some tumours were defined by a combi­nation of microscopic morphologic and molecular and genetic factors, whereas others continue to be defined by morphology alone. Although not offi­cial, there is a role for DTI and MRS in the current evaluaLaslotion of glioblastoma: IDH1 and IDH2 mutations (which are referred collectively as isoci­trate dehydrogenase [IDH] mutation) have become definitional for infiltrating gliomas in adults, with 1p/19q codeletion further characterizing the type.51 Mutation in IDH1 and IDH2 alters the role of the IDHs in the citric acid cycle and leads to accumu­lation of the oncometabolite 2-hydroxyglutarate (2HG) within tumour cells. Although IDH mutants themselves do not present a clear radiologic signa­ture, 2HG can be detected at MR spectroscopy.52 The 1p/19q codeletion is associated with the appar­ent diffusion coefficient value53, which is equiva­lent to the MD24, a DTI metric that had significant Rs in our study. Routine use of advanced MRI in glioblastoma has been incorporated into glioma imaging protocols at some institutions.51 Conclusions A comprehensive understanding of appropriate DTI and MRS biomarkers for each tumour region in glioblastoma would obtain complementary met­abolic and ultrastructural information necessary to preoperatively identify sites of signi.cant tumour in.ltration that appear normal on conventional MRI and in the follow-up of glioblastoma patients. DTI, in combination with MRS, are additional tools of the “biologic targeting” for radiation therapy. DTI and MRS biomarkers answer different ques­tions; peritumoral oedema represents the biggest challenge with at least ten significant correlations between DTI and MRS that need additional stud­ies. The fact that DTI and MRS measures are not specific of one histologic type of tumour broadens their application to a wider variety of intracranial pathologies. 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Song SK, Sun SW, Ramsbottom MJ, Chang C, Russell J, Cross AH. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage 2002; 17: 1429-36. doi: 10.1006/ nimg.2002.1267 42. Zhang X, Sun P, Wang J, Wang Q, Song SK. Diffusion tensor imaging detects retinal ganglion cell axon damage in the mouse model of optic nerve crush. Invest Ophthalmol Vis Sci 2011; 52: 7001-6. doi: 10.1167/iovs.11-7619 408 43. Wang W, Steward CE, Desmond PM. Diffusion tensor imaging in glio­blastoma multiforme and brain metastases: the role of p, q, L, and frac­tional anisotropy. AJNR Am J Neuroradiol 2009; 30: 203-8. do i: ajnr.A1303 [pii]10.3174/ajnr.A1303 44. Lu S, Ahn D, Johnson G, Law M, Zagzag D, Grossman RI. Diffusion-tensor MR imaging of intracranial neoplasia and associated peritumoral edema: introduction of the tumor infiltration index. Radiology 2004; 232: 221-8. doi: 10.1148/radiol.2321030653 45. Barker PB, Bizzi A. Stefano ND, Gullapalli RP, Lin DDM. Clinical MR spectros­copy. Techniques and Applications. Cambridge, UK: Cambridge University Press; 2010. 46. Provencher SW. Automatic quantitation of localized in vivo 1H spectra with LCModel. NMR Biomed 2001; 14: 260-4. doi: 10.1002/nbm.698 47. Naressi A, Couturier C, Castang I, de Beer R, Graveron-Demilly D. Java-based graphical user interface for MRUI, a software package for quantitation of in vivo/medical magnetic resonance spectroscopy signals. Comput Biol Med 2001; 31: 269-8 6. doi: 10.1016/s0010-4825(01)00006-3 48. Yamamoto A, Miki Y, Urayama S, Fushimi Y, Okada T, Hanakawa T, et al. Diffusion tensor fiber tractography of the optic radiation: analysis with 6-, 12-, 40-, and 81-directional motion-probing gradients, a preliminary study. AJNR Am J Neuroradiol 2007; 28: 92-6. 49. Jones DK. The effect of gradient sampling schemes on measures derived from diffusion tensor MRI: a Monte Carlo study. Magn Reson Med 200 4; 51: 807-15. doi: 10.1002/mrm.20033 50. 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A5070 409 research article Adrenal vein sampling for primary aldosteronism: a 15-year national referral center experience Tomaz Kocjan1,2, Mojca Jensterle1,2, Gaj Vidmar2,3,4, Rok Vrckovnik1,2, Pavel Berden2,5, Milenko Stankovic2,5 1 Department of Endocrinology, Diabetes and Metabolic Diseases, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 University Rehabilitation Institute, Ljubljana, Slovenia 4 FAMNIT, University of Primorska, Koper, Slovenia 5 Clinical Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(4): 409-418. Received 14 April 2020 Accepted 12 July 2020 Correspondence to: Prof. Tomaž Kocjan, M.D., Ph.D., Department of Endocrinology, Diabetes and Metabolic Diseases, University Medical Centre Ljubljana, Zaloška 7, SI-1525 Ljubljana, Slovenia. E-mail: tomaz.kocjan@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. Adrenal vein sampling (AVS) is essential for diagnostics of primary aldosteronism, distinguishing uni­lateral from bilateral disease and determining treatment options. We reviewed the performance of AVS for primary aldosteronism at our center during first 15 years, comparing the initial period to the period after the introduction of a dedicated radiologist. Additionally, AVS outcomes were checked against CT findings and the proportion of operated patients with proven unilateral disease was estimated. Patients and methods. A retrospective cross-sectional study conducted at the national endocrine referral center included all patients with primary aldosteronism who underwent AVS after its introduction in 2004 until the end of 2018. AVS was performed sequentially during Synacthen infusion. When the ratio of cortisol concentrations from adrenal vein and inferior vena cava was at least 5, AVS was considered successful. Results. Data from 235 patients were examined (168 men; age 32–73, median 56 years; BMI 18–48, median 30.4 kg/ m2). Average number of annual AVS procedures increased from 7 in the 2004–2011 period to 29 in the 2012–2018 period (p < 0.001). AVS had to be repeated in 10% of procedures; it was successful in 77% of procedures and 86% of patients. The proportion of patients with successful AVS (92% in 2012–2018 vs. 66% in 2004–2011, p < 0.001) and of suc­cessful AVS procedures (82% vs. 61%, p < 0.001) was statistically significantly higher in the recent period. Conclusions. Number of AVS procedures and success rate at our center increased over time. Introduction of a dedicated radiologist and technical advance expanded and improved the AVS practice. Key words: angiography; adrenal gland; endocrine disorders; secondary hypertension Introduction Primary aldosteronism is the most common form of secondary hypertension, with a prevalence of 5.9% among hypertensive patients in primary care practice.1 Autonomous and excessive secretion of aldosterone from one or both adrenal glands in patients with primary aldosteronism causes sig­nificantly higher cardiovascular risk and more pro­nounced renal damage compared to equally severe essential hypertension.1–3 Amongst available tar­geted treatment, the preferred therapeutic option is unilateral laparoscopic adrenalectomy, which can normalize or decrease blood pressure in most patients with proven unilateral disease.4,5 Long­term medical treatment with mineralocorticoid 410 receptor antagonists is not only more expensive and less convenient, but it might also have worse outcomes overall.4,6 Therefore, a crucial part of diagnostic workup in primary aldosteronism is to correctly determine which patients have unilateral disease and could pursue surgical cure. Adrenal computed tomogra­phy (CT) (or magnetic resonance imaging (MRI)) should be the first test in the subtype evaluation of primary aldosteronism and to exclude adrenocor­tical carcinoma.4 However, because of increasing prevalence of nonfunctioning adrenal incidentalo-mas, the reliability of CT in localizing unilateral disease (e.g. an aldosterone producing adenoma) declines with patient age.4,7 In most patients CT cannot accurately distinguish between unilateral and bilateral forms, and may even lead to inappro­priate treatment of primary aldosteronism.8 The only exception are infrequent younger patients be­low 35 years of age with florid disease and a clear one-sided adrenal adenoma with normal contralat­eral gland.4,8,9 All other surgical candidates should proceed to adrenal vein sampling (AVS), which is regarded as the gold standard to demonstrate lateralization and to avoid unnecessary or inappropriate adre­nalectomy. More than 50 years after its introduc­tion, AVS remains controversial as an invasive, expensive and a technically challenging method with successful bilateral catheterization obtained in only about 75% of cases.4,10 Cannulation of the small and short right adrenal vein with direct drainage into the inferior vena cava (IVC) is often the main obstacle to a successful procedure, while the sampling from the left adrenal vein is relatively straightforward. There is substantial inconsistency in how AVS is performed and interpreted. When done by experienced radiologists, the complication rate is low at between 0.2 and 0.9%.11 Only a limit­ed number of referral centers worldwide routinely carry out the procedure.10,12 Recently, the introduc­tion of cone beam CT (CBCT) and other technical developments have further improved the AVS suc­cess rate and reduced the complications.13–16 Primarily, we aimed to review the performance of AVS for primary aldosteronism at our center from its introduction in 2004 up to 2018. The initial period from 2004 to 2011 was compared to the peri­od after the introduction of a dedicated radiologist in 2012. Our secondary objectives were to check the outcomes of AVS against CT findings and to esti­mate the proportion of patients with proven unilat­eral disease who ultimately had surgery. Patients and methods Study design We conducted a retrospective cross-sectional study from AVS introduction in November 2004 to the end of 2018 at the Slovenian national tertiary endo­crine referral center, which serves a country with a population of 2 million inhabitants. All the data originated from the Slovenian AVS database. The data collection and its analysis were approved by the National Medical Ethics Committee. Patients All patients with confirmed primary aldosteron-ism who underwent AVS at our center during the study period were suitable for enrollment. The di­agnostic work-up for primary aldosteronism was done according to the established guidelines4,17, as previously detailed elsewhere.18 Radiological imaging One to three months before the AVS, all patients but one had adrenal imaging with a dual-source computed tomography (CT) scanner (Somatom Dual Source, Siemens, Germany). Our pre-speci­fied adrenal CT protocol included 1 mm axial slices through the abdomen before, and if necessary also Kocjan T et al. / Adrenal vein sampling for primary aldosteronism 411 after, the intravenous administration of 80–100 ml of iodinated contrast (370 mgJ/mL), injected at a rate of 3–4 ml/s via antecubital vein during breath-holding. Contrast-enhanced images were acquired after 60 seconds and 15 minutes. The standard scanning parameters included beam collimation of 64x0.6 mm, 16 slices and gantry rotation time of 0.5 s. Tube voltage was set at 120 kV, while the tube current was variable, optimized for body mass in­dex and size, ranging between 160 and 210 mA. Source images of all phases were reconstructed on the axial plane at 5 mm, and on the coronal planes at 4 mm. Since 2017 a new-generation CT scan­ner (Somatom Force, Siemens, Germany) has been used, which allowed for more precise adaptation to the individual patient body characteristics. The scanning protocol remained essentially the same except for axial reconstructions at 2 mm. In 2014, interdisciplinary meetings dedicated to adrenal pathology were introduced where CT scans were meticulously reassessed with a radiologist, if both adrenals were described as normal. Finally, any thickening of at least 5 mm was deemed abnormal (Figure 1). The interventional radiologist also re­viewed the images, in order to recognize the adre­nal veins, especially on the right side. Adrenal vein sampling AVS was executed after an overnight fast between 8 and 9 AM. All patients were in the recumbent po­sition for at least 1 hour before sampling. Infusion of synthetic adrenocorticotropic hormone (ACTH) Synacthen (50 µg/h) was started 30 min before AVS and continued throughout the procedure. During local anesthesia, a 5 Fr sheath (Avanti+ Introducer, Cordis, USA in the first period; Radiofocus Introducer II, Terumo, Japan in the re­cent period) was introduced into the right femoral vein. AVS was performed sequentially with the right adrenal vein always being cannulated and sampled first, using a 5 Fr Mickelson catheter (Cook Medical Inc., USA) or a 5 Fr Cobra C2 catheter with open-ended tip and two side-holes (Cordis, USA) in the first period. In the recent period a 4 Fr Mickelson the junction of these two veins or selectively from catheter (Cook Medical Inc., USA) was routinely the left adrenal vein above the junction (Figure 3). used on the right side (Figure 2). Catheterization Finally, a microcatheter was removed and the of the left renal vein then followed with the same Mickelson catheter slightly pulled out to sample catheter, which was used as a guide for a 2.7 Fr blood from the infra-renal IVC. On the other hand, Progreat microcatheter (coaxial type with catheter a 4 Fr MPA 2 catheter with open-ended tip and two and guidewire; Terumo Interventional Systems, side-holes (Cordis, USA) was used on the left side USA) to cannulate the common trunk of the left in-in the first period. Standard 0.035-inch guidewire ferior phrenic vein and the left adrenal vein. The (J Tef Guidewire, Kimal, UK) was used in all cases. corresponding blood sample was drawn either at Additionally, 0.035-inch guidewire with J angled 412 tip (Terumo, Japan) was used on the left side in the first period. Blood samples were drawn in 5 ml sy­ringes and sent to laboratory for aldosterone and cortisol measurements. Hemostasis at the puncture site was ensured by manual compression. During the initial period from 2004 to 2011 there were two interventional radiologists perform­ing AVS; from 2012 onwards all procedures were done by a single dedicated interventional radiolo­gist. During the first period, AVS was performed with fluoroscopic guidance by digital subtrac­tion angiography (INTEGRIS V5000; Philips, The Netherlands), which was later changed to single-plane digital subtraction angiography (Allura Xper FD; Philips, The Netherlands). In the majority of cases, small amounts of contrast (90 ml on average per procedure in the first period and 52 ml on av­erage per procedure in the recent period, respec­tively) were injected to better visualize the right adrenal vein. High-resolution CBCT (Phillips Allura XperCT, The Netherlands) acquisition during AVS has been used since 2012 at first sporadically and then more consistently to identify the tip of the catheter ac­curately, in order to differentiate between the right adrenal vein and a hepatic accessory vein or a para-vertebral vein when necessary (Figure 4). The average AVS procedure time decreased from 18.2 minutes in the first period to 16.8 min­utes in the recent period. When the selectivity index (SI), computed as the ratio of concentrations of cortisol from an adrenal vein and the infra-renal IVC, was at least 5, AVS was deemed successful. Lateralization index (LI), defined as the ratio of the higher over the lower cortisol–corrected aldosterone ratio, of more than 4 indicated unilateral aldosterone excess, while the values between 3 and 4 were assumed borderline.19 Suppressed plasma renin activity (PRA) values (< 0.6 ng/mL/h) were used as proof for unlikely stim­ulation of the contralateral adrenal cortex at a level adequate to confound interpretation of lateraliza­ tion.20,21 Assays Serum aldosterone was measured with the Active® Aldosterone RIA (Beckman Coulter, Immunotech, Czech Republic). Serum cortisol was measured with an automated chemiluminescent immunoas­say (CLIA) on the Immulite® 2000 XPi (Siemens Healthcare, Gwynedd, United Kingdom). The re­spective within- and between-assay coefficients of variation were below 4.5% and 9.8% for aldosterone and below 6.8% and 9.4% for cortisol. PRA meas­urements were performed using the Angiotensin I RIA KIT (Beckman Coulter, Immunotech, Czech Republic). The respective within- and between-as­say coefficients of variation were below 11.3% and 20.9%. Statistical analysis Descriptive statistics were calculated. Patient char­acteristics and outcomes were compared between periods or groups using t-test, exact Mann-Whitney test and Fisher’s exact test. Cohen’s kappa was used to assess agreement between diagnostic methods. Statistical analyses were conducted using IBM SPSS Statistics 20 (IBM Corp., Armonk, USA, 2011). Results Data from 235 patients with primary aldosteronism were examined. Their clinical characteristics and laboratory parameters are presented in Table 1. Most of them had a unilateral adrenal abnormal­ity (62%) on CT scan, while bilateral adrenal thick- Kocjan T et al. / Adrenal vein sampling for primary aldosteronism 413 TABLE 1. Clinical characteristics and laboratory parameters of the patients n 235 Male patients 168 (71%) Age (years) 56 (32–73) Body Mass Index (kg/m2) 30.4 (18.3–48.4) Systolic BP at presentation (mm Hg) 155 (145–170) Diastolic BP at presentation (mm Hg) 90 (80–95) Number of antihypertensive agents 3 (2–4) Hypokalemia 172 (73%) eGFR (ml/min/1.73 m2) 88 (71–102) Baseline aldosterone (nmol/L) 0.7 (0.3–8.8) Baseline PRA (ng/mL/h) 0.2 (0.2–0.9) Baseline ARR 4.2 (1.1–43.8) 66 (28%) / CT* normal / bilateral / unilateral 24 (10%) / 144 (62%) Tumor size on CT (mm) 13 (8–19) * = not performed in one patient; ARR = serum aldosterone-to-renin ratio; BP = blood pressure; eGFR = estimated glomerular filtration rate; PRA = plasma renin activity; Descriptive statistics are reported as median (interquartile range) for numeric variables and number (percentage) for categorical variables; ening was present in 10% of the cases. The average adrenal nodules’ size was 13 mm and left-sided lesions were more prevalent than the right-sided ones (62% vs. 38% in total). There were 28 left-sided lesions (60%) in the first period and 91 left-sided lesions (62%) in the recent period. Finally, in 28% of the cases CT scans of both adrenals were consid­ered normal. The average number of AVS procedures per­formed per year increased statistically significantly from 7 in the 2004–2011 period to 29 in the 2012– 2018 period (p < 0.001) (Figure 5). In total, AVS had to be repeated in 10% of the procedures (9% in the first period, 10% in the recent period). AVS was successful (SI = 5 in both adrenal veins) in 86% of the patients and in 77% of the procedures. The overall success rate of left adrenal vein cannulation was significantly higher than that of the right ad­renal vein (p = 0.001). While the success rate on the left side remained unchanged over time (94% vs. 97%; p = 0.434), there was a statistically significant improvement on the right side after the introduc­tion of a single dedicated interventional radiologist in 2012 (66% vs. 94%; p < 0.001). Consequently, the proportion of patients with successful AVS (66% vs. No. of AVS procedures 40 FIGURE 5. Number of adrenal vein sampling procedures per year during the study period. 0% 25% 50% 75% 100% FIGURE 6. Patients with successful adrenal vein sampling (AVS) and successful AVS procedures during the study period. 92%, p < 0.001) and of successful AVS procedures (61% vs. 82%, p < 0.001) was also significantly high­er in the recent period (Figure 6). The right and left median SI values were not statistically significant­ly different (22.3 [interquartile range 18.2] vs. 22.4 [13.1]; p = 0.285). Decreasing the SI to = 3 instead of = 5 would not have improved the AVS perfor­mance on either side. Among previously tested clinical determinants of bilateral AVS success22,23, only younger age proved to be statistically signifi­cant (p = 0.004) in our cohort, whereas higher BMI and male gender did not. Adrenal hemorrhage due to vein rupture occurred during two procedures (0.8% overall), one in the initial period (1 out of 57 procedures, 1.8%) and another in the recent peri­od (1 out of 203 procedures; 0.5%), both resolved conservatively. Primary aldosteronism persisted in both cases and was treated medically. There were no other serious adverse events associated with AVS during the study. CT and AVS results were compared in 181 pa­tients with bilaterally successful AVS, excluding cases with borderline LI values between 3 and 4. The agreement amongst the two diagnostic meth- 414 AVS bilateral AVS left AVS right Total CT bilateral CT left CT right Total 51 (28%) 6 (3%) 14 (8%) 71 39% 36 (20%) 2 (1%) 44 24% 4 (2%) 72 40% 19 (10%) 37 20% 32 (18%) 38 21% 17 (9%) 100 55% N = 181 FIGURE 7. Agreement between adrenal vein sampling (AVS) and CT findings depicted with a variable-width stacked column chart. Patients with normal CT scans are included in the CT bilateral category ods was present in only 59% of cases (kappa = 0.36) (Figure 7). Among the patients with successful AVS, 44% overall (n = 79) had LI > 4 and hence proven unilat­eral disease. The percentage of lateralized cases did not statistically significantly differ between the two study periods (p = 0.248) or between younger (< 40 years) and older patients (p = 0.470). Adrenalectomy was recommended to all the patients with lateral-ized aldosterone secretion, but only 86% of them underwent surgery. All patients below 40 years of age with proven unilateral disease were operated on, but the same was true for only 84% of older subjects. The main reason for not having surgery was patient’s reluctance (n = 9). One patient was diagnosed with liver cirrhosis and was rejected by the surgeon, two patients were lost to follow-up. The proportion of patients with unilateral disease undergoing surgery did not differ statistically sig­nificantly between the periods (89% vs. 85%, p = 1.000). Finally, additional four out of 21 patients with successful AVS and borderline LI values be­tween 3 and 4 also opted for surgery. Three of them had clear unilateral adrenal nodule on CT, whereas the remaining patient had normal glands on imag­ing. All other patients were treated medically. Discussion The present study provides an important insight in the implementation process and continued de­velopment of AVS at the Slovenian national endo­crine referral center over 15 years. The overall suc­cess rate for the AVS procedures during this period was 77%, which is similar to the recently published large multicenter AVS registry study on 1625 pa­tients, where 80% of cases were bilaterally selec­tive.24 Interestingly, the data from German Conn’s Registry revealed that only 31% of their initial AVS studies were successful with later increase of the success rate to 61%.25 On the other hand, the pro­portion of successful AVS procedures at our insti­tution increased from 61% in 2004–2011 to 82% in 2012–2018. With 10% of procedures being repeated overall, the proportion of our patients with suc­cessful AVS rose concurrently from 66% to 92%, which is close to the success rate at the centers of excellence.19,26,27 The observed increment could be partially explained by our decision in 2012 to fol­low the recommendation for low-volume centers and focus the expertise on a single, dedicated in-terventional radiologist.12,28 This decision not only improved, but also expanded the AVS performance at our center (Figure 5). The overall success rate improved due to su­perior cannulation of the right adrenal vein in the recent period (94% vs. 66%), whereas the success rate on the left side remained around 95% and un­changed over time. This was most probably not only due to the learning curve of the radiologist29,30, but mainly due to more regular pre-procedural review of CT images and intra-procedural use of high-resolution CBCT since 2012 to better map the adrenal venous anatomy, especially on the right side. The same approach has been recently used in other centers and allowed not only a better evalu­ation of the selectivity of right-sided adrenal vein cannulation, but also a significant decrease in the fluoroscopy time and quantity of iodine contrast injected in combination with unchanged or even lower radiation exposure.13–16 Recently, another possibility to improve the catheterization success has been offered by using the newly developed ultra-rapid technique for semi-quantitative measurement of the cortisol level in adrenal veins in approximately 5 minutes, thus enabling the radiologist to reposition the catheter if the first result indicates an incorrect position.31 The rapid on-site measurement of the cortisol might be associated with a shorter procedure time and lower radiation dose than CT assisted AVS.32 However, this approach was not available at our center dur­ing the analyzed period. Throughout the study period we strictly fol­lowed the Mayo Clinic protocol and used con­tinuous Synacthen infusion starting 30 min before sampling and continuing throughout the proce­dure during sequential AVS.19 The main rationale Kocjan T et al. / Adrenal vein sampling for primary aldosteronism 415 for ACTH-stimulated AVS is to maximize the cor­tisol gradient between the adrenal veins and VCI. Consequently, SI is increased and so is the propor­tion of diagnostic AVS procedures, which is why such a practice is particularly suitable for less expe­rienced and low-volume centers.20,21 On the other hand, some authors consider the use of ACTH-stimulation as controversial because it might have the undesirable effect of masking the lateralization of aldosterone production, thus rendering some patients with unilateral primary aldosteronism apparently unsuitable for surgery.10 Fortunately, accumulated data overall suggest that surgical out­comes are similar irrespective of whether AVS is done by ACTH stimulation or not.5,33,34 ACTH stimulation also minimizes stress-in­duced variations in aldosterone secretion during sequential sampling19–21, which might otherwise generate artificial between-sides gradients and lower its diagnostic accuracy.35 Additionally, ac­cording to our protocol the right adrenal vein was always being cannulated first to lessen the time lag amid the sides.28 Thereafter, a microcatheter was used to quickly cannulate the left adrenal vein36 and to keep the delay between sequential sampling under 5 minutes in most of our AVS procedures.37 Clearly, AVS studies that are not bilaterally suc­cessful should not be used to establish lateraliza­tion.20 The choice of the correct SI is pivotal for the reported catheterization success rate, diagnostic reliability of the method and clinical outcome.34,38,39 According to the expert consensus the cutoff value for the SI should be = 3.0 during ACTH stimula­tion20, but we consistently applied an even more robust criterion (SI = 5) in order to minimize the chance of misdiagnosing either unilateral or bilat­eral primary aldosteronism.19,21 It is conceivable that there is a progressive decrease in success rate with increasing SI cut-offs, although the recent multicenter study showed this to be less dramatic with ACTH-stimulation.34 Concordantly, decreas­ing the SI to = 3 instead of = 5 in our cohort would not improve the cannulation success rate on either side. Furthermore, the data from the same study showed post-ACTH SI cut-off of 5 to be able to clearly segregate biochemically successful and non-successful studies.34 Actually, our median SI values were much higher than the advocated threshold. There was no usual distinction between higher median right-sided and lower median left-sided SI values7,19, pointing to selective cannula­tion of the left adrenal vein in most cases with the microcatheter. Notably, blood sampling from the common trunk of the inferior phrenic vein and the left adrenal vein might be the preferable method of AVS due to better potential diagnostic accuracy, technical ease, lower cost and lower risk of vein rupture.40 The overall complication rate during the study was low (0.8%). Despite the almost fourfold in­crease of AVS procedures in the recent period, the between periods complication rates were compa­rable, with one adrenal hemorrhage due to vein rupture in each period (1.8% vs. 0.5%). The obser­vation confirmed that the major determinant of the incidence of such events is the number of AVS per­formed by each radiologist.12 When AVS results were used as the gold stand­ard for lateralization in a subgroup with unequivo­cal diagnosis of unilateral (LI > 4) or bilateral (LI < 3) disease, CT misdiagnosed the primary aldo­steronism subtype in 41% of our patients despite reassessment of all normal scans at our interdisci­plinary meetings. If we had relied only on imaging, 20/71 (28%) patients would have been incorrectly denied adrenalectomy and treated medically. In addition, 49/100 (49%) patients with bilateral primary aldosteronism would have been sent to unilateral adrenalectomy, and 6/77 (8%) patients with unilateral primary aldosteronism would have had removed the normal-functioning adrenal (Figure 7). By contrast, Mulatero et al. demonstrat­ed much higher agreement of AVS and CT (77%) when imaging was performed by the same highly motivated radiologist.41 Nevertheless, the propor­tion of discordant AVS and CT results in our co­hort closely resembles the findings of a systematic review of 38 diagnostic studies on 950 patients, where CT (or MRI) might have missed the type of primary aldosteronism in 37.8% of cases.8 Ultimately, 44% of our patients lateralized on AVS, which represents a slightly higher prevalence of unilateral disease than traditionally reported.4,7 Yet this finding was not unexpected, because sev­eral clinical characteristics of our cohort, e.g. high median number of antihypertensives, prevalent spontaneous hypokalemia and higher median al­dosterone values, pointed towards more severe disease, which is consistent with unilateral pri­mary aldosteronism. We used the most stringent LI cut-off (> 4), which is favored by the expert consensus for ACTH-stimulated AVS, in order to avoid false-positives and ensure highest possible cure rates.10,20,21 Only four out of 21 patients with borderline LI values (3–4) were referred to surgery. Use of contralateral gland suppression (e.g. lower aldosterone to cortisol ratio than the same ratio in IVC) might be potentially helpful to determine lat- 416 eralization in intermediate cases8,10,38 but was not employed during the study period. Using our con­servative approach to make surgical decision, close to 100% of operated patients at our center achieved complete biochemical remission of primary aldo­steronism according to the international PASO out­come consensus.5 Adrenalectomy was recommended to all pa­tients who lateralized on AVS, however a substan­tial proportion (14%) was ultimately treated medi­cally. Only patients older than 40 years changed their mind and decided against the operation. These outcomes stress the importance of careful selection of patients for AVS and operation.28 Most appropriate candidates desire surgery and have a high probability of unilateral primary aldosteron-ism. On the other hand, AVS is not needed in indi­viduals who prefer medical therapy and in those who are not suitable for surgery due to comorbidi-ties or age.21,42 A simple clinical prediction criterion could probably identify some patients with bilat­eral primary aldosteronism who should avoid un­necessary AVS and be treated medically.18 Last but not least, the primary aldosteronism surgical out­come predictor might help finding patients who are expected to attain long-term blood pressure control after adrenalectomy to guide preoperative patient counseling and final decision for or against AVS and surgery.43 There are some limitations of the present study. Primarily, the outcomes were deducted from a retrospective analysis. However, all relevant clinical and laboratory data were logged into our AVS database virtually without missing values. Furthermore, discontinuation and/or adjustment of the antihypertensive agents before and during AVS could probably have been more rigorous, especial­ly during the early years. Still, hypokalemia was al­ways corrected, mineralocorticoid antagonists and potassium-wasting diuretics were discontinued on time. Most patients had resistant hypertension, so we mostly followed the expert recommendation that less interfering antihypertensive medications may be used if PRA, which was routinely meas­ured before AVS, remained suppressed.20,21 ACTH stimulation might have the potential to mask lat­eralization of aldosterone production in patients with adenomas simultaneously producing cortisol, which appears more frequently than we thought earlier.10,44 During the study dexamethasone sup­pression testing to detect this entity was recom­mended only in rare patients with relatively large adrenal tumors of = 3 cm and not routinely.4,17 Consequently, another possible source of error might have been unrecognized autonomous corti-sol cosecretion in some patients. Finally, the tech­nical advances in AVS techniques over the 15-year study period and their impact on the AVS success rate might not have been emphasized enough. The main strength of our study is that our results were derived from a relatively large and a well-de­fined national cohort. Management of the patients was standardized and followed the Endocrine Society clinical guidelines whenever feasible4,17, which can significantly decrease the selection bias. Conclusions Based on the present study, we conclude that the introduction of a dedicated radiologist with high­er workload and regular use of intra-procedural CBCT since 2012 have significantly enhanced the AVS performance at our center. In the future, we aim to improve the concordance of AVS results with CT findings by revising our interdisciplinary strategy with radiologists. We will also address the protocols for the selection of appropriate candi­dates for AVS, since we demonstrated that a sub­stantial number of patients with proven unilateral primary aldosteronism did not proceed to surgery. Acknowledgements The authors would like to thank Marjana Turk Jerovsek, M.D. and Barbara Robnik, M.D. for their work with the AVS database. We appreciate the assistance of Vlasta Hocevar and Mateja Adamlje, RNs. References 1. Monticone S, Burrello J, Tizzani D, Bertello C, Viola A, Buffolo F, et al. Prevalence and clinical manifestations of primary aldosteronism encoun­tered in primary care practice. J Am Coll Cardiol 2017; 69: 1811-20. doi: 10.1016/j.jacc.2017.01.052 2. Savard S, Amar L, Plouin PF, Steichen O. Cardiovascular complications as­sociated with primary aldosteronism: a controlled cross-sectional study. Hypertension 2013; 62: 331-6. doi: 10.1161/HYPERTENSIONAHA.113.01060 3. Monticone S, Sconfienza E, D’Ascenzo F, Buffolo F, Satoh F, Sechi LA, et al. Renal damage in primary aldosteronism: a systematic review and meta-analysis. J Hypertens 2020; 38: 3-12. doi: 10.1097/HJH.0000000000002216 4. Funder JW, Carey RM, Mantero F, Murad MH, Reincke M, Shibata H, et al. 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JCI Insight 2017; 2: e93136. doi: 10.1172/jci.insight.93136 419 research article Adnexal masses characterized on 3 tesla magnetic resonance imaging – added value of diffusion techniques Julia Dimova1, Dora Zlatareva1, Rumiana Bakalova2,3,4, Ichio Aoki3,4, George Hadjidekov2,5 1 Department of Diagnostic Imaging, Medical University, Sofia, Bulgaria 2 Department of Physics, Biophysics and Radiology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria 3 Department of Molecular Imaging and Theranostics and 4 Group of Quantum-state Controlled MRI, National Institutes for Quantum and Radiological Science and Technology (QST/ NIRS), Chiba, Japan 5 Department of Radiology, University Hospital “Lozenetz”, Sofia, Bulgaria Radiol Oncol 2020; 54(4): 419-428. Received 19 June 2020 Accepted 14 September 2020 Correspondence to: Prof. George Hadjidekov, M.D., University Hospital Lozenetz, Department of Radiology, Koziak 1 str. 1407 Sofia, Bulgaria, E-mail: jordiman76@yahoo.com Disclosure: No potential conflicts of interest were disclosed. Background. To assess different types of adnexal masses as identified by 3T MRI and to discuss the added value of diffusion techniques compared with conventional sequences. Patients and methods. 174 women age between 13 and 87 underwent an MRI examination of the pelvis for a period of three years. Patients were examined in two radiology departments – 135 of them on 3 Tesla MRI Siemens Verio and 39 on 3 Tesla MRI Philips Ingenia. At least one adnexal mass was diagnosed in 98 patients and they are subject to this study. Some of them were reviewed retrospectively. Data from patients’ history, physical examination and laboratory tests were reviewed as well. Results. 124 ovarian masses in 98 females’ group of average age 47.2 years were detected. Following the MRI criteria, 59.2% of the cases were considered benign, 30.6% malignant and 10.2% borderline. Out of all masses 58.1% were classified as cystic, 12.9% as solid and 29% as mixed. .f histologically proven tumors 74.4% were benign and 25.6% were malignant. All of the malignant tumors had restricted diffusion. 64 out of all patients underwent contrast enhancement. (34 there were a subject of contraindications). 39 (61%) of the masses showed contrast enhancement. Conclusions. Classifying adnexal masses is essential for the preoperative management of the patients. 3T MRI pro­tocols, in particular diffusion techniques, increase significantly the accuracy of the diagnostic assessment. Key words: adnexal masses; 3 Tesla MRI; diagnosis; malignancy; ovarian neoplasms; diffusion restriction Introduction Incidental adnexal masses are commonly detected in daily medical practice due to the frequent lack of clinical manifestation.1 Approximately 9% to 10% of women undergoing ultrasound have ovarian le­sions.2 Although most commonly used, ultrasound has some limitations including the small field of view, low resolution and interference by obesity or by gaseous bowel loops.3 Ultrasound indetermi­nate adnexal masses vary between 5% and 25%.4,5 If furthermore examined with computed tomog­raphy (CT), distant metastases, respectively the staging of the disease could be assessed. Magnetic resonance (MR) has been considered as the most useful imaging technique for characterizing ad-nexal formations. This modality has a key role in the preoperative evaluation and their follow-up, identifying the origin of the mass and the different types of tissue contained in with accuracy of 88% to 93%.6 3 Tesla MRI is superior for examining female pelvis due to its higher resolution and the possibil- 420 ity of providing more detailed images.7-9 MRI tech­niques such as diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) are of an additional benefit differentiating malignant from benign lesions.10-12 The aim of our study is to assess different types of adnexal masses as identified by 3T MRI and to discuss the added value of diffusion and perfusion techniques compared with conventional sequenc­es. Patients and methods 174 women age between 13 and 87 underwent MRI examination of the pelvis for a period of three years. Indications were: sonographically detected pelvic mass; or gynecological complaints; or his­tory of previous adnexal tumor; or family history of ovarian cancer. Six women were examined for other reasons (hips, sigma/colon or perianal ab­scess), nine for uterine pathology, but adnexal mass was detected and the complete gynecologi­cal MRI protocol was performed, too. At least one adnexal mass was diagnosed in 98 patients and they are subject of this study. 51% of them were reviewed retrospectively. Data from patients’ his­tory, physical examination and laboratory tests were reviewed as well. Patients were examined in two radiology depart­ments, 135 of them on 3 Tesla MRI Siemens Verio and the 39 on 3 Tesla MRI Philips Ingenia. The Siemens MRI protocol included: coronal (COR) T1; sagittal (SAG) T2; paracoronal and paratransversal of the uterus T2 with and without fat saturation; SAG T1; transversal T1 Vibe Dixon; DWI and ADC. The Philips MRI protocol included: COR STIR; SAG T2; COR T2; COR T1; axial (AX) T2; AX T2 with fat saturation; DWI and ADC. (Table 1) M easurement of the ADC value was carried out for all ovarian masses in our study. For each tu­mor a region of interest (ROI: 1 cm2) was manually defined. In the mixed malignant formations ROIs were placed on the solid component only. The ADC values are presented as numerical value x 10-3 mm2/s representing quantitative metric. I ntravenous contrast administration was applied when needed and when there were no contraindi­cations. A macrocyclic contrast agent Gadobutrol [1.0 mmol/ml] (Gadovist® 1.0, Bayerhealthcare, Berlin, Germany) was used at a dose of 0.1 mmol/ kg in all contrast-enhanced studies on both MR de­vices. Injection rate of 0.5 mL/sec was performed in order to achieve equimolar amounts of gadolin­ium. Saline flush (25–30 ml) at the same flow rate followed the contrast administration. In part of our cases dynamic contrast enhanced – magnetic resonance imaging (DCE-MRI) was performed and time-signal intensity curve (TIC) was generated using the Mean Curve software package (Philips). A round region of interest (ROI: 1 cm2) was placed at target areas referring to T2W and contrast-enhanced images. Areas with hemor­rhage and necrosis were avoided. The following patterns were evaluated on MR images: – tumor appearance (cystic, solid or mixed) – uni- or bilateral ovaries involvement – size of the mass – adipose tissue presence or not – signal intensity on T2 weighted images – diffusion restriction – wall thickness – presence or not of septa – papillary projections – presence or not of ascites – lymph nodes involvement and metastases Following MR criteria of malignancy, as report­ed in the literature (by Jeong et al.13, Valentini et al.6 and El-Wekil et al.14), are used: – lesion size more than 4 cm – solid components with heterogeneous enhance­ment – papillary projections – septa thick more than 3 mm – areas of necrosis and breaking down – lymph nodes involvement sized more than 1 cm. SPSS Statistics release 21 for Microsoft Windows was used to perform Kolmogorov-Smirnov (2-tailed) test for establishing correlations between malignancy and diffusion restriction and between malignancy and type of mass. Approval was obtaine d from the Institutional Review Board of both University hospitals prior the initiation of the study. Informed written con­sent was obtained from each patient. Personal identity information of all patients was protected. Results In 98 females of ave rage age 47.2 years, a total of 124 ovarian masses were detected. In 16 of the pa­tients (16.3%) additional uterine pathology was found. One case considered as an ovarian cyst was histologically proven to be an inclusion peritoneal Dimova J et al. / 3 Tesla MRI of adnexal masses 421 TABLE 1. 3 Tesla Siemens and 3 Tesla Philips MRI protocols T1 COR 300 390/320 5 500 8.7 0.9×0.9×5.0 01:36 T2 SAG 200 320/320 4 3300 133 0.6×0.6×4.0 03:44 T2 paracor 200 320/320 4 3700 140 0.6×0.6×4.0 03:24 T2 paracor +FS 200 256/256 4 3700 131 0.8×0.8×4.0 01:58 T2 paratra 200 320/320 4 3740 148 0.6×0.6×4.0 03:29 T2 paratra + FS 200 256/256 4 3700 138 0.8×0.8×4.0 02:13 T1 SAG 160 217/192 4 569 12 0.4×0.4×4.0 03:44 T1 vibe dixon AX 380 188/320 3.5 3.92 1.27 0.6×0.6×3.5 00:19 DWI AX (b50-400-800) 360 100/128 5 4700 57 1.4×1.4×5.0 02:49 POST C T1 vibe dixon AX 380 188/320 3.5 3.92 1.27 0.6×0.6×3.5 00:19 T1 SAG 160 217/192 4 569 12 0.4×0.4×4.0 03:44 T1 COR 300 390/320 5 500 8.7 0.9×0.9×5.0 01:36 COR STIR 340 228/186 5 5622 50 1.5×1.5×5.0 03:45 T2 SAG 229 208/208 3 3776 100 1.1×1.1×3.0 03:01 COR T2 315 392/297 5 4846 90 0.8×1.6×5.0 01:56 COR T1 315 392/315 5 483 8 0.8×1.2×5.0 02:11 AX T2 261 328/251 5 4805 100 0.8×1.0×5.0 02:05 AX T2 FS 261 236/208 5 4346 80 1.11×1.25×5.0 02:37 DWI 3b 0,100,800 375 124/106 4 5299 77 3.0×3.0×4.0 01:51 POST C MDixon AX 240 220/222 3,5 5.4 1.96 1.09×1.08×3.5 02:58 AX = axial; COR = coronal; COR STIR = coronal short tau inversion recovery; DWI = diffusion-weighted imaging; FS = fat sat; paracor = paracoronal; SAG = sagital cyst. The results of all ovarian masses according to 58.1% out of all mas ses were classified as cystic, their MRI features are listed in Table 2. 12.9% as solid and 29% as mixed. In four cases both Following the MRI cr iteria, 59.2% of the cases solid and cystic masses were found in the same were considered benign, 30.6% malignant and patient. Of all ovarian tumors 37 (29.8%) had wall 10.2% borderline. The results of DWI sequences thickness greater than 3 mm, 16 (12.9%) had papil-show a statistically significant correlation with the lary projections and 41 (33%) were septated. Only 6 assessment of masses as benign/borderline/malig-masses of all contained fat, 5 of them were histolog­nant. 34.3% of all malignant cases were found in ically proven to be mature teratomas. Kolmogorov-the age group 61–70. Of all patients 32 were tested Smirnov test shows a statistically significant corre-for CA-125 tumor marker and 12 had elevated lev-lation between the type of mass and the assessment els. Only half of those 12 cases were histologically of masses as benign/borderline/malignant. proven malignant. .f histologically proven tumors 74.4% were be- The biggest diameter of all 124 ovarian masses nign and 25.6% were malignant. All masses clas-was measured – the largest one was 216 mm, the sified on MRI as benign were identified correctly. smallest one was under 10 mm. 54% of all tumors Two masses, described as suspicious and malig-had diameter larger than 4 cm. nant, turned out to be benign. All of the malignant FIGURE 1. (A) Box plot presenting ADC values in four different types of adnexal tumors – highest ADC value found in a simple cyst; lowest found in a malignant tumor. (B) Mean apparent diffusion coefficient (ADC) values of twelve patients with histologically proven benign adnexal lesion and twelve patients with histologically proven malignant adnexal lesion. All values are expressed as mean value ± standard deviation (SD) x 10 -3 mm 2 /s. tumors had restricted diffusion. The calculated ADC values of malignant adnexal masses are sig­nificantly lower than the ADC values of benign masses. Exceptions were found for endometrioma (1.01 ± 0.05 x 10-3 mm2/s); mature teratoma (0.80 ± 0.04 x 10–3 mm2/s) and chronic abscess (0.61 ± 0.06 TABLE 2. Results of 124 ovarian masses according to their MRI features Cystic masses Solid masses Mixed masses Cases with one ovary involvement Cases with both ovaries’ involvement Size of the mass (more than 4 cm) Masses with adipose tissue presence Masses with high signal intensity in T2WI Masses with low signal intensity in T2WI Heterogeneous masses Diffusion restriction Wall thickness (more than 3 mm) Presence of septa Papillary projections presented Cases with presence of ascites 5/41 (12.2%) 61/71 (85.9%) 6/12 (50%) 10/41 (24.4%) 4/71 (5.6%) 2/12 (16.6%) 26/41 (63.4%) 6/71 (8.5%) 4/12 (33.4%) 18/30 (60%) 46/58 (79.3%) 9/10 (90%) 12/30 (40%) 12/58 (20.7%) 1/10 (10%) 37/41 (90.2%) 22/71 (31%) 8/12 (66.7%) -5/71 (7%) 1/12 (8.3%) 5/41 (12.2%) 42/71 (59.1%) 5/12 (41.6%) 7/41 (17.1%) 18/71 (25.4%) 1/12 (8.4%) 29/41 (70.7%) 11/71 (15.5%) 6/12 (50%) 39/41 (95.1%) 19/71 (26.8%) 7/12 (58.4%) 20/41 (48.8%) 12/71 (16.9%) 5/12 (41.7%) 25/41 (61%) 11/71 (15.5%) 5/12 (41.7%) 14/41 (34.1%) -2/12 (16.6%) 16/30 (53.3%) 15/58 (25.9%) 2/10 (20%) T2WI = T2 weighted imaging x 10-3 mm2/s) – all of them presenting lower ADC values. (Figure 1) 72.7% of malignant neoplasms were mixed masses, 18.2% were solid and only one (9.1%) was cystic. Compared to them, 75% of be­nign tumors were cystic. 64 out of all 98 patients u nderwent contrast en­hancement. 34 there were a subject of contraindica­tions (history of previous allergic reactions to the contrast agent, elevated levels of serum creatinine or patient refusal). 39 (61%) of the masses showed enhancement. Three were classified as benign and four – as suspicious. 32 of the enhanced tumors were identified as malignant. Ascites was found in 33 of the cases – in 15 of which is located only in the pouch of Douglas. In 15.3% of the cases, enlarged lymph nodes with dif­fusion restriction were found – all in patients with malignant masses and one with a proven chronic inflammatory process. In 15 cases enlarged meta­static locoregional lymph nodes were found. Eight patients had peritoneal deposits; four patients liver metastases; three patients bone metastases, two pa­tients were with urinary bladder invasion and one patient had adrenal metastasis. In all cases with me­tastases three turned out to be from uterine cancer (ovarian masses in these cases were proven benign). Discussion Assessing different types of adnexal lesions is im­portant preoperatively. We find a number of rea­sons about the value of 3 Tesla MRI in such differ­entiation. The MRI gynecological protocols we used concur the ESUR Quick Guide to Female Pelvis Dimova J et al. / 3 Tesla MRI of adnexal masses 423 FIGURE 2. Type III time intensity curve (TIC) of a malignant adnexal mass. Imaging, 1.0 from 2019.15 Classical sequences (T1, T2) combined with post-gadolinium sequences and diffusion techniques provide reliable informa­tion on the nature of the adnexal masses. It is known from previous s tudies that dynamic contrast enhanced MRI (DCE-MRI) is helpful in characterizing adnexal tumors. It could discrimi­nate malignant from benign masses. According to the study of Thomassin-Naggara et al. there are three types of TIC showing benign, borderline and malignant ovarian tumors. Figure 2 demonstrates representative Type III curve of a malignant ad-nexal mass. The number of patients (98) in our study ex­ceeds those of similar ones known from the litera­ture (30 in El-Wekil et al.14 and 58 in Koc et al.16). The average age of patients (47.2 years) as well differs respectably by seven and four years from the cited studies.14,16 The WHO histological classification (according to Foti et al.17) divides primary ovarian masses into three main categories: epithelial, germ cell and sex cord-stromal tumors. Metastatic tumors are clas­sified in a separate category. In 2016 Meinhold-Heerlein et al. revised the WHO classification in­troducing seromucinous tumors as a new entity.18 Our study includes 14 histologically different groups of ovarian masses – ten benign and four TABLE 3. Diffusion MRI appearance of histologically different groups Simple cyst - 5 Inclusion cyst - 1 Abscess 1 - Endometrioma 12 5 Teratoma 5 - Serous cystadenoma - 2 Mucinous cystadenoma 1 1 Serous adenofibroma 1 - Serous cystadenofibroma 1 - Brenner tumor - 1 Seromucinous carcinoma 2 - Serous papillary adenocarcinoma 2 - Adenosarcoma 1 - DWI = diffusion-weighted imaging malignant. Diffusion MRI appearance of histologi­cally different groups is shown in Table 3. Some of the benign formations have diffusion restriction – abscess, endometrioma, mature cystic teratoma 424 FIGURE 3. 45-year old patient with bilateral adnexal masses; serous papillary cystadenoma (arrow) and mucinous cystadenoma (arrowhead); both masses have predominantly high signal intensity on T2WI and T2WI fat sat (A), (B) and low signal intensity on T1WI fat sat (C). and serous adenofibroma. In 88% of cases mature cystic teratomas are filled with sebaceous material and are lined with keratinized squamous epithe­lium19, compared to the most relevant feature – ad­ipose tissue which is presented in only 67–75%.20 Diffusion restriction is caused by the presence of keratin or Rokitansky nodule and fat globules.21 Endometriomas as containing blood and hemosi­derin can show diffusion restriction too.21,22 Solid areas with similar changes can help the detection of malignant transformation. When it comes to an ovarian abscess, diffusion characteristics depend on the content – in more viscous one the signal in­tensity is higher on DWI and lower on ADC map.23 Diffusion techniques could differentiate abscess from cystic or necrotic neoplasm. Neoplasms usu­ally show diffusion restriction peripherally and abscesses centrally.22,24 According to cystic degen­eration, some adenofibromas also could be charac­terized by restriction of the water molecules.22,25,26 In this study adnexal masses are classified based on their morphological appearance, similar to Foti et. al.17 and divided into three main groups – cystic, solid and mixed (cystic and solid). Cystic adnexal masses could be unilocular or multilocular. Some of them have a non-ovarian origin. They are usually benign, with low signal intensity on T1-weighted images and high signal intensity on T2-weighted images. Peritoneal inclusion cysts and hydrosalpinx are the most common extra ovarian lesions. They occur almost exclusively in premenopausal women and at imaging the ovaries are clearly separated from these cystic formations.27,28 Functional ovarian cysts are the most common finding in women of reproductive age. Follicles are up to 20 mm as the dominant one could be 25 mm. Follicular cysts and corpus luteum cysts are larger and tend to increase if there is internal bleeding. This manifests with an increase of the signal on FIGURE 4. 68-year old patient with previous hysterectomy; right serous cystadenofibroma (arrow); complex mass - heterogeneous on T2WI (A) which shows peripheral enhancement on T1WI fat sat with contrast (B). (C) Macroscopic histological preparation of the tumor. Dimova J et al. / 3 Tesla MRI of adnexal masses 425 T1-weighted images.13,17 They do not usually have diffusion restriction and does not change after con­trast administration. Although, in corpus luteum cysts intense wall enhancement may be seen. Serous cystadenoma and mucinous cystadeno-ma are benign tumors with thin walls (Figure 3). Mucinous type is usually larger, septated and has variable intensity on both T1- and T2-weighted images based on different mucin concentration. Some loculi are hyperintense on T1-weighted im­ages, forming a pattern known as “honeycomb” or “stained glass”.17,29,30 Serous cystadenoma is more often bilateral and its wall could contain small nod­ules due to fibrosis or calcification.31,32 Diffusion restriction could be detected in mucinous cystad­enoma due to the dense mucinous material.12 Cystadenofibroma is usually a benign epithelial tumor that can present as a complex cystic mass with thick septa and solid component. It could pre­sent with plaques and nodules that have low signal intensity on T2-weighted images due to fibrous tis­sue (Figure 4).33-35 Endometriomas are part of the cystic lesions containing blood products. In addition to that, they characterize with hyperintensity on T1-weighted images and lower signal intensity on T2-weighted images, called “shading sign”. Sometimes these lesions could have high signal intensity on both T1- and T2-weighed images. They do not change their signal intensity on fat-suppressed sequences.13,31,36,37 Patients having endometriosis are at risk of developing ovarian malignancies.38 Endometriomas usually do not enhance after con­trast administration but could have restricted dif­fusion.11,22 Mixed ovarian masses containing both cystic and solid parts are always suspicious for malig­nant – surface epithelial tumors and metastases. The benign representative of this category is ma­ture cystic teratoma. Mature cystic teratoma is known as the most common ovarian neoplasm that arises from ovar­ian germ cells.13,31 Usually part of this tumor has high signal intensity on T1WI and intermediate on T2WI, fat-fluid or fluid-fluid level, low signal calcification parts and floating debris. It could also have a soft-tissue protuberance called Rokitansky nodule. On fat-suppressed sequences the ar­eas containing fat show drop in signal intensity. Malignant transformation of mature cystic tera­toma is rare.19,37,39,40 Enhancement after contrast ap­plication is not typical. They could represent with restricted diffusion in the areas with keratin and fat globules.22,23 426 Serous and mucinous cystadenocarcinoma are the most common epithelial malignancies of the ovaries – 50% and 10% of malignant lesions.41 Mucinous tumors are larger, lobulated and may be hyperintense on T1WI in addition to the high protein concentration in mucoid material.30,42 Cystadenocarcinomas have thick and irregular walls, septations, solid components and papillary projections that have low signal intensity on T2WI with contrast enhancement after contrast adminis­tration. Serious fluid part demonstrates with high signal intensity on T2WI (Figure 5). Peritoneal in­vasion is sometimes discovered.17,41 In connection with their malignant nature, a pronounced diffu­sion restriction is observed. Ovarian metastases most frequently origi­nate from a primary process in the female genital tract, gastrointestinal tract (Krukenberg tumor) or breast. They are more commonly bilateral and mul­tiloculated. Their solid parts are hypointense on T2WI and enhance after gadolinium administra­tion. Distinguishing them from a primary ovarian process is not easy.17,41,43 Ovarian metastases have high signal intensity on DWI and low on ADC map (Figure 6). Other less common representatives of mixed ovarian neoplasms are endometrioid tumors, yolk sac tumors and granulosa cell tumors. Solid ovarian masses could have benign, border­line and malignant behavior. They include all three main histological types – epithelial, germ cell and sex cord tumors and metastases. The Brenner tumor is a rare epithelial tumor and represents 2% of ovarian neoplasms.44 It is usually benign and has largely homogeneous low signal intensity on T1- and T2-weighed images. Its signal intensity is similar to those of fibromas but no cysts and necrosis are found in Brenner tumor. This ovarian tumor can occur in association with mu-cinous cystadenoma (Figure 7). Mild enhancement is observed after contrast application. Diffusion re­striction is not characteristic of benign representa­tives of this tumor.39,45 Fibromas encounter around 4% of all ovarian tumors. They could mimic malignant neoplasm as their size can vary and may be associated with ascites and pleural effusion (Meig syndrome). Another pathology they should be defined from is pedunculated uterine leiomyoma. These tumors demonstrate low signal intensity on both T1- and T2-weighted images. Scattered areas of high signal intensity could be present on T1WI due to cystic degeneration or edema.13,17,35 In this case diffusion restriction may be found. After contrast adminis­tration minimal enhancement is evident. In this study 72.7% of histologically proven ma­lignant neoplasms were mixed cystic and solid, 18.2% were solid and only one (9.1%) was cystic. That statement disagrees with El-Wekil et al.14 where no solid mass was found but cystic masses were 37.5% of their case series. However, 62.5% of tumors in their study were mixed cystic and solid which roughly coincides with our findings. All of the histologically proven malignant le­sions in this study show restricted diffusion. This confirms the literature data that an adnexal mass with higher signal intensity on DWI and lower on ADC map usually is a malignant lesion. Our results confirm the findings of previous studies in the lit­erature that benign adnexal lesions have higher ADC values than the malignant once. We also found some exceptions of this statement concern­ing endometrioma, mature teratoma and chronic abscess presenting with lower ADC values despite of their benign origin. Borderline ovarian tumors are usually complex masses that have some of the MR characteristics of Dimova J et al. / 3 Tesla MRI of adnexal masses 427 the malignant one. They could show cellular pro­liferation and moderate nuclear atypia but without stromal invasion.46,47 Similar to the study of Bent et. al.46 we identified 11 of the cases as suspicious. All of them demonstrated one or more MRI feature suggestive for malignancy – size more than 4 cm, solid part, cystic part with vegetations and septa-tions, wall thickness more than 3 mm; contrast en­hancement. In our study only one of the 10 suspi­cious cases were bilateral. CA-125 is established tumor marker for ovar­ian cancer.13,48 Limitation of this study is the small number of CA-125 tests performed before magnetic resonance imaging. Of these, elevated levels of CA­125 were found in 12 patients. Similarly, to other studies, over 60% of our patients with elevated CA­125 levels have proven malignant ovarian lesions. Concerning unilateral or bilateral adnexal masses, we found malignant to be more often bi­lateral. Unilateral lesions are more often found in the right adnexa and in younger patients. This study as well as the Zhang et al. one49 suggests that large sizes and atypical signal intensity may influ­ence the correct assessment of the type of ovarian lesions. The main limitations of our study include the retrospective reviewing of patients with some clinical missing, as well as surgical missing find­ings in patients who underwent surgery in anoth­er hospital. Conclusions Classifying adnexal masses is essential for the pre­operative management of the patients. 3T MRI pro­tocols, in particular diffusion techniques, increase significantly the accuracy of the diagnostic assess­ment. Further studies correlated with histological validation would support the role of MRI as a man­datory part of the patients’ management. Acknowledgements This study was partially supported by the Japanese Society for the Promotion of Science (JSPS) (Grand-in-aid “Kakenhi C" granted to R.B. and the Japanese Agency for Medical Research and Development (AMED) (Project for Cancer Research and Therapeutic Evolution, P-CREATE, no. 16 cm0106202h0001). References 1. Expert Panel on Women’s Imaging, Atri M, Alabousi A, Reinhold C, Akin EA, Benson CB, Bhosale PR, et al. ACR Appropriateness CriteriaR clinically suspected adnexal mass, no acute symptoms. J Am Coll Radiol 2019; 16: S77-93. doi: 10.1016/j.jacr.2019.02.011 2. Sharma A, Apostolidou S, Burnell M, Campbell S, Habib M, Gentry-Maharaj A, et al. Risk of epithelial ovarian cancer in asymptomatic women with ultrasound-detected ovarian masses: a prospective cohort study within the UK collaborative trial of ovarian cancer screening (UKCTOCS). Ultrasound Obstet Gynecol 2012. 40: 338-44. doi: 10.1002/uog.12270 3. Biggs WS, Marks ST. Diagnosis and management of adnexal masses. 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Evaluation of primary ad-nexal masses by 3T MRI: categorization with conventional MR imaging and diffusion-weighted imaging. J Ovarian Res 2012; 5: 33. doi: 10.1186/1757­2215-5-33 429 research article The influence of genetic variability in IL1B and MIR146A on the risk of pleural plaques and malignant mesothelioma Petra Piber1, Neza Vavpetic1, Katja Goricar2, Vita Dolzan2, Viljem Kovac1,3, Alenka Franko1,4 1 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 2 Pharmacogenetics Laboratory, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Clinical Institute of Occupational Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(4): 429-436. Received 7 July 2020 Accepted 6 August 2020 Correspondence to: Assoc. Prof. Alenka Franko, M.D., Ph.D., Clinical Institute of Occupational Medicine, University Medical Centre Ljubljana, Poljanski nasip 58, SI-1000 Ljubljana, Slovenia. Email: alenka.franko@siol.net Petra Piber and Neza Vavpetic contributed equally. Disclosure: No potential conflict of interest were disclosed. Background. Asbestos exposure is associated with the development of pleural plaques as well as malignant meso­thelioma (MM). Asbestos fibres activate macrophages, leading to the release of inflammatory mediators including interleukin 1 beta (IL-1ß). The expression of IL-1ß may be influenced by genetic variability of IL1B gene or regulatory microRNAs (miRNAs). This study investigated the effect of polymorphisms in IL1B and MIR146A genes on the risk of developing pleural plaques and MM. Subjects and methods. In total, 394 patients with pleural plaques, 277 patients with MM, and 175 healthy control subjects were genotyped for IL1B and MIR146A polymorphisms. Logistic regression was used in statistical analysis. Results. We found no association between MIR146A and IL1B genotypes, and the risk of pleural plaques. MIR146A rs2910164 was significantly associated with a decreased risk of MM (OR = 0.31, 95% CI = 0.13–0.73, p = 0.008). Carriers of two polymorphic alleles had a lower risk of developing MM, even after adjustment for gender and age (OR = 0.34, 95% CI = 0.14–0.85, p = 0.020). Among patients with known asbestos exposure, carriers of at least one polymorphic IL1B rs1143623 allele also had a lower risk of MM in multivariable analysis (OR = 0.50, 95% CI = 0.28–0.92, p = 0.025). The inter­action between IL1B rs1143623 and IL1B rs1071676 was significantly associated with an increased risk of MM (p = 0.050). Conclusions. Our findings suggest that genetic variability of inflammatory mediator IL-1ß could contribute to the risk of developing MM, but not pleural plaques. Key words: asbestos; genetic variation; malignant mesothelioma; miRNA; pleural plaques Introduction Asbestos exposure is related to several pleural diseases, such as pleural plaques, diffuse pleu­ral thickenings, pleural effusions and malignant mesothelioma (MM). MM is an aggressive form of cancer found on the mesothelium, generally on the pleura (65%), peritoneum (30%) or other serosal membranes (1%).1,2 MM is often diagnosed in its later stages, is rarely operable and can respond poorly to con­ventional chemotherapy.3 Clinical signs and symptoms are uncharacteristic and reminiscent of many other pulmonary diseases. Patients often experience dyspnea, chest pain, weight loss and fa­tigue. Only a small proportion of MM patients are asymptomatic at the time of diagnosis.2,4 Average life expectancy is around 7 months with support 430 therapy and 12 months with chemotherapy.5 MM most often occurs in patients older than 65 years.2,6 Epidemiological studies have shown that the main cause of MM is asbestos exposure, with the inci­dence of this cancer still increasing due to the long latent period.7 Genetic factors have also been sug­gested to influence the development of MM; pa­tients often have mutations in tumour suppressor genes, such as BAP1, CDKN2A and NF2.8,9 Along with MM, asbestos exposure is also related to the development of pleural plaques. Pleural plaques are white and yellow thicken­ings of pleura, often asymmetrical and bilateral. Histologically, they are acellular, composed of hyalinised collagen, which is covered by one lay­er of mesothelial cells. Half of the patients with a history of asbestos exposure develop pleural plaques, typically 20 to 30 years after exposure. The risk of pleural plaques rises with the length of asbestos exposure.10 It has been proposed that in­flammation caused by asbestos is involved in the pathogenesis of both pleural plaques and MM.3,11 Asbestos fibres are known to trigger the release of inflammatory mediators, which leads to the downregulation of apoptosis.3 After inhalation, asbestos fibres reach pleural space and are deposited in mesothelial cells.12 This leads to local inflammatory response and prolif­eration of mesothelial cells. In vitro studies have shown that the fibres induce inflammation and ap­optosis and most of the tissue damage is related to elevated interleukin 1 beta (IL-1ß).3 Macrophages accumulate near asbestos deposits and release cy­tokines, such as IL-1ß and tumour necrosis factor alpha (TNF-a).8 In response to asbestos, an intrinsic inflamma­tory mechanism triggers inflammation via inflam­masome NLRP3, which is NLR family pyrin do­main containing 3, activated by danger-associated molecular patterns (DAMP) or pathogen-associat­ed molecular patterns (PAMP).13,14 NLRP3 inflam­masome is a protein complex of NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1, found in macrophages, which triggers a type of apoptosis, known as piroptosis.11,13,15 The activation of NLRP3 inflammasome increases the production of IL-1ß from its precursor, mediated by caspase-1 and pro-inflammatory mediators from macrophag­es.8,15,16 IL-1ß, coded by IL1B gene, is an inflamma­tory mediator, found during chronic inflammation and a key player in carcinogenesis.17,18 It promotes neutrophil recruitment and transcription of NF-.B (nuclear factor kappa B), the latter being known to influence tumour growth and response to chemo­therapy.18 In vitro studies showed that IL-1ß plays an important part in increasing proliferation, lead­ing to a malignant transformation.8 IL-1ß release can also be regulated by miRNAs, 21-23 nucleotides long non-coding RNAs, which inhibit translation by binding to the 3'-untrans­lated region (3'-UTR) of mRNA.19 miRNAs are involved in networks of gene regulation and their expression often changes in cancerous tissue, in­cluding MM.20-25 A key miRNA, influencing the ex­pression of IL1B, is miRNA-146. Two human vari­ants are found; miRNA-146a and miRNA-146b, both assumed to play a role in toll-like receptor (TLR) based signalling and cytokine response.21,22,26 Previous studies found that miRNA-146a has an anti-inflammatory function, with its silencing lead­ing to an increase in IL-1. and its induction having the opposite effect.22,26,27 Genetic factors, such as single nucleotide poly­morphisms (SNPs), may influence protein ex­pression.17,28,29 IL1B rs16944 (c.-511C>T), located in 5’ untranslated region (UTR), influences the binding of transcription factors.30 Higher levels of IL-1ß were found in homozygotes with polymor­phic allele, leading to a higher risk of developing chronic inflammation-related diseases, such as diabetes mellitus type 2 and breast cancer.31,32 IL1B rs1143623 (-1464G>C) is also located in 5’ UTR and affects the binding of transcription factors.30 Its polymorphic C allele was associated with a lower risk of developing lung and colorectal carcinoma, 28,29 due to a lower production and release of IL-1ß.The relationship between IL1B rs1071676, located in 3’UTR, and carcinogenesis has not yet been es­tablished, but as it affects miRNA binding site, it could also influence IL1B expression. SNPs have also been found in genes coding for miRNAs, such as MIR146A rs2910164, which has been related to both higher33 and lower risks34 of malignant trans­formations, according to previous research.35 To the best of our knowledge, the role of IL1B and MIR146A genetic variability in the develop­ment of asbestos-related diseases has not been evaluated so far. The aim of the present study was therefore to evaluate the influence of IL1B and MIR146A polymorphisms on the risk of develop­ing pleural plaques and MM. Subjects and methods Subjects The retrospective case-control study included 277 patients with histologically confirmed pleu- Piber et al. / IL1B and MIR146A SNPs in malignant mesothelioma 431 ral or peritoneal MM, treated at the Institute of Oncology Ljubljana between 1 January 2001 and 30 September 2018, 394 patients with pleural plaques and 175 healthy control subjects, all of whom were previously exposed to asbestos. The control group and those with pleural plaques were occupation­ally exposed to asbestos by working in the factory Salonit Anhovo, Slovenia, and were presented at the State Board for the Recognition of Occupational Asbestos Diseases between January 1999 and December 2003. In 2018, the subjects from the con­trol group were found not to have any asbestos-related disease. The study was approved by the Slovenian Ethics Committee for Research in Medicine and was car­ried out according to the Declaration of Helsinki. Clinical diagnosis Patients with pleural plaques have been diagnosed based on X-ray and high-resolution computed to­mography (HRCT), while MM diagnosis was con­firmed by a pathologist based on the histopathol­ogy of samples gathered thoracoscopically in the case of the pleural and laparoscopically in the case of the peritoneal type of MM.2,36,37 Asbestos exposure and smoking A semiquantative method was used to assess the asbestos exposure. The data on cumulative asbes­tos exposure expressed in fibres/cm3-years were available for all control subjects, all subjects with pleural plaques except for 6, and for 40 subjects with MM. Based on these data, the asbestos expo­sure in these subjects was categorised into three groups: low (< 11 fibres/cm3-years), medium (11–20 fibres/cm3-years) and high (> 20 fibres/cm3-years) asbestos exposure. For additional 49 subjects with MM who lacked the data on cumulative asbestos exposure a thorough work history was obtained by an interview performed by a single expert ex­perienced in asbestos exposure assessment. Their exposures were compared with the exposures from the group of patients with known cumulative as­bestos exposure and were categorized accordingly into three groups with presumed low, medium and high asbestos exposure.2 For the remaining 188 MM patients, exact data on asbestos exposure were not available. An interview based on a standardized question­naire was conducted with the control group and patients with pleural plaques to collect data on smoking, while the medical documentation of the Institute of Oncology of Ljubljana was used to ob­tain this piece of data for patients with MM.2,38 Single nucleotide polymorphism (SNP) selection Using LD Tag SNP Selection,30 dbSNP,39 Ensembl40 and LDlink41 we identified IL1B SNPs, which had the minor allele frequency (MAF) greater than 0.05 in the European population, could influence the expression of IL1B and were located less than 5000 base pairs up- or downstream from the gene. Polymorphism rs1071676, located in 3’UTR as well as rs16944 and rs1146323, located in 5’UTR matched our criteria. Based on miRDB,42 miRTarBase43 and Variation Viewer we identified miRNAs, that could influence IL1B expression and SNPs in the genes coding for these miRNAs. Based on the inclusion cri­teria, we selected rs2910164, a SNP in miRNA-146a. Molecular genetic analysis We isolated DNA from venous blood of 44 pa­tients with MM using E.Z.N.A.® SQ II Blood DNA Kit (Omega Bio-tek, Inc., Norcross, Georgia, USA) following the manufacturer’s instructions. DNA samples of all other subjects had been isolated dur­ing previous studies.44 Genotyping was performed using competitive allele-specific PCR (KASP), the KASP Master mix (LGC, Middlesex, UK) and cus­tom KASP Genotyping Assay (LGC, Middlesex, UK) according to the manufacturer’s instructions. Statistics Median and interquartile range were used to de­scribe continuous variables, while frequencies were used for categorical variables. To compare the distribution of categorical variables, Fisher’s exact test was performed, while non-parametric Kruskal-Wallis test was used for continuous vari­ables. Deviation from the Hardy-Weinberg equilib­rium (HWE) was evaluated using chi-square test. Both additive and dominant genetic models were used in statistical analyses. Univariable and mul­tivariable logistic regression was used to analyse the association between genotypes and asbestos-related diseases (pleural plaques and MM). For the analysis of multiplicative interactions between genotypes, logistic regression models using dum­my variables were used. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 21.0 (IBM Corporation, Armonk, NY, USA). Results Characteristics of patients with MM and pleural plaques as well as the control group are shown in Table 1. There was a statistically significant dif­ference between the groups in respect to age (p < 0.001) and asbestos exposure (p < 0.001). MM patients were significantly older than the control group or patients with pleural plaques. Among the subjects with known asbestos exposure, 51.7% of patients with MM had medium or high exposure compared to 23.4% of the control group and 28.4 % of patients with pleural plaques. There was no statistically significant difference between groups regarding gender (p = 0.410) and smoking status (p = 0.267) (Table 1). A further analysis of asbestos exposure showed that medium and high levels of asbestos exposure were associated with an increased risk of MM com­pared both to the control group (odds ratio [OR] = 3.50; 95% confidence interval [CI] = 2.03–6.02; p < 0.001) and patients with pleural plaques (OR = 2.70; 95% CI = 1.69–4.33; p < 0.001). Among the patients with MM, 19 (6.9 %) had stage I MM, 61 (22.1 %) were in stage II of the dis­ease, 83 (30.1 %) had stage III and 81 (29.3 %) stage IV MM. Thirty-two patients (11.6 %) had the peri­toneal subtype of MM, where stage was not deter­mined and in one patient, the MM stage could not be determined. In our cohort, the most prevalent histological subtype of MM was the epithelioid subtype (206; 74.4 %); however some of the patients had either the biphasic (26; 9.4 %) or sarcomatoid subtype (26; 9.4 %) and in the case of a few patients (19; 6.6 %), the histological subtype was not deter­mined. TABLE 1. Characteristics of subjects included in the study A comparison of patients with pleural plaques and healthy controls revealed no statistically sig­nificant influence on the risk of pleural plaques for any of the selected polymorphisms, neither in univariable analysis nor after adjustments for age, gender and asbestos exposure (Supplementary Table 1). The analysis of the association between genetic polymorphisms and MM has shown statistically significant influence of polymorphism MIR146A rs2910164 on the risk of developing MM. Carriers of two polymorphic alleles (genotype CC) had a lower risk of developing MM (OR = 0.31; 95% CI = 0.13–0.73; p = 0.008). There was no influence of other genetic polymorphisms on the development of MM (Table 2). In multivariable analysis, polymorphism MIR146A rs2910164 remained associated with a decreased risk of developing MM after adjustment for age and gender (OR = 0.34; 95% CI = 0.14–0.85; p = 0.020). However, in the subgroup with asbestos exposure data, MIR146A rs2910164 polymorphism no longer showed statistically significant influence on the risk of MM after adjustment for age, gender and asbestos exposure (OR = 0.39; 95% CI = 0.11– 1.38; p = 0.144) (Table 2). Carriers of at least one polymorphic IL1B rs1143623 (genotype GC or CC) showed a significantly decreased risk of MM after adjustment for age, gender and asbestos exposure (OR = 0.50; 95% CI = 0.28–0.92; p = 0.025) (Table 2). A comparison of patients with MM and pleu­ral plaques showed that polymorphism MIR146A rs2910164 was statistically significantly associated with the risk of the development of MM compared to pleural plaques (Table 3). Patients that had two polymorphic MIR146A rs2910164 alleles (geno- Gender Male, N (%) 119 (68.0) 271 (68.8) 202 (72.9) 1.757a 0.410 Female, N (%) 56 (32.0) 123 (31.3) 75 (27.1) Age Median (25%–75%) 55.3 (48.6–63.7) 54.9 (48.8–62.7) 66.0 (59.0–73.0) 151.666b < 0.001 Low, N (%) 134 (76.6) 278 (71.6) [6] 43 (48.3) [188] 26.891a < 0.001 Asbestos exposure Medium, N (%) 13 (7.4) 41 (10.6) 24 (27.0) High, N (%) 28 (16.0) 69 (17.8) 22 (24.7) No, N (%) 94 (53.7) 194 (49.4) [1] 150 (55.6) [7] 2.640a 0.267 a calculated using Fisher exact test; b calculated using Kruskal-Wallis test; number of missing data is presented in [] brackets Piber et al. / IL1B and MIR146A SNPs in malignant mesothelioma 433 TABLE 2. Association between selected polymorphisms and the risk of developing malignant mesothelioma GG 88 (50.3) 152 (54.9) reference reference IL1B rs1143623 GC 67 (38.3) 97 (35.0) 0.84 (0.56–1.26) 0.396 0.82 (0.52–1.29) 0.388 0.56 (0.29–1.05) 0.072 CC 20 (11.4) 28 (10.1) 0.81 (0.43–1.52) 0.514 0.69 (0.35–1.38) 0.294 0.34 (0.11–1.04) 0.060 GC+CC 87 (49.7) 125 (45.1) 0.83 (0.57–1.22) 0.341 0.79 (0.52–1.20) 0.266 0.50 (0.28–0.92) 0.025 TT 21 (12.0) 36 (13.0) 1.10 (0.60–2.02) 0.756 0.94 (0.48–1.82) 0.849 0.53 (0.20–1.43) 0.210 IL1B rs16944 TC 75 (42.9) 118 (42.6) 1.01 (0.67–1.51) 0.960 0.98 (0.63–1.52) 0.911 0.67 (0.36–1.24) 0.198 CC 79 (45.1) 123 (44.4) reference reference TC+TT 96 (54.9) 154 (55.6) 1.03 (0.70–1.51) 0.878 0.97 (0.64–1.47) 0.873 0.63 (0.35–1.13) 0.122 GG 105 (60.0) 165 (59.6) reference reference IL1B rs1071676 GC 60 (34.3) 98 (35.4) 1.04 (0.69–1.56) 0.851 0.97 (0.62–1.51) 0.895 1.00 (0.53–1.89) 0.993 CC 10 (5.7) 14 (5.1) 0.89 (0.38–2.08) 0.789 0.96 (0.38–2.41) 0.931 2.03 (0.70–5.90) 0.194 GC+CC 70 (40.0) 112 (40.4) 1.02 (0.69–1.50) 0.927 0.97 (0.64–1.48) 0.885 1.15 (0.64–2.08) 0.632 GG 94 (53.7) 158 (57.0) reference reference GC 64 (36.6) 110 (39.7) 1.02 (0.69–1.53) 0.913 0.91 (0.59–1.41) 0.672 0.67 (0.36–1.25) 0.209 CC 17 (9.7) 9 (3.2) 0.31 (0.13–0.73) 0.008 0.34 (0.14–0.85) 0.020 0.39 (0.11–1.38) 0.144 adj1 = adjustment for age and gender; adj2 = adjustment for age, gender and asbestos exposure; CI = confidence interval; MM = malignant mesothelioma; OR = odds ratio; SNP = single nucleotide polymorphism TABLE 3. Association between selected polymorphisms and the risk of developing malignant mesothelioma compared to pleural plaques GG 205 (52.0) 152 (54.9) reference reference IL1B rs1143623 GC 157 (39.8) 97 (35.0) 0.83 (0.60–1.16) 0.277 0.88 (0.61–1.27) 0.499 0.67 (0.39–1.15) 0.151 CC 32 (8.1) 28 (10.1) 1.18 (0.68–2.04) 0.554 1.07 (0.58–1.99) 0.827 0.65 (0.22–1.86) 0.418 GC+CC 189 (48.0) 125 (45.1) 0.89 (0.66–1.21) 0.467 0.92 (0.65–1.29) 0.617 0.67 (0.40–1.11) 0.123 TT 50 (12.7) 36 (13.0) 1.02 (0.63–1.67) 0.923 0.96 (0.56–1.67) 0.897 0.54 (0.22–1.34) 0.184 IL1B rs16944 TC 169 (42.9) 118 (42.6) 0.99 (0.71–1.38) 0.969 0.98 (0.68–1.42) 0.929 0.70 (0.41–1.19) 0.186 CC 175 (44.4) 123 (44.4) reference reference TC+TT 219 (55.6) 154 (55.6) 1.00 (0.73–1.36) 0.998 0.98 (0.69–1.38) 0.905 0.66 (0.40–1.10) 0.110 GG 233 (59.1) 165 (59.6) reference reference IL1B rs1071676 GC 145 (36.8) 98 (35.4) 0.95 (0.69–1.32) 0.778 0.93 (0.65–1.34) 0.708 0.92 (0.53–1.60) 0.774 CC 16 (4.1) 14 (5.1) 1.24 (0.59–2.60) 0.578 1.29 (0.56–2.97) 0.553 2.83 (1.04–7.71) 0.042 GC+CC 161 (40.9) 112 (40.4) 0.98 (0.72–1.34) 0.911 0.97 (0.68–1.37) 0.849 1.09 (0.66–1.82) 0.731 GG 196 (49.7) 158 (57.0) reference reference GC 163 (41.4) 110 (39.7) 0.84 (0.61–1.15) 0.276 0.84 (0.58–1.20) 0.326 0.65 (0.38–1.11) 0.118 CC 35 (8.9) 9 (3.2) 0.32 (0.15–0.68) 0.003 0.33 (0.15–0.75) 0.008 0.34 (0.11–1.09) 0.069 adj1 = adjustment for age and gender; adj2 = adjustment for age, gender and asbestos exposure; CI = confidence interval; MM = malignant mesothelioma; OR = odds ratio; SNP = single nucleotide polymorphism 434 type CC) had a significantly decreased risk of MM compared to pleural plaques (OR = 0.32; 95% CI = 0.15–0.68; p = 0.003). Similarly, after adjustment for gender and age, patients who were homozygotes for polymorphic MIR146A rs2910164 still showed a lower risk of developing MM (OR = 0.33; 95% CI = 0.15–0.75; p = 0.008). In the subgroup with avail­able asbestos exposure data, MIR146A rs2910164 was significantly associated with a decreased MM risk only in the dominant model (OR = 0.59; 95% CI = 0.36–0.99; p = 0.046) (Table 3). Additionally, patients that were homozygotes for the IL1B rs1071676 polymorphism (CC genotype) had an increased risk of developing MM when patients with pleural plaques were used as a control group and the analysis was adjusted for age, gender and asbestos exposure (OR = 2.83; 95% CI = 1.04–7.71; p = 0.042) (Table 3). In further logistic regression modelling, the in­teractions between polymorphisms showed no significant influence on the risk of pleural plaques (data not shown). The analysis of the influence of the interaction between IL1B rs1143623 and IL1B rs1071676 polymorphisms showed significant in­fluence on the increased MM risk (OR = 2.24, 95% CI = 1.00–5.00, p = 0.050). No other interactions between polymorphisms had a statistically signifi­cant influence on the risk of MM (Supplementary Table 2). Discussion The association between MM and asbestos expo­sure has first been described in 1960 and, although very few genetic factors have been studied, multi­ple factors have since then been considered to in­fluence the pathogenesis of MM.45 In the present study, we evaluated the effect of polymorphisms of IL-1ß and miRNA-146a genes on the risk of de­veloping MM and pleural plaques. The key finding of the present study was the association between MIR146A rs2910164 and lower risk of the develop­ment of MM. Consistent with the previous studies, the aver­age age of MM patients was found to be higher than that of the patients with pleural plaques or the con­trol group, probably due to the long latency period between the first asbestos exposure and MM.2,6,44 Our study showed no significant association be­tween smoking and MM, which is in agreement with previous findings.2,44,46 Subjects with high or medium exposure to asbestos had a higher risk of developing MM, compared to the group with pleu­ral plaques or the control group. Regardless of that, almost half (48.3%) of MM patients were exposed to low levels of asbestos, which is consistent with previous studies claiming there is no threshold lev­el for the development of MM.47,48 It is not yet clear to what an extent the pleural plaques present a risk factor for MM. The studies performed so far suggested that pleural plaques are more a sign of asbestos exposure, than a carci­nogenic factor.49,50 This hypothesis is in agreement with the findings of this study as the genotype frequency distribution of patients with pleural plaques was found to be more similar to that of the control group, rather than the genotype frequency distribution of patients with MM. Compared to both the control group and the patients with pleural plaques, homozygotes with polymorphic MIR146A rs2910164 C allele were at a lower risk of developing MM, even after adjust­ment for age and gender. In the subgroup of pa­tients with known asbestos exposure, carriers of at least one polymorphic MIR146A rs2910164 allele had a lower risk of MM in comparison to patients with pleural plaques. According to our knowledge, the relation be­tween MIR146A rs2910164 and MM has not yet been studied, but the polymorphism itself has already been associated with several other malig­nant diseases. Previous studies suggested that the polymorphic allele C had a protective function in the oncogenesis of melanoma51 and non-small cell lung carcinoma33, while the same was found for the G allele in case of papillary thyroid tumour.52 The association of rs2910164 with the pathogenesis of MM could be explained with its influence in miR-NA expression: CC genotype was previously as­sociated with a greater production of miRNA-146a in cancerous tissue.53-55 Increased expression of miRNA-146a in turn leads to the suppression of in­flammatory pathways, reducing the expression of proinflammatory cytokines IL-1., IL-6 and TNFa,56 while miRNA-146a inhibition has been shown to increase production of those cytokines, resulting in greater inflammatory response to asbestos, pro­moting carcinogenesis and increasing the risk of MM.17,18 The role and expression of miRNA-146a in carcinogenesis is still unclear, as some studies found the levels of miRNA-146a to be decreased in cancerous tissue of the lung57 and stomach carci-noma,58 while other studies found increased levels in the cases of melanoma,51 cervical cancer59 and papillary thyroid cancer.52 It is possible that miR­NA-146a has a tissue-specific function, so further studies are required. Piber et al. / IL1B and MIR146A SNPs in malignant mesothelioma 435 Another important finding of this study has been the association between the polymorphic IL1B rs1143623 allele and a lower risk of develop­ing MM. In the subgroup of subjects with known asbestos exposure, subjects with at least one poly­morphic C allele had a lower risk of developing MM compared to the control group. IL1B rs1143623 is located at the biding site of the transcription factors and can lower the expression of IL1B.30,39 Lower levels of IL-1ß result in a less intensive in­flammatory reaction caused by the asbestos fibres, which could have a protective effect. The former is in agreement with the studies that showed subjects with one polymorphic C allele having a lower risk of developing lung cancer28 and homozygotes for the polymorphic C allele having a lower risk of de­veloping colorectal cancer.29 Finally, this study has shown that the interac­tion between IL1B rs1143623 and IL1B rs1071676 is associated with a higher risk of developing MM, even though IL1B rs1071676 independently had no effect on the risk of MM, while IL1B rs1143623 was associated with a lower risk of MM within the sub­group of subjects with known asbestos exposure. IL1B rs1143623 was associated with a lower risk of MM only among carriers of two wild type IL1B rs1071676 alleles. As IL1B rs1143623 can influence the binding of transcription factors and rs1071676 can influence the binding of miRNA, the interac­tion of both polymorphisms could result in a great­er expression of IL-1ß, however this has not been studied yet.30 Further studies are needed to explain the role of IL1B rs1143623 and its interactions with other polymorphisms and environmental factors in MM. Lack of asbestos exposure information for all the subjects has been identified as the limitation of our study. Therefore, the subgroup for which as­bestos exposure has been taken into consideration, was smaller than the overall sample. This could account for the discrepancy between the results of the analysis adjusted for asbestos exposure and the results of the analysis that did not take asbestos ex­posure into account. The strength of this study is its large sample size. To the best of our knowledge, this is also the first study researching the effect of IL1B and MIR146A polymorphisms on the risk of developing MM. In conclusion, our results suggest that IL1B and MIR146A polymorphisms may contribute to the risk of MM development. Further studies, possibly evaluating serum or tissue protein expression, are needed to confirm these associations in independ­ent patient cohorts and elucidate the role of IL-1. and miRNA-146a in the development of asbestos-related diseases. Acknowledgements This work was financially supported by the Slovenian Research Agency (ARRS Grants No. P1­0170 and L3-8203). References 1. Kovac V, Dodic-Fikfak M, Arneric N, Dolzan V, Franko A. Fibulin-3 as a bio-marker of response to treatment in malignant mesothelioma. Radiol Oncol 2015; 49: 279-85. doi: 10.1515/raon-2015-0019 2. Franko A, Kotnik N, Goricar K, Kovac V, Dodic-Fikfak M, Dolzan V. The influ­ence of genetic variability on the risk of developing malignant mesothe­lioma. 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PLoS One 2008; 3: e2557. doi: 10.1371/journal. pone.0002557 437 research article Neutrophil-to-lymphocyte ratio can predict outcome in extensive-stage small cell lung cancer Gordana Drpa1, Maja Sutic2, Jurica Baranasic2, Marko Jakopovic1, Miroslav Samarzija1, Suzana Kukulj1, Jelena Knezevic2 1 Department for Lung Diseases Jordanovac, University Hospital Center Zagreb, Zagreb, Croatia 2 Laboratory for Advanced Genomics, Division of Molecular Medicine, Ruder Boškovic Institute, Zagreb, Croatia Radiol Oncol 2020; 54(4): 437-446. Received 18 February 2020 Accepted 22 July 2020 Correspondence to: Gordana Drpa, Jordanovac 104, 10000 Zagreb, Croatia. E-mail: gordana.drpa@kbc-zagreb.hr Disclosure: The authors declare no conflict of interest. Background. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to­monocyte ratio (LMR) were analyzed in various carcinomas and their potential prognostic significance was deter­mined. The objective of present study was to determine the correlation between these parameters and the survival of patients with small cell lung cancer (SCLC), since very few studies have been published on this type of carcinoma. Patients and methods. One hundred and forty patients diagnosed with SCLC at University Hospital Center Zagreb, between 2012 and 2016 were retrospectively analyzed. Extensive-stage disease (ED) was verified in 80 patients and limited-stage disease (LD) in 60 patients. We analyzed the potential prognostic significance of various laboratory pa­rameters, including NLR, PLR, and LMR, measured before the start of treatment. Results. Disease extension, response to therapy, chest irradiation and prophylactic cranial irradiation (PCI), as well as hemoglobin, monocyte count, C-reactive protein (CRP), and lactate dehydrogenase (LDH) showed a prognostic significance in all patients. When we analyzed the patients separately, depending on the disease extension, we found that only skin metastases as well as LDH and NLR values, regardless of the cut-off value, had a prognostic significance in ED. Meanwhile, the ECOG performance status, chest irradiation, PCI, and hemoglobin and creatinine values had a prognostic significance in LD. Conclusions. NLR calculated before the start of the treatment had a prognostic significance for ED, while PLR and LMR had no prognostic significance in any of the analyzed groups of patients. Key words: small cell lung cancer; hematological markers; neutrophil-to-lymphocyte ratio; platelet-to-lymphocyte ratio; lymphocyte-to-monocyte ratio Introduction Lung cancer is still one of the most malignant dis­eases nowadays. It is the most commonly occur­ring cancer in men and the second most commonly occurring cancer in women according to the lat­est data by the International Agency for Research on Cancer (IACR).1 At the same time, lung cancer is the leading cause of cancer death among both men and women. For the purposes of comparison, breast cancer in women occurs three times more of­ten than lung cancer, while the mortality is almost equal. Moreover, prostate cancer and lung cancer have almost the same incidence in men, but the lung cancer mortality rate is four times higher than the prostate cancer mortality rate.1,2 Small cell lung cancer (SCLC) is the most aggres­sive subtype of lung cancer. Nowadays, small cell lung cancer makes up about 15% of all lung can­cers and occurs almost only in smokers. The inci­dence of this lung cancer subtype has decreased in the last few decades, but primarily in developed 438 countries.3,4 There are no global data on SCLC prev­alence. In Croatia, there are no separate data on SCLC either, and the available epidemiological data relate to lung cancer as an entity. In the last twenty years, a slight reduction in the share of SCLC in re­lation to the total number of lung cancer patients has been observed at our institution, which is the largest thoracic oncology center in the country. According to literature there are differences in survival rates for various tumors, including small cell lung cancer, depending on ethnic origin.5 Therefore, the results of epidemiological and clini­cal studies in one geographic area are not applica­ble to some other geographic areas. The main characteristics of small cell lung can­cer are its rapid growth and early spread to distal body parts. This is the reason why in most cases this carcinoma is diagnosed late, when metastatic disease has already developed.6 Surgical treat­ment is therefore rarely possible, but in the last few years it has been recommended for certain pa­tients with early-stage disease.7 Before the intro­duction of platinum-based antineoplastic drugs for the treatment of malignant disease, the median survival of patients diagnosed with small cell lung cancer was two to three months.8,9 The survival rate has increased four to five times with chemo­therapy, but for most patients with extensive-stage disease it does not exceed ten months. In fact, this tumor is extremely chemosensitive and usually responds to chemotherapy very well. However, it recurs very rapidly and most patients die after a relapse. Despite numerous clinical trials, progress in the treatment of small cell lung cancer has been modest. However, as treatment of limited disease (LD) became more successful with the introduc­tion of thoracic radiotherapy and prophylactic cranial irradiation (PCI), concurrent chemoradio-therapy has been a standard in the treatment of LD for a long time now.6 The optimal radiation therapy protocol has remained controversial un­til this day, although it has been established that there are no differences in either survival or tox­icity between hyperfractionated and normofrac­tionated radiotherapy.10,11 The application of con­solidation radiotherapy in selected patients with extensive-stage disease (ED) and a good initial re­sponse to chemotherapy have partly contributed to the improved survival rate, but application has been very inconsistent.12,13 Immunotherapy has resulted in significant progress in the treatment of numerous malignant diseases, including non-small cell lung cancer (NSCLC). Expectations for the treatment of small cell lung cancer were high as well. For the time being, adding checkpoint in­hibitors to first-line chemotherapy in ED has re­sulted in a slight increase of overall survival and progression-free survival, but the results are far from expected.14-16 It is well known that infection and deregulated inflammatory response are associated with the oc­currence and progression of almost all chronic dis­eases, including cancers.17 In the last few decades, a great number of researches investigating the role of different inflammatory markers in cancer de­velopment and outcome have been published.18-20 Usually the investigated inflammatory markers in­clude C-reactive protein (CRP), lactate dehydroge­nase (LDH), erythrocyte sedimentation rate, plate­let (Pc) and neutrophil counts.21-23 In most cases, it has been found that elevated levels of these param­eters are associated with poorer outcome of vari­ous cancers, including small cell lung cancer.24,25 On the other hand, the lymphocyte count reflects the immunological status of a host, thus a low lym­phocyte count is a predictor of poorer outcome.26 The prognostic value of combinations of these and other parameters has also been extensively investi­gated. Among them, the neutrophil-to-lymphocyte ratio in various chronic diseases, including numer­ous malignant diseases, has been investigated the most.27-29 In this study, we have investigated CRP, LDH, Pc, hemoglobin (Hb), creatinine, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) and their impact on the outcome of patients with SCLC. To the best of our knowledge, this is the first study carried out exclusively on a European population which investigated the prognostic sig­nificance of all three mentioned ratios in patients with limited-stage and extensive-stage small cell lung cancer.29-31 Patients and methods Patients For research purposes, we analyzed the medical records of 438 patients diagnosed with small cell lung cancer admitted to the University Hospital Center, Department for Lung Diseases Jordanovac between 2012 and 2016. We included only patients whose disease was verified by histopathological analysis and who had undergone first-line chemo­therapy or chemoradiotherapy. Some additional criteria needed to be met in order to be included in the research: documented laboratory test results Drpa G et al./ Prognostic parameters for survival in SCLC 439 with the investigated parameters measured up to three weeks before the first chemotherapy, as well as data on performance status, follow up, and out­come. The following patient categories were ex­cluded from further research: surgically treated patients, patients with combined small cell lung carcinoma, patients with one or more synchronous tumors, patients who received no therapy, patients without the required medical records, and patients lost to follow-up. After exclusion of the mentioned groups, 140 patients remained who met all the re­quired inclusion and exclusion criteria for further investigation. Out of the total number of patients, 80 were diagnosed with extensive-stage disease and 60 with limited-stage disease. The patients’ performance status was measured before the start of the treatment and defined according to the Eastern Cooperative Oncology Group Performance Status (ECOG) scale.32 Regarding the ECOG status, the patients were divided into two groups: good ECOG status (0–1) and poor ECOG status (2–3). All patients underwent a thoracic and abdominal computed tomography (CT) scan before the start of the treatment. Skeletal scintigraphy was done only in cases with a clinical indication, because it was not routinely performed at our Department. The same applied to brain CT scanning. Disease exten­sion was defined according to the staging system established by the International Association for the Study of Lung Cancer (IASLC) in 1989, which divides SCLC into two stages, “limited-stage dis­ease” and “extensive-stage disease”.33 The patients underwent follow-up chest X-ray scans after every two chemotherapy cycles. A follow-up CT scan was performed after the treatment was completed, especially in cases of initial limited-stage disease. Regression of a primary tumor and metastasis or stable disease was marked as response to therapy what was in fact disease control after initial thera­py, whereas progression of the disease was marked as no-response. Response to therapy was assessed radiologically and clinically (e.g., if a patient had subcutaneous metastases or palpable lymph nodes in a region which had not been examined by CT). In our institution, patients usually receive 4–6 cycles of the first-line platinum-doublet chemo­therapy. Patients who received a minimum of two and a maximum of six cycles of the mentioned chemotherapy, with or without radiotherapy, were included in the study. A concomitant or sequential radiotherapy protocol was carried out, primarily in patients with limited-stage disease or as pal­liative treatment in patients with extensive-stage disease and a good response to chemotherapy. Prophylactic cranial irradiation was mainly per­formed in patients with limited-stage disease. Data collection and ethical consideration Data were collected by using the electronic infor­mation database, based on good clinical practice and complying with international standards in­cluding the Helsinki Declaration on Patient Safety. We obtained approval for data collection and anal­ysis by the Ethics Committee of our institution. Since this was a retrospective study, informed con­sent was not required. Demographic, laboratory, cytological, histo-pathological, clinical, and treatment data were collected on the patients included in the study. Laboratory test results obtained shortly before the start of treatment, that is, a maximum of three weeks before the first chemotherapy, were included in the study. Among all the hematological results, the following parameters were analyzed: leukocyte count, lymphocyte count, neutrophil count, mono-cyte count, platelets, hemoglobin, CRP, creatinine, and LDH. The neutrophil-to-lymphocyte ratio was calculated by dividing the total neutrophil count by the total lymphocyte count. The platelet-to-lym­phocyte and lymphocyte-to-monocyte ratios were calculated in the same way. Overall survival (OS) was defined as the length of time from the date of diagnosis to death from any cause, or the last follow-up for patients who were still alive. Progression-free survival (PFS) was defined as the length of time from diagnosis to pro­gression or death, depending on what happened first. Statistical analysis For the analysis of demographic and clinical da­ta, we used descriptive and inferential statistical methods. Parameters are indicated as sum and per­centage, arithmetic mean +/- standard deviation, or as interquartile range limits with the median as a measure of the central tendency. Differences among the ranked parameters, i.e., the investigated values, were calculated by using the Mann–Whitney U test. Differences among categorical data were test­ed by using the Chi-square test with Fisher’s exact test for smaller samples. Intercorrelation among the variables was tested by using Spearman’s rank correlation coefficient varying within the closed in­terval –1 = r = +1. For survival analysis, the Kaplan– Meier estimator was used, and the Log-rank test (Mantel-Cox) was used as a test of significance. The 440 TABLE 1. Patient characteristics regarding the disease stage or for clinically relevant parameters. All P values were two-tailed. The level of significance was set at Alpha = 0.05. Statistical analysis was performed by using IBM SPSS Statistics for Windows, Version 21.0 (IBM SPSS Inc, Chicago, IL, USA). Cut-off values suggested by the literature were Gender Male55 (68.8%) 34 (56.7%) 0.159 used for testing the potential prognostic value of Female 25 (31.2%) 26 (43.4%) the investigated ratios, since the ROC curves of the Smoking Yes77 (96.2%) 57 (95.0%) 1.000 investigated ratios did not have a statistical signifi- No 3 (3.8%) 3 (5.0%) cance. All ratios were tested regarding two cut-off PS (ECOG) 0–164 (80.0%) 52 (86.7%) 0.368 values. The cut-off values for NLR were 4 and 5, 2–3 16 (20.0%) 8 (13.3%) those for PLR were 150 and 250, and those for LMR Chest irradiation were 2.64 and 4.19.34-39 Yes9 (11.2%) 36 (60.0%) < 0.0001 No 71 (88.8%) 24 (40.0%) PCI Yes2 (2.5%) 10 (16.7%) 0.004 Results No 78 (97.5%) 50 (83.3%) Disease control Patient characteristics Yes63 (78.8%) 56 (93.3%) 0.018 No 17 (21.2%) 4 (6.7%) Patient characteristics regarding the disease stage PFS (weeks) x (SD) 30.1 (14.5) 60.3 (57.9) < 0.0001 are shown in Table 1. Out of 438 patients diag-OS (weeks)nosed with small cell lung cancer or mixed neu- < 0.0001 x (SD) 48.3 (23.4) 83.3 (59.3) roendocrine carcinoma between 2012 and 2016, Outcome 140 met the inclusion and exclusion criteria and dead79 (98.8%) 46 (76.7%) 0.013 alive 1 (1.2%) 14 (23.3%) were included in the study. Of those 140 patients, WBC count (x 109/l)0.686 80 were diagnosed with extensive-stage disease x (SD) 9.1 (3.7) 9.2 (3.3) and 60 with limited-stage disease. The mean pa- Platelet count (x 109/l) 0.249 tient age was 63.1 years with a mean deviation of 293 (119) 304 (95) x (SD) 9.2 years (42–87 years of age). Slightly more males Hemoglobin (g/l) 0.540 than females were involved in the study (89 or x (SD) 130.9 (17.8) 133.0 (16.8) 63.6%). The majority of the patients were smokers CRP (mg/l) 0.048 x (SD) 34.2 (44.6) 21.2 (26.6) (95.7%), of good performance status, 0–1 according Creatinine (umol/l)0.443 to the ECOG scale (82.9%). Only 14 patients (10%) x (SD) 81.9 (20.0) 82.3 (29.6) received less than 4 chemotherapy cycles. Forty- LDH (U/l) 0.004 five patients (32%) underwent radiotherapy, most Lymphocytes (x of whom were in the limited-stage disease group. 109/l)0.202 x (SD) 336.2 (193.5) 311.1 (607.3) 1.6 (0.8) 1.7 (0.7) Only twelve patients underwent PCI (8.6%), again x (SD) significantly more in the limited-stage disease Neutrophils (x 109/l) 0.812 x (SD) 6.6 (3.4) 6.6 (3.2) group. Disease control was observed in 119 patients Monocytes (x109/l)0.700 (85%). After two years, 125 patients (89.2%) died. x (SD) 0.7 (0.3) 0.7 (0.3) Fifteen out of the total number of patients included NLR 0.485 in the analysis (10.7%) survived for more than 2 PLRyears, and all of them belonged to the limited-stage x (SD) 5.1 (3.6) 4.6 (3.4) 0.714 x (SD) 217.9 (119.9) 213.4 (123.3) disease group. According to the statistical analysis, disease control, PFS, OS, and outcome were signifi­cantly better in the limited-stage disease group. Of CRP = C-reactive protein; ECOG = Eastern Cooperative Oncology Group; ED-SCLC = extensive- the laboratory parameters, a significant statistical stage disease small cell lung cancer; LDH = lactate dehydrogenase; LD-SCLC = limited-stage disease small cell lung cancer; LMR = lymphocyte-to-monocyte ratio; NLR = neutrophil-to-difference regarding the disease stage was only ob-lymphocyte ratio; OS = overall survival; SD = standard deviation; PCI = prophylactic cranial irradiation; PFS = progression- free survival; PLR = platelet-to-lymphocyte ratio; PS = performance served for CRP and LDH. The mean NLR and PLR status; WBC = white blood cells; x = arithmetic mean values were higher in the extensive-stage disease group of patients, while the mean LMR value was higher in the limited-stage disease group, but the Cox regression was used for determining possible difference was not statistically significant. In the multiple interactions among the parameters. The extensive-stage disease group, a statistically signif-Cox regression was performed in the case of p < 0.3 icant difference of LMR values regarding patient Drpa G et al./ Prognostic parameters for survival in SCLC 441 age was observed, i.e., higher LMR values were ob­served in the younger age group. Survival analysis The median survival time for all patients was 52.6 weeks (95% confidence interval [CI] 47.5–57.7). The median survival time for the ED group of pa­tients was 45.7 weeks (95% confidence interval [CI] 42.3–49.2) and for the LD patient group it was 64.1 weeks (95% confidence interval [CI] 56.70–71.6). According to the Kaplan-Meier estimator, sur­vival analysis of all 140 patients showed a statisti­cally significant difference in the overall survival regarding disease extension, radiotherapy to the primary tumor, prophylactic brain irradiation and disease control. Therefore, patients with lim­ited-stage disease, patients with disease control, irradiated patients and patients who underwent PCI had a better survival. Of the laboratory pa­rameters, a statistically significant difference in the overall survival was observed regarding the hemoglobin, CRP, LDH, and boundary monocyte values, whereas a statistically significant differ­ence in the overall survival regarding the ECOG status, NLR, PLR, and LMR was not observed (Table 2). Separate testing showed a statistically signifi­cant difference in overall survival in patients with extensive-stage disease, considering the presence of skin metastases and laboratory parameters in­cluding LDH and NLR, regardless of the cut-off values. Therefore, a better overall survival was observed in the patients who did not have skin metastases and had lower LDH and NLR values (Table 3). No positive correlation between overall survival and ECOG status, number of metastatic sites, and disease control was observed in the sub­jects with metastatic disease. A statistically significant difference in overall survival, regarding the ECOG status, radiotherapy of the primary tumor, prophylactic cranial irra­diation, and laboratory values such as hemoglobin and creatinine levels, was determined in the limit-ed-stage disease group of patients (Table 4). As we have already mentioned, Cox regres­sion was used for determining possible multiple interactions among the variables. Thus, all statisti­cally significant parameters from the Kaplan-Meier analysis were included in the multiple regression model. In this model LDH became the most signifi­cant prognostic factor in extensive-stage disease, while the ECOG performance status became the TABLE 2. Prognostic parameters for survival – all patients Extent of disease LD ED 60 80 64.1 (56.7–71.6) 45.7 (42.3–49.2) < 0.0001 Chest irradiation Yes No 45 95 69.1 (63.3–75.0) 45.3 (39.0–51.6) < 0.0001 PCI Yes No 12 128 69.0 (12.3–125.7) 49.1 (43.4–54.9) 0.003 Disease control Yes No 119 21 53.4 (49.5–57.3) 36.4 (25.3–47.5) 0.013 Hemoglobin (g/l) M = 138 < 138 F = 119< 119 78 62 57.1 (50.6–63.6) 40.6 (28.9–52.3) 0.006 CRP (mg/l) < 5.0 = 5.0 35 104 57.1 (48.9–65.4) 47.9 (41.4–54.3) 0.026 LDH (U/l) < 241 = 241 55 56 63.0 (53.0–73.0) 37.0 (27.7–46.3) 0.002 CRP = C-reactive protein; ED = extensive-stage disease; LD = limited-stage disease; LDH - lactate dehydrogenase; PCI = prophylactic cranial irradiation TABLE 3. Prognostic parameters for survival – extensive-stage disease (ED) Skin metastases Yes No 4 76 15.9 (0.7–31.0) 46.9 (42.7–51.0) < 0.0001 LDH (U/l) < 241 = 241 26 36 54.0 (45.4–62.6) 33.7 (22.8–44.6) 0.017 NLR < 4 = 4 40 40 50.1 (43.5–56.8) 44.7 (37.4–52.0) 0.026 LDH = lactate dehydrogenase; NLR = neutrophil-to-lymphocyte ratio most powerful one in limited-stage disease. The data are presented in Table 5. Discussion Numerous prognostic factors were investigated in various cancer types in order to find the factor which would most accurately define the patient groups that could benefit from a certain therapy and consequently expect a better survival.39 The established fact about the important role inflam­mation plays in the process of carcinogenesis has led to research into the prognostic significance of various inflammatory markers. In the past decade numerous papers have been published on such research in relation to non-small cell lung can­cer29,31, but, very few studies of this kind have been done for small cell lung cancer. The present study 442 TABLE 4. Prognostic parameters for survival – limited-stage disease (LD) limited-stage disease patient group, which can be explained by the fact that it was possibly assessed more accurately in this patient group. As a matter of fact, performance status assessment is a subjec­0–1 52 66.3 (57.6–75.0) tive method and in retrospective studies there is PS (ECOG) 0.007 2–3 8 35.9 (8.3–63.4) always a possibility that the criteria for certain pa- Chest Yes 36 70.7 (51.8–89.6) irradiation No 24 36.7 (16.1–57.3) 0.003 tients varied. Unlike in other neoplasms, age did Yes 10 102.0 (0.0–209.6) not have a prognostic significance in most of the PCI 0.032 No 50 58.3 (46.7–69.8) studies regarding SCLC, which was confirmed in Hemoglobin M = 138 F = 11935 71.9 (57.0–86.8) 0.033 our study, too.23 Neither gender nor smoking sta- (g/l) < 138 < 119 25 54.3 (17.7–90.9) Creatinine M < 125 F < 107 57 66.3 (58.6–74.0) tus had a prognostic significance, but, it is note­ 0.001 (umol/l) = 125 = 107 3 32.9 (27.8–37.9) worthy that the number of non-smokers in the study was negligible. Of all the variables, radio- ECOG = Eastern Cooperative Oncology Group; PCI = prophylactic cranial irradiation; PS = performance status therapy, PCI and disease control had a survival im­pact in the whole research patient group. When we separated the patients with extensive-stage from TABLE 5. Results of Cox regression analysis those with limited-stage disease, radiotherapy and PCI retained a survival impact in the patients with limited-stage disease, as we expected. However, disease control showed prognostic value neither in Skin metastases Yes vs No 0.034 0.006 0.192 0.000 LD nor in ED. LDH < 241 vs. = 241 1.691 1.130 2.530 0.011 In the last few decade various laboratory param- Monocytes = 0.84 vs. > 0.84 1.057 0.675 1.655 0.809 eters regarding prognostic value have been inves­tigated. Their ratios have also been investigated NLR < 4 vs. = 4 1.497 0.757 2.961 0.246 recently. Some studies verified a prognostic signifi- NLR < 5 vs. = 5 0.795 0.391 1.615 0.525 cance of hemoglobin, leukocyte count, CRP, LDH, LD-SCLC and serum sodium concentration in SCLC.23,25,40 ECOG 0–1 vs. 2–3 2.865 1.032 7.953 0.043 The prognostic significance of hemoglobin and Chest irradiation Yes vs. No 1.558 0.793 3.047 0.195 LDH was confirmed in our patients, along with a lower significance of CRP and monocyte count as PCI Yes vs. No 2.038 0.893 4.654 0.091 prognostic factors. When we excluded disease ex- Hemoglobin Normal vs. Anemia 1.439 0.773 2.678 0.251 tension from the analysis, LDH retained a prognos- Creatinine Normal vs. Elevated 1.432 0.155 13.198 0.751 tic significance in the ED group, while hemoglobin retained a prognostic significance in the LD group CI = confidence interval; ECOG = Eastern Cooperative Oncology Group, ED-SCLC = extensive-stage disease small cell lung cancer; HR = hazard ratio; LDH = lactate dehydrogenase; LD-SCLC of patients. Besides, creatinine level occurred as = limited-stage disease small cell lung cancer; NLR = neutrophil-to-lymphocyte ratio; PCI = prophylactic cranial irradiation an independent prognostic factor for survival in the LD group of patients, but again only in the ex­ tremely small number of patients with increased was conducted with the intention to determine creatinine levels. potential prognostic parameters of survival in a Although the combinations of various labora- European population of patients diagnosed with tory indicators, including NLR, PLR, and LMR, SCLC. Survival parameters were identified for the have already been examined as prognostic factors whole population of patients, as well as separately in SCLC, a relatively small number of studies have for patients with extensive-stage and those with been published regarding this type of cancer. Most limited-stage disease, in order to determine differ-of the published papers investigating the predic­ ences between these two groups. tive significance of these parameters in patients As disease extension and performance status are with lung cancer address non-small cell lung car-generally among the most investigated prognostic cinoma.29-31 Consulting the literature in English parameters, they were verified as the most impor-until May, 2020, we found a total of twenty stud-tant for SCLC as well.23 Our study also showed that ies, seven of which had been published in 2019, disease extension was a significant prognostic fac-which investigated one or more of these three tor, and certainly the most significant predictor of ratios in patients with small cell lung cancer. It is longer survival. On the other hand, performance interesting to note that most of the studies relate status showed a prognostic value only for the to the Asian population. For example, the prog- Drpa G et al./ Prognostic parameters for survival in SCLC 443 nostic significance of LMR in SCLC was only in­vestigated in two studies, both conducted in the Asian population.38,41 Out of twelve studies which investigated the prognostic value of PLR alone or in combination with NLR, only one was done in Europe.35 NLR, as the most researched ratio, was the subject of investigation in seventeen studies, of which only three were European.25,35,42 There are only two studies investigating the prognostic role of NLR and/or PLR exclusively in the ED group of patients.34,43 To our knowledge, to date neither of these two parameters have been investigated on a European population in cases of extended SCLC. As race has been determined as a significant prognostic factor in SCLC patients, in the sense that being Caucasian represents a favorable inde­pendent prognostic factor, we were interested in whether our results would differ from the ones ob­tained elsewhere so far.5 It is important to mention that the results of the former studies are inconsistent, that is, some stud­ies showed a statistically significant correlation between the NLR and PLR ratios and overall sur­vival of the patients, while others did not yield a statistical significance. In fact, some studies didn’t investigate these ratios in correlation with survival at all.44-46 The only prospective study conducted in the USA on more than 900 patients verified that NLR was a prognostic parameter for OS only in the extensive-stage disease group of patients, which is consistent with our results.47 The same study estab­lished that PLR was a prognostic parameter for OS only in limited-stage disease, which was different from our results. There are no prospective studies for LMR. Most retrospective studies which inves­tigated NLR established its prognostic value, re­gardless of whether it was investigated in LD, ED, or simultaneously in both patient groups. Among twelve retrospective studies investigating PLR, only three showed a prognostic significance of this parameter.48-50 Out of the two studies investigating LMR, only one showed a prognostic significance of this parameter.38 In the prospective study mentioned above, among other things it was established that NLR and PLR were statistically significantly greater in patients with extended disease.47 In our study, the mean values of NLR and PLR were also higher in ED patients, while LMR was higher in LD, al­though the difference was not statistically signifi­cant. On the other hand, we found statistically sig­nificant differences in LMR values in correlation with patient age in the ED group, i.e., higher LMR values in the younger age group of these patients. In spite of the fact that some of our results were consistent with those from the only prospective study, our study had numerous limitations. In eve­ry study where data are collected from available re­cords, there is a possibility that some of it may not be reliable, particularly data undergoing subjective assessment. As mentioned earlier, performance status is one of such parameters, thus making it more difficult for analysis in retrospective studies. A similar situation may arise in the assessment of peripheral lymph node regression during patient follow up and evaluation of the response to treat­ment. Furthermore, in the determination of disease ex­tent, especially in concomitant chemoradiotherapy candidates, assessment based only on clinical ex­amination, bronchoscopy, and CT is not sufficient. Since this type of carcinoma is characterized by rapid spread, complete staging should be done prior to treatment, including brain CT and bone scintigraphy. This is the standard procedure at our 444 institution today, but was not always possible in the past for technical reasons. The relatively small number of subjects enrolled in the study was also a limitation. However, two published studies enrolled approximately the same number of patients.51,52 Also, some of the published studies were conducted in even smaller groups of participants.44,51,53 Although some studies had a large number of patients, they didn’t analyze patients separately considering disease extension.54 It is important to note that the number of pa­tients enrolled in the study was probably not ade­quate for the analysis of certain variables. Namely, only a very small number of patients with skin metastases and increased creatinine participated in the study, as well as very few patients with a low performance status. This presents a problem for many studies, since low-performance status patients are usually not candidates for differential treatment and are rarely included in clinical stud­ies. The same applies for kidney failure patients. On the other hand, since the skin is an uncommon metastatic site, such patients are rare. Considering the confidence interval, it is clear that according to this study skin metastases are not a favorable in­dicator of survival. On the contrary, creatinine can be considered a favorable indicator of survival de­spite the small number of patients. As far as the investigated treatment procedures and their prognostic values are concerned, there are certain limitations as well. In the group of all patients, statistically significant differences were found for survival in relation to PCI and thoracic irradiation. However, when the patients were an­alyzed separately in relation to the extent of the disease, those differences disappeared in the ED group. This is due to the fact that disease extent is one of the most important prognostic factors for SCLC, which was established in 2003 in a pro­spective study involving 436 patients.23 Therefore, these two patient groups should always be in­vestigated separately, because the differences in their prognoses entail different modes and aims of treatment. In our study, PCI remained prognosti­cally valuable in the LD patient group, but with an insufficient number of subjects for the result to be considered reliable. This treatment procedure has always been controversial, presenting an issue for confrontation and opposing research.55 The prog­nostic value of PCI was certainly not the primary aim of our study. In spite of its limitations, we be­lieve that our study will contribute to the elucida­tion of small cell lung cancer, as well as stimulate further research on this type of carcinoma, which has somehow always remained in the margins of lung cancer research. Conclusions The objective of this study was to determine a potential prognostic value of the neutrophil-to-lymphocyte, platelet-to-lymphocyte, and lym­phocyte-to-monocyte ratios in patients diagnosed with extensive-stage and limited-stage small cell lung cancer. To the best of our knowledge, this is the first study carried out on a European popula­tion which analyzed all three of the mentioned ra­tios. According to the study, NLR could be a good prognostic marker in patients with extensive-stage SCLC. Further prospective studies are definitely needed for this type of cancer. Acknowledgments This research was supported by the grant awarded to JK (Croatian Scientific Foundation; IP-06-2016­1441). This research did not receive any other spe­cific grant from funding agencies in the public, commercial, or not-for-profit sectors. We wish to thank the Croatian Scientific Foundation for its financial support. 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BMC Cancer 2019; 19: 95. doi: 10.1186/s12885-018-5251-3 447 research article Treatment patterns and real-world evidence for stage III non-small cell lung cancer in Central and Eastern Europe Milada Zemanova1, Marko Jakopovic2, Karmen Stanic3,4, Malgorzata Lazar-Poniatowska5, Martina Vrankar3,4, Petronela Rusu6, Tudor Ciuleanu6, Davorin Radosavljevic7, Krisztina Bogos8, Sergiusz Nawrocki9 1 1st Faculty of Medicine of Charles University, Prague, Czech Republic 2 Department for Respiratory Diseases Jordanovac, University Hospital Centre Zagreb, School of Medicine, University of Zagreb, Zagreb, Croatia 3 Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 5 Department of Oncology and Radiotherapy, Medical University of Gdansk, Gdansk, Poland 6 Institute of Oncology “Prof. Dr. Ion Chiricuta”, Cluj-Napoca, Romania 7 Institute for Oncology and Radiology of Serbia, Belgrade, Serbia 8 National Koranyi Institute of TB and Pulmonology, Budapest, Hungary 9 Department of Oncology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland Radiol Oncol 2020; 54(4): 447-454. Received 9 June 2020 Accepted 10 July 2020 Correspondence to: Assist. Prof. Karmen Stanic, P.D., Ph.D., Institute of Oncology Ljubljana, Zaloška 2, 1000 Ljubljana, Slovenia. E-mail: kstanic@onko-i.si Disclosure: Dr. Zemanova reports other from AstraZeneca, during the conduct of the study; grants from AstraZeneca, grants and personal fees from BristolMyersSquibb, personal fees from Boehrinegr Ingelheim, personal fees from MSD, personal fees from Roche, grants from Novartis, outside the submitted work; Dr. Jakopovic reports personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Roche, personal fees and non-financial support from MSD, personal fees and non-financial support from Pfizer, personal fees and non-financial support from Boehringer Ingelheim, outside the submitted work; Dr. Stanic reports personal fees and non-financial support from AstraZeneca, during the conduct of the study; Dr. Vrankar reports personal fees from AstraZeneca, Roche, Boehringer Ingelheim, Merck Sharp & Dohme, other from AstraZeneca, Roche, Boehringer Ingelheim, outside the submitted work; Dr. Rusu reports personal fees from AstraZeneca, personal fees from AD-pharma, outside the submitted work; Dr. Ciuleanu reports consultancy fees from Astellas, Janssen, Bristol-Myers Squibb, Merck Serono, Amgen, Roche, Pfizer, Boehringer Ingelheim, Lilly, AstraZeneca, MSD, Sanofi, Novartis, Servier, AD Pharma; Dr. Radosavljevic reports personal fees and non-financial support from Astra Zeneca, personal fees and non-financial support from MSD, personal fees and non-financial support from Roche, personal fees and non-financial support from Pfizer, personal fees from Boerhinger Ingelheim, personal fees and non-financial support from Merck, personal fees and non-financial support from Amicus, outside the submitted work; other authors have nothing to disclose. Background. The aim of this project was to collect real-world evidence and describe treatment patterns for stage III non-small cell lung cancer in Central and Eastern Europe. Based on real-world evidence, an expert opinion was developed, and the unmet needs and quality indicators were identified. Patients and methods. A systematic literature search and a multidisciplinary expert panel of 10 physicians from 7 countries used a modified Delphi process to identify quality indicators and unmet needs in patients with stage III non-small cell lung cancer. The profound questionnaire was used to characterize treatment patterns used for stage III non-small cell lung cancer, and a systematic review identified patterns in Central and Eastern Europe. The first questionnaire was completed by a group of medical oncologists, radiation oncologists and pneumologists. The panel of experts attended an in-person meeting to review the results of the questionnaire and to process a second round Delphi. An additional survey was then compiled and completed by the panel. Results. A complete consensus was reached by the panel of experts on a set of evidence-based clinical recommen­dations. The experience-based questionnaire generated a highly variable map of treatment patterns within the region. A list of unmet needs and barriers to quality care were developed with near-unanimous consent of the panel of experts. Conclusions. The current landscape of diagnostic and therapeutic approaches in Central and Eastern European countries is highly variable. We identified several significant barriers, mainly related to the availability of diagnostic and imaging methods and low rates of chemoradiotherapy with curative intention as initial treatment for unresectable stage III NSCLC. Key words: stage III non-small cell lung cancer; treatment patterns; Delphi method; quality of care; expert panel; real-world evidence 448 Introduction Lung cancer is the leading cause of cancer mortality worldwide, with over 2 million newly diagnosed cases annually. Lung cancer constituted 11.6% of all cancer cases diagnosed in 2018, according to the International Agency for Research on Cancer and worldwide numbers are still rising.1 There were over 1.8 million deaths caused by lung cancer in 2018.1 In Central and Eastern Europe (CEE)2, the lung cancer incidence was almost 150,000 newly diagnosed cases in 2018, with over 131,000 deaths caused by lung cancer in the region.3 The most common form of lung cancer is non-small cell lung cancer (NSCLC), accounting for 80%–85% of all cases.4 Stage III non-small cell lung cancer comprises approximately one-third of NSCLC patients and is very heterogeneous with a variable, although mostly poor, prognosis.4 Due to its heterogeneity, a general schematic management approach is not appropriate and is recommended that the decision about the treatment is reached through multidisciplinary tumor board. Usually, a combination of local therapy with systemic plat-inum-based doublet chemotherapy and, recently added, immune therapy is used.4 According to the TNM 8 staging system, stage III NSCLC is subclassified into stage IIIA, IIIB, and IIIC.5,6 Lung cancer symptoms occur mostly late in the disease, so the majority of patients with NSCLC present with advanced metastatic disease that is in­curable with currently available therapy, therefore, patient prognosis is critically dependent on early diagnosis and early treatment. European Society for Medical Oncology (ESMO) guidelines, which were updated in 2017, directs the treatment of locally advanced NSCLC as follows: concurrent chemoradiotherapy is con­sidered the preferred treatment for patients who are in good condition in stage IIIA, IIIB and IIIC. If chemoradiotherapy is not possible, then se­quential chemotherapy followed by definitive radiotherapy represents a valid and effective al­ternative.7 Results from the PACIFIC trial show improvement in overall survival (OS) and pro-gression-free survival (PFS) using a combination of chemoradiotherapy and immunotherapy (rep­resented by durvalumab in this case).8,9 In this ran­domized trial, the 36-month OS rate was 57.0% in the durvalumab group and 43.5% in the placebo group.10 PFS was reported as a median duration of 17.2 months in the durvalumab group and 5.6 months in the placebo group (p < 0.001) according to the study report from 2018.9 Real-world data on treatment patterns of locally advanced NSCLC in CEE are limited. Therefore, we aimed to: 1. Generate a real-world matrix on treatment patterns in CEE based on an extensive litera­ture search. 2. Generate a summary of treatment patterns used in stage III NSCLC in CEE based on clin­ical practice, find the main barriers to treat­ments, and formulate a set of quality of care indicators. 3. Develop a consensus on evidence-based clini­cal recommendations for Stage III NSCLC in CEE in cooperation with a panel of experts (henceforth referred to as the expert panel [EP]) from the region. The Delphi method was used as a technique for consensus development. Patients and methods Study design The study consisted of five parts: (1) an extensive literature search with a focus on real-world evi­dence (RWE); (2) development of a questionnaire; (3) selection of an expert panel; (4) an online sur­vey; and (5) analyzing and discussing the results during the expert panel meeting. This study con­sisted of a survey of expert opinions, and no pa­tient data were collected, so no specific independ­ent ethical approval was necessary. Expert panel The expert panel was composed of 10 members from CEE countries, including Croatia, Czech Republic, Hungary, Poland, Romania, Serbia, and Slovenia. Due to the multidisciplinary nature of NSCLC therapy, representatives from a variety of disciplines were nominated to be on the expert panel, which ultimately consisted of medical on­cology, radiation oncology and pneumology. Each of the panelists was an authority in the particular area of expertise in her or his country. Literature search Web of Science, PubMed, and the Cochrane li­brary were thoroughly searched. A total of ten hits was considered relevant, and from those, a map of treatment patterns in CEE was generated. The goal was to identify synthesized research evidence 449 TABLE 1. List of real-world evidence literature from the Central and Eastern Europe region Zemanová et al., 202018 Vrankar et al., 201822 Ramlau et al., 201723 Podmaniczky et al., 201524 Jeremic, 201525 Georgieva el at., 201426 Zielinski et al., 201327 Kolodziejczyk et al., 201128 Jeremic et al., 201129 Registry, Czechia, Austria, Latvia, Serbia, Hungary, Poland Observational, Slovenia Registry, Poland Observational, Hungary Review, Serbia Observational, Bulgaria Retrospective observational study, Poland Prospective study, Poland Toxicity studies, Serbia Surgery 23%, RT 55%, CT 80% Induction CT in 3 cycles, + CCRT, 2 cycles Surgery 27%, 14% RT, 80% systemic therapy Platinum-based neoadjuvant CT Standard treatment options NA Staging Radical RT, neoadjuvant CT 46% CCRT IIIA 55%, IIIB 45% IIIA 57%, IIIB 43% IIIA 12%, IIIB 15% IIIA 60%, IIIB 20% NA III 2.4%, IIIA 12%, IIIB 2.4% NA IIIA 31%, IIIB 39% NA Squamous 53%, adenoc. 38%, not specified 6%, other 3% Squamous 58%, adenoc. 22%, large cell 6%, other 14% Adenoc. 37%, Squamous 59%, adenoc. 41% NA Squamous 22%, adenoc. 55%, non-small 14%, other 10% NA Squamous 41%, adenoc. 8%, large cell 2%, no specification 45%, no histology 4% NA 583 p., 78% males 102 p., 79% males 696 p., 60% males 46 p., 63% males NA 42 p., 57% males 899 p. 100 p., 78% males 600 p. CCRT = concurrent chemoradiotherapy; CT = chemotherapy; NA = not available; NSCLC = non-small cell lung cancer; p. = patients; RT = radiotherapy including clinical practice, systematic reviews, me-ta-analyses, and conference proceedings. Articles were included in the RWE map if they were fully published in English. Online survey The online expert questionnaire was divided into three parts. These parts covered expert experienc­es in diagnosis, therapy, and organization of the care of patients with stage III NSCLC. The expert panel members entered the rates of utilization in each category or other specific counts according to clinical practice in their medical center. Some of the outcomes, e.g. the number of specialized oncology centers in the country, were determined as counts in the country of panelists. Delphi panel The Delphi technique is a method for collecting data from respondents within their domain of ex­pertise.11 The aim is to achieve a convergence of opinion on a specific medical issue (in this case, NSCLC stage III therapy). There have been sev­eral published cases using the Delphi method to study lung cancer.12–17 The consensus part of the study was carried out using a modified Delphi method. The first round of the Delphi consensus was built as a set of 12 evidence-based recommen­dations extracted from ESMO clinical guidelines.7 Responses were collected on a 5-point Likert scale. In the 1st round, each panelist responded using the following answers: (1) strongly disagree; (2) basi­cally disagree; (3) doubtful; (4) basically agree; or (5) strongly agree. A Delphi consensus was reached when the mean of all values was > 4.0. If the mean of all values was 5.0, the consensus was considered unanimous. All statements then under­went a second Delphi round. The second Delphi round was held as an in-person meeting, and all 12 statements were discussed. In the meeting, it was possible to vote for or against each statement. A consensus was defined as > 80% of the responses were in favor of the statement. The overall deci­sion was then distributed via email for any sub­sequent comments by the expert panel. A total of nine panelists responded to the first round and ten panelists responded to the second round. The first round took place from November 11–23, 2019 and was performed via an online survey. The second round took place in Prague, Czech Republic, on November 29, 2019. Statistical analysis Descriptive analytical methods were used to analyze continuous and categorical variables. Continuous variables were reported as mean, standard deviation, minimum, and maximum. Categorical variables were reported as count and rate. MS Excel was used for the analysis. Manuscript preparation Based on the input of expert panel, the draft man­uscript was prepared by medical writing agency. This project began in August 2019 and ended in March 2020. During the drafting of the article, new­ly published literature was reviewed to analyze the clinical implications of any new data in patients with stage III NSCLC. Results A literature search of RWE in CEE There was a limited number of RWE-based litera­ture on population diagnosed with stage III NSCLC from the CEE region. Table 1 presents a list of ana­lyzed literature from 2011–2020. This includes data on patient registries and observational and toxicity studies. The only relevant literature with the texts written completely in English related to Bulgaria, Czech Republic, Hungary, Poland, Slovenia and Serbia. Mostly, these publications presented data on treatment, diagnostic methods and staging. Treatment patterns based on clinical experience Table 2 presents the data collected in the area of staging and diagnosis of stage III NSCLC. Concerning the staging of NSCLC, 32% (± 13%) of NSCLC patients in any particular medical center were diagnosed with stage III NSCLC, and most of those were in stage IIIB (45% ± 12%). Good consist­ency in the field of imaging was observed. The most common diagnostic procedures, i.e. X-Ray, chest computed tomography (CT) (including the CT of upper abdomen area), and bronchoscopy, were provided to at least 93% of patients with stage III NSCLC. Differences in the percentages of treated patients who received abdominal CT, brain CT, en- T ABLE 2. Patterns in stage III non-small cell lung cancer diagnosis in Central and Eastern Europe region; % of patients treated in the medical center of particular panelists Staging All stage III 9 32% (± 13%) 20%–65% Stage IIIA 9 37% (± 14%) 20%–60% Stage IIIB 9 45% (± 12%) 30%–60% Stage IIIC 9 18% (± 11%) 6%–40% Imaging X-Ray 9 99% (± 3%) 90%–100% Chest CT 9 98% (± 4%) 90%–100% Abdominal CT 9 87% (± 19%) 50%–100% Brain CT 9 58% (± 33%) 12%–100% Bronchoscopy 9 93% (± 10%) 75%–100% EBUS 9 37% (± 29%) 9%–80% PET-CT 9 54% (± 30%) 20%–80% Bone scan 9 15% (± 16%) 0%–40% Brain MRI 9 14% (± 7%) 2%–20% Biomarkers PD-L1 reflex testing 9 50% (±40%) 2%–100% * Rates of PD-L1 results available of PD-L1 tests performed; CT = computed tomography; EBUS = endobronchial ultrasound; MRI = magnetic resonance imaging; PET-CT = positron emission tomography-computed tomography; SD = standard deviation dobronchial ultrasound (EBUS), and PET-CT were evaluated. Bone scans and brain MRIs were pro­vided at lower rates to patients (14%–15%). Also, PD-L1 was tested at various rates within the CEE. The mean rate of patients undergoing the PD-L1 reflex testing was 50% (± 40%). The mean rate of available PD-L1 results was 56% (± 31%), as pro­portion of patients was tested on demand. The most heterogeneous set of responses was obtained for the descriptions of initial patient therapy, which reflects high variability in treat­ment approaches across the region (Table 3). About two-thirds of patients initially received radical treatment, and a mean of 30% of patients were treated palliative. When looking at the initial radical treatment modalities, with the intention to cure, showed that clinical practice was heterogene­ous from country to country, and even individual clinical centers within countries had their own ap­proach. As reported, in mean, chemotherapy alone was administered in 13% and cumulatively, con­current and sequential chemoradiotherapy was ad- 451 ministered in more than 50% of patients in mean. TABLE 3. Patterns in stage III non-small cell lung cancer diagnosis therapy; % of patients treated in the medical center of the particular panelist Palliative radiotherapy was provided in 60% of pa­tients intended for palliative treatment. Table 4 describes how the care of patients with stage III NSCLC is organized. Usually, the first contact physician is a general practitioner (54% ± Radical treatment 9 70% (±20%) 30%–96% 27%) or a pneumologist (35% ± 29%). Other, less Palliative treatment 9 30% (±20%) 4%–70% common, first contact variants included other pro­fessionals such as medical oncologists and radia-Radical treatmenttion oncologists. In four out of nine countries lung Surgery 9 17% (±6%) 10%–25%cancer patients are referred to specialized oncology Chemotherapy 8 13% (±16%) 0%–48%centers, where the patient has access to innovative Radiotherapy 8 15% (±9%) 5%–25% oncology treatment options, while in other five Concurrent chemoradiotherapy 8 21% (±12%) 0%–30% countries, lung cancer is treated in local hospitals. Sequential chemoradiotherapy 8 34% (±14%) 18%–50% The number of specialized oncology centers per country varies from 3 to 50, according to the popu-Palliative treatmentlation size of the particular country. Most respond- Palliative radiotherapy 8 60% (±33%) 3%–90%ents (89%) reported that patients diagnosed with stage III of NSCLC were referred to a multidisci­plinary team. In most cases, medical oncologist is supervising the follow-up after initial therapy for unresectable stage III patients. TABLE 4. Patterns in stage III non-small cell lung cancer diagnosis organization of care; % of patients treated in the medical centers of particular panelists Evidence-based clinical recommendations General practitioner 9 54% (± 27%) 20%–90% Table 5 shows the level of consensus for the twelve Pneumologist 9 35% (± 29%) 10%–95% evidence-based clinical recommendations relative Medical oncologist 9 9% (± 13%) 0%–30% to stage III NSCLC. There was a high level of con- Radiation oncologist 9 3% (± 5%) 0%–10% sensus and sometimes even unanimity with many of the twelve statements. Other 9 5% (± 5%) 0%–10% Main barriers and quality indicators Table 6 presents the final list of the main barriers to treatment of stage III NSCLC identified and con­sensually agreed on by the expert panel. Rates of chemoradiotherapy (CRT) are low due to the long waiting times for radiotherapy and especially for advanced radiotherapy techniques. The reason for the low CRT rates could also be caused by provid­ing radiotherapy and chemotherapy in different institutions. Another barrier is a long referral pro­cess among different physician specialties. Next, awareness of lung cancer symptoms, risk factors, and treatment options among patients is affected by health literacy and the influence of social status. Finally, late access to diagnostic and imaging pro­cedures is also combined with long waiting times and low capacity. The list of the agreed quality of care indicators is presented in Table 7. The proportion of patients treated with chemoradiotherapeutic radical treat­ment intention was described as the most signifi­cant indicator of quality of care, followed by the improved survival over time. Discussion The main scope of the project was to explore the treatment patterns in stage III NSCLC in the CEE region since the current information on this topic is very limited. Data were gathered through a sys­tematic literature search, an online survey of lead­ing experts, and a modified Delphi consensus. It should be noted that the abovementioned data on treatment patterns represent the particular medi­cal centers associated with the respondents and that situations in particular countries could differ slightly. The literature search generated a limited amount of real-world data from the CEE region and only represented a subset of the countries participating in the study. This is mainly because most of the 452 TABLE 5. Evidence based clinical recommendations consensus All patients planned for stage III NSCLC treatment should undergo a diagnostic contrast-enhanced CT scan of 1. the chest and upper abdomen followed by a PET or a combined PET-CT using a CT technique with adequately 4.8 Consensus high resolution for initial staging purposes. 2. All patients planned for curative stage III NSCLC treatment should receive brain imaging for initial staging. 4.8 Consensus 3. Concurrent CRT is the treatment of choice in patients evaluated as unresectable in stage IIIa, IIIb, and IIIc. 4.6 Consensus If concurrent CRT is not possible - for any reason - sequential ChT followed by definitive RT represents a valid and 4. 4.8 Consensus effective alternative. An experienced multidisciplinary team is of paramount importance in any complex multimodality treatment 5. 4.9 Consensus strategy decision. In the absence of contraindications, the optimal ChT to be combined with radiation in stage III NSCLC should 6. 4.3 Consensus be platinum-based therapy. When delivered perioperatively, platinum-based combinations are considered the treatment of choice, in the 7. 4.6 Consensus absence of contraindications. 8. In the stage III disease CRT strategy, two to four cycles of concomitant ChT should be delivered. 4.9 Consensus 9. In the perioperative setting, three to four cycles of platinum-based ChT are recommended. 4.8 Consensus 60–66 Gy in 30–33 daily fractions is recommended for concurrent CRT. The maximum overall treatment time 10. 5.0 Unanimity should not exceed 7 weeks. 11. In sequential approaches, RT delivered over a short overall treatment time is recommended. 4.3 Consensus Adjuvant anti PD-L1 checkpoint inhibitor durvalumab is indicated for unresectable NSCLC with PD-L1 = 1% without progression after chemoradiotherapy with a platinum-based regime. ChT = chemotherapy; CRT = chemoradiotherapy; CT = computed tomography; NSCLC = non-small cell lung cancer; PET-CT = positron emission tomography-computed tomography; RT = radiotherapy TABLE 6. Main barriers in the treatment of stage III non-small cell TABLE 7. Quality of care indicators in stage III non-small cell lung cancer found by our panel of experts lung cancer found by our panel of experts Low chemoradiotherapy rates due to long waiting times for radiotherapy, especially for advanced RT 1. techniques and/or radiotherapy and chemotherapy performed by different institutions. Long referral process among different specialities 2. (general practitioner, pneumologist, medical oncologist, radiotherapist). Poor health literacy and social status of patients 3. influence awareness of lung cancer symptoms, risk factors and treatment. Late access to imaging and diagnostic procedures, 4. especially PET-CT – long waiting times, low capacity. Barriers to implementing targeted population 5. screening programs. PET-CT = positron emission tomography-computed tomography; RT = radiotherapy published data were not published in English or in the indexed literature. It is worth noting that much of the literature was published in recent years, which may reflect the availability of new therapeu­tic modalities. A comparison of the expert panel consensus with the ESMO guidelines and the current prac­tice in staging, diagnosis, and treatment of stage III NSCLC revealed differences.7 This fact was well described in the survey of the main barri- The proportion of patients treated with 1. chemoradiotherapy in radical treatment intention. Improved survival (median OS, 5 years survival) over 2. time. Time from first symptoms to first contact with a lung 3. cancer specialist, time from first contact with a lung cancer specialist to first treatment. The proportion of patients with full histopathological/ 4. molecular confirmation of the diagnosis – PET-CT, brain imaging, PD-L1. The proportion of treatment decisions confirmed by 5. a multidisciplinary team. OS = overall survival; PET-CT = positron emission tomography-computed tomography ers and quality of care indicators among the pan­elists. There was great agreement regarding the evidence-based recommendations extracted from ESMO clinical guidelines in the treatment of stage III NSCLC, even though the experience-based survey revealed considerable differences in cur­rent treatment patterns. The list of quality of care indicators produced by the expert panel agreed in part with the list produced in other countries (e.g., the United Kingdom and China), especially with 453 regard to the proportions of patients intended to various treatments or histopathological diagnostic procedures, but our list also proposed several new indicators relative to the decision making role of multidisciplinary teams.14,16 Expert panel agreed a new era for unresectable stage III NSCLC pa­tients in CEE is coming and expert panel agreed to reevaluate the 1–2 year the treatment improvement based on the indicators. Significantly, our survey found a great deal of heterogeneity in therapy organization and treat­ment modalities offered (available) in different medical centers within CEE. The heterogeneity was found in almost all parts of the survey, excluding consent rates of patients in long-term established diagnostic procedures such as X-Ray and CT. The more specific the procedure, e.g., histopathologi-cal diagnostic procedures, the greater the variance in rates of utilization in patients. It is agreed, that reflex PD-L1 testing and brain MRI rates should be improved. Moreover, patterns of initial radi­cal treatment showed great variability among the panelists. This fact was also observed in a recent publication by Zemanová et al., which mapped these patterns in the same region.18 It is important to focus on improving the rates of chemoradio-therapy provided to patients. Yet in 2007, the posi­tive impact of concurrent chemoradiotherapy with vinorelbine and platinum based compounds fol­lowed by consolidation chemotherapy was proven by Rusu et al.19 This study reported a 15 months median OS in patients with stage III NSCLC and well tolerability of the treatment. Importantly, the expert panel unanimously agreed that adjuvant anti PD-L1 checkpoint in­hibitor durvalumab is indicated for unresectable NSCLC with PD-L1 = 1% without progression after chemoradiotherapy with a platinum-based regime (Table 5). Chemoradiotherapy followed by the im­munotherapy is new standard of care according to the National Comprehensive Cancer Network (NCCN) guidelines and The National Institute for Health and Care Excellence (NICE) guidance.20,21 Improving the chemoradiotherapy rates, PD-L1 testing and gaining access to durvalumab are the next needed steps to be implemented in CEE in or­der to treat the stage III unresectable patients ac­cording to new standard of care. Conclusions The current landscape of diagnostic and therapeu­tic approaches in CEE countries is highly variable, and relevant real-world data are missing. We iden­tified several significant barriers, mainly related to the availability of diagnostic and imaging methods and low rates of chemoradiotherapy with curative intention as initial treatment for unresectable stage III NSCLC. Improving CRT rates will also enable consolidative treatment with durvalumab to fur­ther improve the OS of stage III unresectable pa­tient population. The way forward will involve an agreement to establish a set of quality of care indicators with rou­tine monitoring and assessment within the clinical practice framework. The panel of experts agreed on future monitoring of improvement in the stand­ards of care for stage III unresectable NSCLC. Acknowledgem ents This manuscript presents independent work of the expert panel. The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reflect the overall situation in specific country. The project was funded by AstraZeneca for lo­gistical and medical writing support. Tina Jeric, M. Pharm. and Lenka Mikasová, Ph. D. of AstraZeneca contributed to the project design and participated at the expert panel meeting. Medical writing support, which was in accord­ance with good publication practice guidelines, was provided by Tomaš Doležal and Tereza Tumova, of Value outcomes (Prague, Czech Republic). References 1. International Agency for Research on Cancer. World Health Organization. Press release N. 263. [cited 2020 Feb 13]. Available at: https://www.who. int/cancer/PRGlobocanFinal.pdf 2. Publications Office of the EU. Central and Eastern Europe. [cited 2020 Feb 14]. Available at: https://op.europa.eu/en/web/eu-vocabularies/th­concept/-/resource/eurovoc/914 3. International Agency for Research on Cancer. 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Development, validation and results from the impact of treatment evolu­tion in non-small cell lung cancer (iTEN) model. Lung Cancer 2020; 139: 185-94. doi: 10.1016/j.lungcan.2019.10.019 16. Wang X, Su S, Li S, Bao H, Zhang M, Liu D, et al. Development of quality indicators for non-small cell lung cancer care: a first step toward assessing and improving quality of cancer care in China. BMC Cancer 2017; 17: 306. doi: 10.1186/s12885-017-3602-0 17. Provencio M, Carcereny E, Artal Á. Consensus on the use of immune-related response criteria to evaluate the efficacy of immunotherapy in non-small cell lung cancer. Clin Transl Oncol 2019; 21: 1464-71. doi: 10.1007/s12094­019-02072-4 18. Milada Zemanová, Pirker R, Petruzelka L, Zbozínkova Z, Jovanovic D, Rajer M, et al. Care of patients with non-small-cell lung cancer Stage III – the Central European real-world experience. Radiol Oncol 2020; 54(4): 447­454.; 54: 209-20. doi: 10.2478/raon-2020-0026 19. Rusu P, Ciuleanu TE, Cernea D, Pelau D, Gaal V, Cebotaru C, et al. Concurrent chemoradiotherapy with vinorelbine and a platinum compound followed by consolidation chemotherapy for unresectable stage III non-small cell lung cancer: preliminary results of a phase II study. J BUON 2007; 12: 33-9. 20. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology - Non-Small Cell Lung Cancer. [Internet]. Version 5.2020. [cited 2020 May 27]. Avaiable at: https://www.nccn.org/professionals/physi­cian_gls/pdf/nscl.pdf 21. Durvalumab for treating locally advanced unresectable non-small-cell lung cancer after platinum-based chemoradiation | Guidance | NICE [Internet]. [cited 2020 Feb 27]. Available at: https://www.nice.org.uk/guidance/ta578/ chapter/1-Recommendations 22. Vrankar M, Stanic K. Long-term survival of locally advanced stage III non-small cell lung cancer patients treated with chemoradiotherapy and per­spectives for the treatment with immunotherapy. Radiol Oncol 2018; 52: 281-8. doi: 10.2478/raon-2018-0009 23. Ramlau R, Krawczyk P, Dziadziuszko R, Chmielewska I, Milanowski J, Olszewski W, et al. Predictors of EGFR mutation and factors associated with clinical tumor stage at diagnosis: Experience of the INSIGHT study in Poland. Oncol Lett 2017; 14: 5611-8. doi: 10.3892/ol.2017.6907 24. Podmaniczky E, Fábián K, Pápay J, Puskás R, Gyulai M, Furák J, et al. Decreased ERCC1 Expression After Platinum-Based Neoadjuvant Chemotherapy in non-Small Cell Lung Cancer. Pathol Oncol Res 2015; 21: 423-31. doi: 10.1007/s12253-014-9839-x 25. Jeremic B. Standard treatment option in stage III non-small-cell lung cancer: case against trimodal therapy and consolidation drug therapy. Clin Lung Cancer 2015; 16: 80-5. doi: 10.1016/j.cllc.2014.08.003 26. Georgieva N, Bochev P, Dancheva Z, Chaushev B, Balev B, Klisarova A, et al. PET/CT in NSCLC with brain metastases. Rentgenol Radiol 2014; 53: 204-10. 27. Zielinski M, Szlubowski A, Kolodziej M, Orzechowski S, Laczynska E, Pankowski J, et al. Comparison of endobronchial ultrasound and/or endoe­sophageal ultrasound with transcervical extended mediastinal lymphad­enectomy for staging and restaging of non-small-cell lung cancer. J Thorac Oncol 2013 ; 8: 630-6. doi: 10.1097/JTO.0b013e318287c0ce 28. Kolodziejczyk M, Kepka L, Dziuk M, Zawadzka A, Szalus N, Gizewska A, et al. Impact of [18F]fluorodeoxyglucose PET-CT staging on treatment planning in radiotherapy incorporating elective nodal irradiation for non-small-cell lung cancer: a prospective study. Int J Radiat Oncol Biol Phys 2011; 80: 1008-14. doi: 10.1016/j.ijrobp.2010.04.018 29. Jeremic B, Milicic B, Milisavljevic S. Toxicity of concurrent hyperfractionated radiation therapy and chemotherapy in locally advanced (stage III) non-small cell lung cancer (NSCLC): single institution experience in 600 patients. Clin Transl Oncol 2012; 14: 613-8. doi: 10.1007/s12094-012-0848-5 30. Kepka L, Bujko K, Orlowski TM, Jagiello R, Salata A, Matecka-Nowak M, et al. Cardiopulmonary morbidity and quality of life in non-small cell lung cancer patients treated with or without postoperative radiotherapy. Radiother Oncol 2011; 98: 238-43. doi: 10.1016/j.radonc.2010.09.020 455 research article Treatment of rhabdomyosarcoma in children and adolescent from four low health expenditures average rates countries Maja Cesen Mazic1, Aleksandra Bonevski2, Martina Mikeskova3, Emilia Mihut4, Gianni Bisogno5, Janez Jazbec1,6 1 University Children’s Hospital Ljubljana, Slovenia 2 Children’s Hospital Zagreb, Croatia 3 University Children’s Hospital Bratislava, Slovakia 4 Oncology Institute Cluj-Napoca, Romania 5 Hematology Oncology Division, Department of Women’s and Children’s Health, University of Padova, Italy 6 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(4): 455-460. Received 18 June 2020 Accepted 8 September 2020 Correspondence to: Maja Cesen Mazic, M.D., University Children’s Hospital Ljubljana, Bohoriceva 20, 1000 Ljubljana, Slovenia. Phone: 00 386 1 522 9215; Fax: 00 386 1 522 4036; E-mail: maja.cesenmazic@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. Survival of children with cancer in Eastern and Central Europe is 10–20% lower than in high income European countries. We evaluated outcome of children and adolescents with rhabdomyosarcoma (RMS) in Slovenia, Croatia, Slovakia and in Romania. Patients and methods. We retrospectively analysed event-free survival (EFS) and overall survival (OS) for all patients treated in Slovenia and Croatia. Slovakia included patients from two centers, representing half of expected cases. Romania included patients from single institution, representing only 10% of expected patients. Joint database for analysis was established. Results. One hundred seventy-eight children and adolescent with RMS diagnosed from January 2000 to December 2015 were included. Mean patient age at diagnosis was 7.7 years, one third was older than 10 years. Twenty-five per­cent had alveolar histology and 72% unfavorable location. Higher than expected proportion of patients had nodal involvement (24%) or metastatic disease (27%). All patients received systemic chemotherapy, 57% had radiotherapy and 63% surgery as local control. Kaplan- Meier estimates for 5-year EFS and OS were 50.7% and 59.6%, respectively. Five-year OS for patients with localised disease was 72% compared to 24% for metastatic disease. Conclusions. Children with RMS treated in Eastern and Central Europe have inferior outcome compared to their counterparts treated in high income European countries. Active participation of low health expenditures average rates (LHEAR) countries in international clinical trials may improve outcome of paediatric oncology patients. Key words: rhabdomyosarcoma; low income country; outcome Introduction Cancer is diagnosed in more than 35.000 chil­dren and young adults across Europe each year.1 Despite great improvement in treatment and care over last decades, cancer is still leading cause of death due to disease in this population. While 5-year survival rates are around 80% with best available therapy2, survival in Eastern and Central Europe is 10–20% lower than in high income European countries.3 European Society of Pediatric Oncology (SIOPE) study confirmed lack of health care resources to ensure minimal standards of care for children with cancer as one of the main reasons for existing in­equalities.4 456 ALL IC BFM 2002 trial is a role model for inter­national collaboration between centers with limited resources and lower level of experience. Improved management of children with acute lymphoblastic leukemia was achieved with detailed treatment agenda and supportive care guidelines.5 Outcome of patients with soft tissue sarcomas is improved, if patients are treated according to es­tablished guidelines and in expert centers.6 Quality of local control is critical point in therapy and is in­creased in specialized centers with high expertise.6 We report outcome of children and adolescent with rhabdomyosarcoma in four low health expendi­ture average rate (LHEAR) countries. Patients and methods Patients Study presents data from 178 patients aged 0–17 years with rhabdomyosarcoma treated in four LHEAR countries (Slovenia, Slovakia, Croatia, Romania) from January 2000 to December 2015. Data from Slovenia were extracted from National Cancer Registry and are population based. Data from Croatia were collected from all pediatric on­cology hospitals in Croatia (two in Zagreb, Rijeka, Split) and cover entire population. Slovakia provid­ed data from two centers (Department of Pediatric Oncology/Hematology, Children University Hospital, Bratislava and Kosice) and Romania from single center (Oncology Institute Cluj Napoca). Data were collected by first author in joint database. We estimated expected number of patients from WHO International Incidence of Childhood Cancer 3 report (IICC-3).7 In IICC-3 Slovenia has an annual average of 2.2; Croatia an annual average of 4.9; Slovakia an annual average of 5.9 and Romania an annual average of 28 children with soft-tissue sarco­ma. Present study includes 25 cases from Slovenia (annual average 1.6), 73 cases from Croatia (annual average 4.5), 40 cases from Slovakia (annual aver­age 2.5) and 39 cases from Romania (annual aver­age 2.4). Number of cases reported in study from Croatia and Slovenia correspond to expected in population, approximately 50% of cases are report­ed from Slovakia and 10% from Romania. Slovenia has 2 million, Croatia 4 million, Slovakia 5,4 mil­lion and Romania 19 million inhabitants. Patients were eligible for this analysis, if diagnosis of rhab­domyosarcoma was confirmed by local patholo­gist. Disease staging included postsurgical tumor stage (IRS), age and size, histology, site, presence of nodal involvement or distant metastasis. Treatment in Slovenia was based on Cooperative Weichteilsarcom Studiengruppe protocol (CWS); from 2011–2015 patients were enrolled in European Pediatric Soft Tissue Sarcoma Group protocol (5 cases) (EpSSG RMS 2005). Patients from Slovakia were treated according to International Society of Pediatric Oncology malignant Mesenchymal Tumor Group (MMT) guidelines; from 2006 pa­tients were enrolled in EpSSG RMS 2005 protocol (16 cases). Croatia and Romania treated patients according to CWS protocols. Five patients from Croatia were treated according to RMS 2005 rec­ommendations and 3 patients from Romania ac­cording to MMT protocols. Patients from Romania and Croatia did not participate in clinical trials. Statistical methods Disease staging included postsurgical tumor stage (primary complete resection (R0), microscopic residual (R1) or macroscopic residual/biopsy only (R2)), patient age and tumor size (favora­ble = tumor size < 5 cm and age < 10 years, unfa­vorable = tumor size > 5 cm and age > 10 years or < 1 year), histology (favorable = embryonal, spin­dle cell, botryoid, unfavorable = alveolar), primary tumor site (favorable = orbit, para-testicular, vagi-na/uterus, head/neck, unfavorable = para-menin­geal, extremities, genitourinary bladder/prostate and other), presence of nodal involvement or dis­tant metastases. Treatment included surgery (yes/ no) and quality of resection (complete resection [R0], microscopic residual [R1] or macroscopic residual [R2]), chemotherapy (yes/no) and radio­therapy (yes/no). Follow up was performed by pediatric oncolo­gist at least 5 years after completed therapy or until 18 years old, whatever comes later. Five-year overall survival (OS) and event-free survival (EFS) were estimated using Kaplan-Meier method with Pandas, Phyton data analysis library. The statistical significance of each variable was tested by log-rank test. Results Local pathologists classified 111 tumors (62%) as embryonal, 45 (25%) as alveolar, 10 (5%) spindle or botryoid RMS; for 12 cases histology subtype was unknown. Fusion status was determined in 21 (11%) tumors. Mean patient age at diagnosis was 7.7 years (range 3 months to 17.9 years) (Figure 1). Fifty- 457 eight patients (32%) were older than 10 years, 9 were infants. Sixty-three patients (35%) presented with small tumors (< 5 cm) and favorable age. Nodal involve­ment was present in 67 patients (37%), 31 had lo­calized disease. Metastases were diagnosed in 48 patients (27%), 9 had pulmonary metastases only. Eleven patients had head/neck (6%), 50 para-meningeal (28%), 13 orbit (7%), 21 extremity (12%), 15 thoracic (8%), 30 abdominal (16%), 14 bladder/ prostate (8%), 21 para-testicular (12%) and 3 vagi­nal primary tumors (Figure 2). All patients received chemotherapy. Biopsy was only surgical procedure for 124 pa­tients (70%) at diagnosis. Remaining 54 patients (30%) had primary surgery; 33 primary complete resection, 15 microscopic and 6 macroscopic resid­ual disease. Secondary surgery was performed in 66 patients, 62 had biopsy at diagnosis, 4 patients had primary R1 resection. Complete resection was achieved in 24 patients, 23 had microscopic and 15 macroscopic residual disease. For 4 patients result of surgery is not known. Complete resection was most commonly achieved in patients with para-tes­ticular, prostate/bladder, extremity and head and neck primary. Radiotherapy was part of primary treatment in 102 patients (57%). Radiotherapy was omitted in 17/21 patients with para-testicular, 8/13 orbit, 11/14 bladder/prostate and 6/11 head/neck primary. Fourteen metastatic patients and fifteen (50%) with abdominal primary had no radiotherapy. Local control with radiotherapy was applied in 27 pa­tients for the first time at relapse. Patient and dis­ease characteristics are shown in Table 1. For whole group 5-year OS was 59.6% (95% CI 51.8–66.6%) and 5-year EFS 50,7% (95% CI Site distribuon parameningeal orbita extremity thorax abdomen bladder/prostate paratescular vagina/uterus FIGURE 2. Site distribution. Event-free and overall survival of our sample 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 1 2 3 45 Years event free survival overall survival FIGURE 3. Event-free (EFS) and overall survival (OS) of investigated sample. 43.0–58.0%) (Figure 3). At median follow up of 5 years 106 patients were alive, 69 dead due to dis­ease relapse or progression and 3 from toxic death. Patients with bladder/prostate (92%), orbit (72%) and para-testicular (81%) primary had highest OS and those with thoracic primary poorest outcome (40%). Survival in patients with head and neck (54%), para-meningeal (55%), extremity (47%), ab­dominal and pelvic primaries (48%) was around 50%. In the study 15/21 of children with para-testicu­lar rhabdomyosarcoma were older than 10 years and had additional poor prognostic signs (6 alveo­lar histology, 9 lymph node involvement, 4 meta­static disease). Only 6 children with para-testicular RMS fulfilled criteria for low risk group and had 458 TABLE 1. Patient and disease characteristics tion was not reported. Last 3 patients in the study patients were managed in expert foreign centre with combined organ preservation surgery and brachytherapy. Number (pt) 25 72 40 41 Patients with embryonal histology (5-year OS Age < 1 year 1233 62% embryonal vs. 48% alveolar, P = 0.003) and fa- Age > 10 years 8 21 12 17 vorable size and age (5-year OS 76% favorable vs. Favourable histology 19 41 31 30 51% unfavorable, P = .001) had better outcome. Unfavourable histology 5 21 8 11 5-year OS for 130 patients with localized disease was 72% compared to 24% for metastatic patients. Primary surgery Survival of subgroup of patients with pulmonary Biopsy 20 50 28 26 metastases only was better compared to other met­ R0 219 4 8 astatic patients (33% vs. 21%, P = 0.02). All meta­ R1 1383 static patients treated without radiotherapy died. R2 2NA NA 4 Twelve patients out of 27 irradiated as salvage Siteat relapse survived; patients had either orbit, head and neck, para-meningeal, para-testicular, blad- Orbit 2371 der/prostate or vaginal primary. Paratesticular 1 6 8 6 5-year OS of patients with localized disease Vagina/uterus 1 1 1 NA treated with or without radiotherapy (77% vs. 72%, Head/neck NA 3 2 6P = 0.53) was similar. Outcome of patients with sur- Parameningeal 12 22 3 13gical resection was better (5-year OS 70 vs. 48%, P Extremity NA 7 9 5= 0.001) and depended on quality of surgical resec­ tion (R0 90% vs. R1 66% and R2 50% 5-year OS, P Abdomen NA 19 6 5 = 0.001). Complete resection was most commonly Thorax 7512 achieved in patients with para-testicular, prostate/ Bladder/prostate 2 6 3 3 bladder, extremity and head/neck primary. There Size (> 5 cm) 13 44 32 24 are no data regarding mutilation after surgery. Nodes + 6 31 12 18 Metastases + 6 24 13 5 Discussion RT 21 (84%) 39 (54%) 21 (52%) 21 (51%) RTsalvage 213 5 7 Our study supports previous reports of lower sur- Secondary surgery 7 29 18 12vival of pediatric cancer patients in Eastern and R0 111 9 3Central Europe.3 OS for the whole group was close R1 5648to 60%, with high proportion (27%) of metastatic patients and patients with advanced localized dis­ R2 112 1 1 ease (24%). Nodal involvement in localized disease Primary+secondary surgery 12 (48%) 51 (70%) 26 (65%) 27 (65%) was almost two times higher compared to MMT Alive 16 (64%) 44 (61%) 23 (57%) 23 (56%) 89 (13%)8 and RMS 2005 study (15%).9 In large co­horts approximately 15% of patients with rhabdo- Pt = patients; RT = radiotherapy; RTsalvage = salvage radiotherapy myosarcoma had metastases.10,11 Survival rate of patients with metastatic disease in our study was also lower from published analysis of pooled meta­static patients from Europe and United States (24% excellent 5-year OS (100%). Local control in para-vs. 34% 5-year OS).12 meningeal primaries was not optimal, since no-Survival of patients with localised disease (72%) body had surgery and 10/50 had no radiotherapy. is almost 10% lower than in recently presented Radiotherapy was not part of primary therapy in RMS 2005 study, where 5-year OS reached 80%.9 8/13 of patients with orbital RMS. Radiotherapy Patients with localized disease have comparable at relapse salvaged only 1/4 patient, 3 died de-survival to high-risk patients treated in recent RMS spite further treatment with chemoradiotherapy. 2005 study13 or with survival in MMT 89 study Outcome for patients with bladder/prostate prima-(5-year OS 71%) where 50% of survivors were ry was excellent, but extent of surgery and mutila-treated without significant local therapy.8 459 Less than two thirds of the patients (57%) were treated with radiotherapy as part of primary treat­ment. Results from previous studies and recent RMS 2005 show that about 30% of children with RMS can be cured without radiotherapy.11,14-17 Omitting radiotherapy in patients with para-tes­ticular, bladder/prostate, head/neck and orbital primary was in line with MMT protocols. Patients with abdominal, thoracic or para-meningeal pri­mary and metastases were not eligible for radio­therapy due to progressive disease, unacceptable toxicity for local radiotherapist or unavailable gen­eral anesthesia for small children. Without radio­therapy adequate local control cannot be achieved in substantial proportion of children with soft tis­sue sarcoma.14-17 There is difference between par­ticipating countries in number of patients irradi­ated (Table 1). High percentage (84%) of irradiated patients in Slovenia is consequence of longstand­ing tradition of pediatric radiotherapy and site distribution. Multidisciplinary team for pediatric cancer patients in Slovenia (pediatricians, radia­tion oncologist, cytologist/pathologist, surgeons) was established in 1960s by founding member of SIOP, prof. B Jereb.18 Small proportion of children with orbital, head and neck, para-meningeal, testicular, bladder/ prostate and vaginal primary tumor were salvaged with use of radiotherapy at relapse. Majority of patients had biopsy only at diagno­sis. Primary surgery was less common than second­ary. Two thirds had surgery for local control at any point of treatment. Most patients with unfavorable site (para-meningeal and trunk location) had no surgery. Surgical resection was performed in 2/3 of patients in Croatia, Slovakia and Romania and only in half of patients in Slovenia; unfavorable site distribution precluding surgery (80% para-menin­geal and thoracic primary). Quality of resection, with 84% of patients achieving R0 or R1 resection, is comparable with data in MMT 89 study.8 Para-testicular, prostate/bladder, extremity and head/ neck primaries were most common accessible for complete excision. Distribution according to site was as expected, with half of the patients presenting with tumor in head and neck region, majority being para-menin­geal. Genitourinary region was second most fre­quent site (20%). Outcome of patients with para-testicular, orbit and para-meningeal primary was lower than expected. Children with para-testicular RMS in our study were older, with unfavourable histology and disseminated disease. Those in low risk group had excellent survival as expected.19,20 Poor local control compromised outcome of pa­tients with para-meningeal RMS.21-23 In RMS 2005 study there was substantial gap between EFS (77%) and OS (94%) for patients with orbit primary, most patients were salvaged by additional chemoradio-therapy. This was not repeated in our study, since only 1/4 patients survived. Outcome for bladder/ prostate primary was comparable to published results, probably more mutilating surgeries were performed without concomitant radiotherapy.24 Relation of local control modality on outcome was not assessed for other variables, such as tumor site and size, nodal or metastatic disease and is thus of limited value. This study has major limitations. Data for Slovakia and Romania are not population based and are thus source of selection bias, precluding firm conclusions. Lack of standardized diagnostic and therapeutic protocol reflects in poor quality of the data and therefore suboptimal statistical analy­sis. Improved outcome for patients with rhabdo­myosarcoma observed in high income European countries over the last three decades is the result of well-designed protocols based on a multidisci­plinary approach and prospective data collection25, which results in standardization of diagnostic pro­cedures, chemotherapy protocols, radiotherapeutic and surgical guidelines and supportive measures. Unfortunately substantial number of children from member states of European Union are not included in academic (therapy optimization) trials. This re­sults not only in inferior treatment outcomes, but also loss from scientific standpoint as data from this group of patients are not used for therapy optimization trials. Our retrospective analysis of data from four countries should be seen as a step towards activation and motivation of pediatric on­cology centers in LHEAR European countries to more active participation and involvement in clini­cal research work in the field of pediatric oncology. 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Intergroup rhabdomyosarcoma study-IV: results for patients with nonmetastatic disease. J Clin Oncol 2001; 19: 3091-102. doi: 10.1200/ JCO.2001.19.12.309 15. Flamant F, Rodary C, Rey A, Praquin MT, Sommelet D, Quintana, et al. Treatment of non-metastatic rhabdomyosarcomas in childhood and adoles­cence. Results of the second study of the International Society of Paediatric Oncology MMT84. Eur J Cancer 1998; 34: 1050-62. doi: 10.1016/s0959­8049(98)00024-0 16. Koscielniak E, Harms D, Henze G, Jgens H, Gadner H, Herbst M, et al. Results of treatment for soft tissue sarcoma in childhood and adolescence: a final report of the German Cooperative Soft Tissue Sarcoma Study CWS-86. J Clin Oncol 1999; 17: 3706-19. doi: 10.1200/JCO.1999.17.12.3706 17. Cecchetto G, Carli M, Sotti G, Bisogno G, Dall’Igna P, C Boglino, et al. Importance of local treatment in pediatric soft tissue sarcomas with mi­croscopic residual after primary surgery: results of the Italian Cooperative Study RMS-88. Med Pediatr Oncol 2000; 34: 97-101. doi: 10.1002/ (sici)1096-911x(200002)34:2<97::aid-mpo4>3.0.co;2-8 18. Jereb B, Anžic J. Pediatric oncology in Slovenia. Pediatric Hematol Oncol 1996; 13: 401-4. doi: 10.3109/08880019609030851 19. Ferrari A, Bisogno G, Casanova M, Meazza C, Piva L, Cecchetto G, et al. Paratesticular rhabdomyosarcoma: report from the Italian and German Cooperative Group. J Clin Oncol 2002; 20: 449-55. doi: 10.1200/ JCO.2002.20.2.449 20. Stewart RJ, Martelli H, Oberlin O, Rey A, Bouvet N, Spicer RD, et al. Treatment of children with nonmetastatic paratesticular rhabdomyosar-coma: results of the Malignant Mesenchymal Tumors studies (MMT 84 and MMT 89) of the International Society of Pediatric Oncology. J Clin Oncol 2003; 21: 793-8. doi: 10.1200/JCO.2003.06.040 21. Merks JH, De Salvo GL, Bergeron C, Bisogno G, De Paoli A, Ferrari A, et al. Parameningeal rhabdomyosarcoma in pediatric age: results of a pooled analysis from North American and European cooperative groups. Ann Oncol 2014; 25: 231-6. doi: 10.1093/annonc/mdt426 22. Bisogno G, De Rossi C, Gamboa Y, Sotti G, Ferrari A, Dallorso S, et al. Improved survival for children with parameningeal rhabdomyosarcoma: Results from the AIEOP soft tissue sarcoma committee. Pediatr Blood Cancer 2008; 50: 1154-8. doi: 10.1002/pbc.2152 23. Minard-Colin V, Kolb F, Saint-Rose C, Fayard F, Janot F, Rey A, et al. Impact of extensive surgery in multidisciplinary approach of pterygopalatine/ infratemporal fossa soft tissue sarcoma. Pediatr Blood Cancer 2013; 60: 928-34. doi: 10.1002/pbc.24374 24. Chargari C, Haie-Meder C, Guérin F, Minard-Colin V, de Lambert G, Mazeron R, et al. Brachytherapy combined with surgery for conservative treatment of children with bladder neck and/or prostate rhabdomyosarcoma. Int J Rad Oncol 2017; 98: 352-9. doi: 10.1016/j.ijrobp.2017.02.026 25. Bisogno G, Pastore G, Perilongo G, Sotti G, Cecchetto G, Dallorso S, et al. Long-term results in childhood rhabdomyosarcoma: A report from the Italian cooperative study RMS 79. Pediatr Blood Cancer 2012; 58: 872-6. doi: 10.1002/pbc.23292 461 research article Influence of concurrent capecitabine based chemoradiotherapy with bevacizumab on the survival rate, late toxicity and health-related quality of life in locally advanced rectal cancer: a prospective phase II CRAB trial Vaneja Velenik1,5, Vesna Zadnik2,5, Mirko Omejc3,5, Jan Grosek3,5, Mojca Tuta4,5 1 Division of Radiotherapy, Institute of Oncology, Ljubljana, Slovenia 2 Division of Epidemiology, Institute of Oncology, Ljubljana, Slovenia 3 Division of Surgery, University Medical Center Ljubljana, Ljubljana, Slovenia 4 Division of Radiology, Institute of Oncology, Ljubljana, Slovenia 5 Medical Faculty, University of Ljubljana, 1000 Ljubljana Radiol Oncol 2020; 54(4): 461-469. Received 1 March 2020 Accepted 13 June 2020 Correspondence to: Assoc. prof. Vaneja Velenik, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: vvelenik@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Few studies reported early results on efficacy, toxicity of combined modality treatment for locally advanced rectal cancer (LARC) by adding bevacizumab to preoperative chemoradiotherapy, but long-term data on survival, and late complications are lacking. Further, none of the studies reported on the assessment of quality of life (QOL). Patients and methods. After more than 5 years of follow-up, we updated the results of our previous phase II trial in 61 patients with LARC treated with neoadjuvant capecitabine, radiotherapy and bevacizumab (CRAB study) before surgery and adjuvant chemotherapy. Secondary endpoints of updated analysis were local control (LC), disease free (DFS) and overall survival (OS), late toxicity and longitudinal health related QOL (before starting the treatment and one year after the treatment) with questionnaire EORTC QLQ-C30 and EORTC QLQ-CR38. Results. Median follow-up was 67 months. During the follow-up period, 16 patients (26.7%) died. The 5-year OS, DFS and LC rate were 72.2%, 70% and 92.4%. Patients with pathological positive nodes or pathological T3–4 tumors had significantly worse survival than patients with pathological negative nodes or T0–2 tumors. Nine patients (14.8%) developed grade ł 3 late complications of combined modality treatment, first event 12 months and last 87 months after operation (median time 48 months). Based on EORTC QLQ-C30 scores one year after treatment there were no significant changes in global QOL and three symptoms (pain, insomnia and diarrhea), but physical and social func­tioning significantly decreased. Based on QLQ-CR38 scores body image scores significantly increase, problems with weight loss significantly decrease, but sexual dysfunction in men and chemotherapy side effects significantly increase. Conclusions. Patients with LARC and high risk factors, such as positive pathological lymph nodes and high patho­logical T stage, deserve more aggressive treatment in the light of improving long-term survival results. Patients after multimodality treatment should be given greater attention to the regulation of individual aspects of quality of life and the occurrence of late side effects. Key words: rectal cancer; bevacizumab; preoperative chemoradiotherapy 462 Introduction Colorectal cancer (CRC) represents a major public health problem in developed countries, especially in parts of Europe (Hungary, Slovenia, Slovakia, the Netherlands and Norway).1 In Slovenia CRC is most frequently diagnosed among age group 50–74.2 The standard treatment for LARC con­sists of capecitabine-based chemoradiation (CRT) followed by counseling surgery and adjuvant chemotherapy (ChT). Advances in multimodality treatments have significantly reduced 5-year local recurrence rates to less than 10% but this fact is not reflected in better survival.3,4 High rate of distant metastases (30% at 10 years) represents the main problem in achieving even better results of rectal cancer treatment.5 Thus, to achieve better control of systemic disease and consequently better survival, intensified systemic therapy is warranted. The main guideline in developing the most opti­mal rectal cancer treatment regimen is elimination of subclinical micrometastases or/and interruption of the metastatic cascade. Angiogenesis plays a significant role in tumor growth, invasion and me­tastasis. The benefit of antiangiogenic inhibitors on better survival is already known in the treatment of metastatic colorectal cancer.6 Pre-clinical experi­ments in a variety of tumor models have shown encouraging results with the combination of anti-angiogenic strategies with cytotoxic agents such as chemotherapy, ionizing radiation, or both in rectal cancer.7,8 Although bevacizumab, a recombinant humanized monoclonal antibody against vascular endothelial cell growth factor (VEGF), is widely tested in the preoperative treatment of LARC, there are only few studies evaluating survival benefit.9-11 Consequently, safety and efficacy of adding beva­cizumab in the preoperative treatment of LARC remain unclear. Significant progress in various approaches to rectal cancer treatment has led to the fact that long­term results and assessment of a patient’s quality of life (QOL) has become increasingly important for offering patient optimal treatment. The European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30) and its tumor-specific 38-item questionnaire mod­ule Quality of Life Questionnaire Colorectal Cancer Module (QLQ-CR38), that later got its successor QLQ-CR29 with revised and fewer questions, were the first questionnaires introduced specifically for CRC.12-14 In addition to these QOL assessment tools for CRC, the Functional Assessment of Cancer Therapy-Colorectal (FACT-C) is also widely used.13 The FACT-C places a larger emphasis on emotional aspects of QOL while the QLQ-CR38 and QLQ­CR29 have greater dominance in the assessment of disease- and treatment-related symptoms.13 Our phase II trial was originally designed to de­termine the pathologic complete response rate of CRT and bevacizumab as a part of a combined mo­dality approach. This report includes the long-term outcome, late complications and health related QOL of patients treated in CRAB study. Patients and methods The trial design, eligibility criteria, treatment and assessments have been published previously in detail.15 All patients provided signed informed consent based on international standards. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia (Number 173/07/08) and was in agreement with the Declaration of Helsinki. It was registered in the ClinicalTrials.gov database (NCT 00842686). Patient selection In brief, patients with histologically proven stage II/III adenocarcinoma of the rectum within 15 cm of the anal verge and without contraindications for ChT or targeted agents were included. Local extend of the disease was determined by magnetic resonance imaging. Treatment Radiotherapy (RT) was delivered using three-di­mensional conformal computed tomography (CT)­based treatment planning. Four-field box technique with all fields treated daily was used. Patients re­ceived 45 Gy to the pelvis plus 5.4 Gy as a boost to the primary tumor in 1.8 Gy over 5.5 weeks. Capecitabine 825 mg/m2 twice daily was admin­istered concomitantly continuously throughout of RT without interruptions on weekends. Patients received bevacizumab intravenously at a dose 5 mg/kg 14 days prior and on days 1, 15 and 29 dur­ing chemoradiotherapy (CRT). Resection (low an­terior resection or abdominoperineal amputation with total mesorectal excision) was performed 6–8 weeks after the completion of CRT. Patients with histopathological R0 resection received 6 cycles of adjuvant chemotherapy with capecitabine, while in those with R1 additional 2 cycles were given. Pathologic response after CRT with bevacizumab Velenik V et al. / Long term results of adding bevacizumab to radiochemotherapy in rectal cancer 463 was determined according to Dworak tumor re­gression grade (TRG) system. The pathological complete response (pCR) was defined as TRG 4, meaning no tumor cells in surgical specimen. Statistical methods Results for primary endpoint of this prospective phase II study, the pCR rate, and on some second­ary endpoints (pathological response rate, rate of sphincter-sparing surgical procedure, radical resection rate, acute and perioperative toxicity of combined modality treatment) have been reported previously.15 Here we report results on other sec­ondary endpoints of this updated analysis: local control (LC), disease free (DFS) recurrence-free (RFS) and overall survival (OS), late toxicity and health related QOL. Survival rates were calculated using Kaplan-Meier technique. All time intervals were calculating from the date of inclusion. The end day for LC was the date of last follow-up or recurrence in the pelvis; for OS the date of last fol­low up or death from any cause; for DFS the date of relapse, secondary cancer, death for any cause or the last follow-up. Separate analysis was performed for health re­lated QLQ applying the questionnaires launched by the EORTC. For this study the core question­naire EORTC QLQ-C3016 adjoined with the colo-rectal module EORTC QLQ-CR3812 was delivered to the patients twice: before starting the treatment and one year after the treatment was finished. The EORTC QLQ-C30 is a questionnaire assessing in­dividual HRQL during the previous week. The EORTC QLQ-C30 has 30 items and is divided into five function scales (physical, role, cognitive, emo­tional and social functions); three symptom scales (fatigue, pain, nausea or vomiting) and one global health-status/quality of life dimension. The six sin­gle items address specific symptoms: dyspnoea, appetite loss, insomnia, constipation, diarrhoea and a question addressing the financial impact of the disease. The EORTC QLQ-CR38 has 38 ques­tions and is divided into four functional and seven scales of symptoms/problems. The answers record­ed by the questionnaires were transformed into di­mensions ranged 0–100 according to the EORTC scoring instructions.17 For functional scales and single items higher scores represent a higher level of functioning, but for symptom scales and single items, a higher score represents a higher level of symptoms. To examine the statistically significant changes in QLQ scores over time the Wilcoxon signed-rank test was applied. All statistical analysis was performed using the SPSS statistical software package, version 24 (SPSS Inc, Chicago, IL, USA). Values of p < 0.05 were con­sidered statistically significant. Results Between February 2009 and March 2010, 61 pa­tients entered the study protocol at our institution. The detailed characteristics of the patients have been presented previously.15 Baseline assessment included complete history, physical examination, full blood count, serum biochemistry, carcinoem­bryonic antigen, chest radiography, ultrasonogra­phy and/or computed tomography (CT) scan of the whole abdomen. Twelve pts (19.7%) presented with stage II and all other with stage III of dis­ease. In 28 patients (45.9%) the tumor invaded the mesorectal fascia. Radical resection was achieved in 57 pts (95%). Sixty patients were eligible for ef­ficacy analysis. TRG 4 (pCR) was recorded in 8 pts (13.3%) and TRG 3 in 9 pts (15%). Fifty-one pts (83.6%) received capecitabine postoperatively. An intention-to-treat analysis was performed on 60/61 pts as one was misdiagnosed. Median follow-up was 67 months (range, 7 to 79 months). During the follow-up period, 16 patients (26.7%) died. A total of 13 (21.7%) of these deaths were a consequence of rectal cancer progression and the remaining 3 due to unrelated causes. Survival analysis The 5-year OS was 72.2% (95% CI 58.2–84). Recurrences were observed in 14 patients (23.3%) and in one secondary cancer occurred. The latest distant recurrence was observed 54 months after the operation. The 5-years recurrence-free survival and DFS were 75.6% (95% CI 64.6–81.3) and 70% (95% CI 58.5–81.5), respectively. Local recurrence as any component of first failure occurred in 4 pa­tients (6.7%), with isolated local recurrence in 1 (1.7%). The 5-year LC rate was 92.4% (95% CI 85.4– 99.4). Figure 1 illustrates OS, DFS and recurrence-free survival and LC of patients treated in CRAB trial. Based on the Cox proportional hazards regres­sion model, there were no significant association between OS or DFS and gender, age, performance status, cT, cN, TRG and adjuvant ChT. Patients with pathological positive nodes or pathological T3–4 tumors had significantly worse survival than 464 TABLE 1. Overall survival (OS) and disease free survival (DSF) according to pTumor patients with pathological negative nodes or T0–2 and pNodal stage on univariate analysis tumor (Table 1 and Figure 2). pTumor stage pT0-2 85% 85.7% pT3-4 60.9% p=0.043 61.8% p=0.044 pNodal stage pN0 81% 81% pN+ 37.5% 41.7% p=0.003 FIGURE 1. Overall survival (OS), disease free survival (DSF), recurrence-free survival and local control (LC) of patients treated in CRAB trial. Late complications Nin e patients (14.8%) developed grade 3 late complications of combined modality treatment (Common Terminology Criteria for Adverse Events version 4.0) for which hospitalization and/or ur­gent intervention was needed. We recorded the first event 12 months and the last 87 months after the operation with the median time to occurrence of 48 months. All these complications, time to oc­currence after surgery of primary cancer and pro­cedures needed are presented in Table 2. Among patients without mentioned complications, we ob­served permanent defecation complications in 11: constipation in 4, defecation urgency in 3 and fecal incontinence in 4 colostomy free patients. Two pa­tients with stoma experienced permanent urinary complications: 1 incompleted bladder emptying and 1 urinary incontinence. Health related quality of life The results of the health related QOL analysis is summarized in Table 3. One year after treatment there were no significant changes in global quality of life, however the physical and social functioning significantly decreased. On the other hand, there was a significant decrease in three symptoms: pain, insomnia and diarrhea. Based on QLQ-CR38 scores there were no sig­nificant change on three functional scales (i.e. future perspectives, sexual functioning and en­joyment), but the body image scores significantly increase. One year after treatment has been com­pleted our patients reported significantly less problems with weight loss, but there was a sig­nificant increase in ChT side effects (dry mouth, thin or lifeless hair and different taste) and sexual dysfunction in men. Discussion The final results of CRAB study show that preop­erative CRT with bevacizumab and capecitabine is feasible with good compliance and acceptable tox­icity.15 The pCR rate of 13% was similar to an earli­er phase II study by our group examining neoadju­vant single-agent capecitabine plus RT in LARC.18 Direct comparison of long term results could only be possible with the study from Gasparini et Velenik V et al. / Long term results of adding bevacizumab to radiochemotherapy in rectal cancer 465 TABLE 2. Late grade ł 3 adverse events in CRAB trial Fistula rectovaginalis 1 1 12 months 31 months Hartman operation Abdominoperineal excision Fistula enteroperinealis 1 74 months No action due to local and distant progression of the disease Fistula enteroglutealis 1 54 months Incision, drain Fistula uretroperinealis 1 87 months Conservative Fistula enteroperinealis 1 54 months No action due to poor performance status Abscessus perinealis 1 48 months Incision, drain Abscessus presacralis 1 36 months Incision, transversostomy Stenosis ureteri bill. 1 43 months J splint bill al, as they reported comparable characteristics of patients and the same treatment regimen (neoadju­vant bevacizumab 5 mg/kg on days -14, and 1, 15, 29, and concomitant CRT with capecitabine 825/ mg/m2/bid with total radiation dose of 50.4 Gy), but such data were not published.19 Only one study from Willett et al. reported 5-years survival data for LARC, treated with concurrent bevacizumab and preoperative capecitabine-based CRT.10 In com­parison to our study Willet et al. reported compa­rable pCR rate of 16%, although their study had slightly different treatment regimen (neoadjuvant bevacizumab 5–10 mg/kg on days 1,8,15,22 and concomitant CRT with 4 cycles of fluorouracil 225 mg/m2/24h with total radiation dose of 50.4 Gy). Additionally, our is the largest study on the long term efficacy and the only one evaluating late tox­icity for neoadjuvant bevacizumab in LARC (Willet et al. vs. CRAB, 32 patients vs. 61 patients).10 To our knowledge our study is one of the first evaluating longitudinal health related QOL of rectal cancer patients after combined modality treatment and the only one with bevacizumab in this specific group of patients.20 Survival Compared to our study, Willett et al. reported bet­ter 5-year LC rate (92,4% vs. 100%) and OS (72.2% vs. 100%).10 However, no difference in 5-year DFS (Willett et al. vs. CRAB, 75% vs. 75.6%) was ob­served. Pathologic complete response rate and gen­der ratio between studies were comparable. Some differences were seen regarding pathological nodal stage and age of patients. Our results revealed that pathological positive nodes and pathological T3/4 tumors were significantly associated with worse survival (Table 1 and Figure 2). Among patients with pathological positive nodes and pathological T3/4 tumors, the 5-year OS and DFS were 37.5% and 60.9%, 41.7% and 61.8%, respectively. These poor results for high-risk subgroup suggest that more aggressive approach is needed for such a patient. Proportion of patient included in CRAB study with pathological negative nodes was lower than in American study (19.7% vs. 28%).10 This fact could affected poorer survival results in CRAB study. Other studies have also shown that patho­logic nodal status may represent a superior predic­tor of better survival for patients with LARC.21,22 Furthermore, patient characteristics (sex and age) predict survival. In the largest analysis exam­ining the impact of demographic characteristics on the survival of patients with rectal cancer, older age and male gender are associated with worse cancer-specific and OS.23 One of the possible causes for our worse OS could be in the older population that was included in the CRAB study compared to American study (median age with range; 60 years [31–80] vs. 51 years [35–72]).10 A comparison between neoadjuvant single-agent capecitabine CRT and the current study re­vealed promising oncologic outcome with adding bevacizumab in the standard neoadjuvant treat­ment of LARC in Slovenia (5-year OS, DFS and LC; 61.4, 52.4% and 87.4 vs. 72.2, 75.6 and 92.4%).24 Thus, this difference is not statistically significant and we cannot state with certainty that the im­provement depends only on adding bevacizumab to standard neoadjuvant capecitabine-based CRT due to differences in the prescribed radiation dose. In CRAB study we used additionally concomitant radiation boost for which it is known that might have a positive effect on OS.25 466 TABLE 3. Health related quality of life analysis: Comparisons of mean scores with standard deviations (SD) before and 1 year after completed treatment for all scales of EORTC QLQ-C30 and EORTC QLQ-CR38 Global health status/quality of life 29,30 50 62.5 (20.8) 68.0 (19.7) 0.087 Functional scales Physical functioning 1 to 5 50 89.9 (15.3) 84.7 (14.9) 0.008 Role functioning 6.7 50 85.3 (23.4) 81.3 (24.4) 0.557 Emotional functioning 21 to 24 50 80.6 (19.0) 83.0 (20.8) 0.259 Cognitive functioning 20,25 50 89.4 (17.1) 86.7 (20.2) 0.346 Social functioning 26,27 50 86.7 (16.2) 77.7 (21.7) 0.007 Symptom scales Fatigue 10,12,18 50 21.5 (22.0) 20.7 (18.8) 0.607 Nausea and vomiting 14,15 50 2,5 (6.0) 2.3 (6.7) 1.000 Pain 9,19 50 19.7 (27.5) 11.7 (15.9) 0.017 Dyspnoea 8 50 3.3 (11.8) 4.7 (13.5) 0.782 Insomnia 11 50 24.4 (25.9) 15.3 (22.5) 0.044 Appetite loss 13 50 7.8 (20.7) 6.7 (17.8) 0.726 Constipation 16 50 4.4 (14.3) 12.0 (23.1) 0.126 Diarrhoea 17 50 31.1 (30.6) 12.0 (18.8) 0.002 Functional scales Body image 13,14,15 50 8.8 (16.1) 23.1 (24.9) 0.001 Sexual functioning 17,18 47 28.7 (22.9) 29.4 (26.5) 0.859 Future perspective 16 50 54.0 (30.5) 49.7 (30.1) 0.420 Sexual enjoyment 19 22 44.4 (24.6) 48.1 (29.7) 0.527 Symptom scales Chemotherapy side effects 10,11,12 51 7.3 (11.8) 10.7 (13.7) 0.021 General gastrointestinal symptoms 4 to 8 51 22.1 (19.3) 18.0 (18.3) 0.084 Defecation problems 25 to 31 26 27.5 (21.1) 30.2 (16.4) 0.456 Stoma-related problems 32 to 38 0 28.0 (12.3) Sexual dysfunction of men 20,21 23 15.3 (18.0) 42.6 (35.6) 0.006 Sexual dysfunction of women 22,23 4 16.7 (18.6) 11.1 (27.2) 1.000 Radiation-induced effects micturition 1,2,3 51 21.1 (18.3) 21.4 (19.0) 0.945 Weight loss 9 50 19.9 (27.4) 8.5 (18.7) 0.016 *statistically significant values (p < 0.050) are bolded Late toxicities Bevacizumab may cause severe late side effects in metastatic setting but late side effects are relatively rare.26 Most often described bevacizumab related late adverse events are spontaneous intestinal per­foration and delayed anastomotic leak.27,28 The ad­dition of bevacizumab to standard CRT could be one of the possible causes for a higher proportion of fistulas in our study. Only few studies analyzed the incidence of the late anastomotic leakage after low anterior resection Velenik V et al. / Long term results of adding bevacizumab to radiochemotherapy in rectal cancer 467 (LAR). Delayed anastomotic leakages that develop after 30 days after surgery are not uncommon. The incidence is between 0.3% and 9.8%.29-35 It is not clear whether early and late anastomotic leakage af­ter LAR are different entities because some patients with late anastomotic leakage may show unevent­ful postoperative clinical recovery.29 Late leakages more frequently involve the fistula type (22–100%) than early postoperative leakage.29-32,34,35 Pelvic ab­scess and anastomotic-vaginal fistula were the most common causes of delayed complications.35 In our study only one female patient developed anastomotic vaginal fistula 12 months after rectal surgery. In the two studies with higher number of female patients included, the overall rate of anas-tomotic-vaginal fistula after LAR was higher, 3% (11/371) and 5.1% (20/390).36,37 Anastomotic-vaginal fistulas in both studies were diagnosed mostly late, on median postoperative day 83 (15–766) in the first study and 25 (5–172) in the second. Despite that on­ly 3% of the female patients in the first study were treated with preoperative ChT and no one with RT, pre-op ChT was one of the independent risk factor for anastomotic-vaginal fistula formation.36 In the second study, risk factors for anastomotic-vaginal fistula were preoperative radiotherapy, anastomo­sis < 5 cm above the anal verge and cancer stage IV.37 The incidence of the delayed anastomotic leak­age in present study was high (13.1%), but it is important that only three fistulas were diagnosed before 5 years of follow up. Comparing the inci­dence of the delayed anastomotic leakage in our study with other studies is controversial. Follow up in our study was the longest than it was in other published studies. The longest interval between the surgery and fistula formation we found in the literature was 5.7 years and in our study 7.25 years. RT and ChT were identified as an independent risk factor for the development of the late anastomotic complications.32,34,35 In all published studies pa­tients with very mixed type of neo-adjuvant and adjuvant treatment or without treatment were in­cluded. On the other side, in our study all patients were treated with neo-adjuvant CRT. According to previous facts, it is important that we pay attention to the appearance of early signs of the fistula dur­ing follow up in the patient previous treated with bevacizumab even several years after the comple­tion of treatment. Health related QOL The global health status (mean 68), which refers to the general assessment of the health and qual­ity of life of the last seven days, was similar over time, and also comparable to the Slovenian general (mean 71.1) and CRC patients (mean 68.3).38,39 The values of social and physical functioning indicated by patients were significantly influenced by the time between diagnosis and 1 year after treatment indicating a high incidence of problems in this area. On the other hand, 1 year after treat­ment patients reported less pain, insomnia and di­arrhea. These results are consistent with QLQ-C30 scores of Slovenian CRC patients after surgical treatment.39 However, compared above mentioned items with the general Slovenian population, pa­tients 1 year after treatment report more diarrhea, lower physical and social functioning, but less pain and insomnia. The population-based QOL refer­ence values should be taken into account in the interpretation of disease progress and treatment effects. For Slovenian general population it was shown that gender, age and self-rated social class are important confounders in the QOL scores de­scriptions.38 Our long-term trends in longitudinal QOL are in agreement with study published from Couwenberg et al. including rectal cancer patients treated with CRT and surgery.20 They demonstrat­ed that treatment of rectal cancer has larger impact on QOL decline within 3–6 months after the start of treatment, but still gradually improves within 1 year after treatment. Moreover, within two years all scores normalize towards pretreatment levels, although compared to general population lower functioning, more insomnia and fatigue persist for more than 2 years from diagnosis. Based on QLQ-CR38 significantly more ChT side effects and sexual dysfunction of men were observed after 1 year of treatment. However, body image has improved and patients reported less weight loss. Male sexual dysfunction is common and remains high after multimodality treatment for rectal cancer, more precisely, surgical nerve damage remain the main cause.40 Patients treated with well-defined and standardized technique of total mesorectal excision together with minimally invasive techniques experienced less sexual dys­function compared to conventional surgery.41 In addition, laparoscopic resection contributes more to the maintenance of the nerves.40 Limitations The main limitations of this study include the sin­gle center design of the study with small number of patients, which limits statistical power. To assess 468 the QOL we have chosen a QLQ-CR38 and not re­vised successor QLQ-CR29, because the latter was published later in 2007. Consequently, our results are less comparable to others that have been used QLQ-CR29. Conclusions The optimal treatment strategy for patients with LARC is still controversial. Neoadjuvant bevaci­zumab with standard CRT in LARC is acceptable strategy. Further studies of its effect on better long­term outcome are warranted. Patients with LARC with high risk factors, such as positive pathologi­cal lymph nodes and high pathological T stage, deserve more aggressive treatment in the light of improving long-term survival results. While advances in multimodality treatment of CRC are enormous, some of the QOL aspects and long-term safety are often not published and inadequately discussed with patients. LARC treatment may no longer be standardized, but adjusted to the wishes, needs and characteristics of an individual patient. References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018 : GLOBOCAN Estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394-424. doi: 10.3322/caac.21492 2. Zadnik V, Primic Zakelj M, Lokar K, Jarm K, Ivanus U, Zagar T. Cancer burden in Slovenia with the time trends analysis. Radiol Oncol 2017; 51: 47-55. doi: 10.1515/raon-2017-0008 3. Ludmir EB, Palta M, Willett CG, Czito BG. Total neoadjuvant therapy for rec­tal cancer: An emerging option. Cancer 2017; 123: 1497-506. doi: 10.1002/ cncr.30600 4. Sauer R, Becker H, Hohenberger W, Rel C, Wittekind C, Fietkau R, et al. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med 2004; 351: 1731-40. doi: 10.1056/NEJMoa040694 5. Sauer R, Liersch T, Merkel S, Fietkau R, Hohenberger W, Hess C, et al. Preoperative versus postoperative chemoradiotherapy for locally advanced rectal cancer: results of the German CAO/ARO/AIO-94 randomized phase III trial after a median follow-up of 11 years. J Clin Oncol 2012; 30: 1926-33. doi: 10.1200/JCO.2011.40.1836 6. Mody K, Baldeo C, Bekaii-Saab T. Antiangiogenic therapy in colorectal can­cer. Cancer J 2018; 24: 165-70. doi: 10.1097/PPO.0000000000000328 7. Nieder C, Wiedenmann N, Andratschke NH, Astner ST, Molls M. Radiation therapy plus angiogenesis inhibition with bevacizumab : ra­tionale and initial experience. Rev Recent Clin Trials 2007; 2: 163-8. doi: 10.2174/157488707781662733 8. Willett CG, Boucher Y, Duda DG, di Tomaso E, Munn LL, Tong RT, et al. Surrogate markers for antiangiogenic therapy and dose-limiting toxicities for bevacizumab with radiation and chemotherapy: continued experience of a phase I trial in rectal cancer patients. J Clin Oncol 2005; 23: 8136-9. doi: 10.1200/JCO.2005.03.5881 9. Fornaro L, Caparello C, Vivaldi C, Rotella V, Musettini G, Falcone A, et al. Bevacizumab in the pre-operative treatment of locally advanced rectal cancer : a systematic review. World J Gastroenterol 2014; 20: 6081-91. doi: 10.3748/wjg.v20.i20.6081 10. Willett CG, Duda DG, Di Tomaso E, Boucher Y, Ancukiewicz M, Sahani D V, et al. Efficacy, safety, and biomarkers of neoadjuvant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: multidisciplinary phase II study. J Clin Oncol 2009; 27: 3020-6. doi: 10.1200/JCO.2008.21.1771 11. García M, Martinez-Villacampa M, Santos C, Navarro V, Teule A, Losa F, et al. Phase II study of preoperative bevacizumab, capecitabine and radiotherapy for resectable locally-advanced rectal cancer. BMC Cancer 2015; 15: 59. doi: 10.1186/s12885-015-1052-0 12. Sprangers MA, Te Velde A, Aaronson NK. The construction and testing of the EORTC colorectal cancer-specific quality of life questionnaire module (QLQ­CR38). Eur J Cancer 1999; 35: 238-47. doi: 10.1016/S0959-8049(98)00357-8 13. Gujral S, Conroy T, Fleissner C, Sezer O, King PM, Avery KN, et al. Assessing quality of life in patients with colorectal cancer : an update of the EORTC quality of life questionnaire. Eur J Cancer 2007; 43: 1564-73. doi: 10.1016/j. ejca.2007.04.005 14. 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Brussels: European Organisation for Research and Treatment of Cancer; 2001. 18. Velenik V, Anderluh F, Oblak I, Strojan P, Zakotnik B. Capecitabine as a radio-sensitizing agent in neoadjuvant treatment of locally advanced resectable rectal cancer: prospective phase II trial. Croat Med J 2006; 47: 693-700. PMID: 17042060 19. Gasparini G, Torino F, Ueno T, Cascinu S, Troiani T, Ballestrero A, et al. A phase II study of neoadjuvant bevacizumab plus capecitabine and con­comitant radiotherapy in patients with locally advanced rectal cancer. Angiogenesis 2012; 15: 141-50. doi: 10.1007/s10456-011-9250-0 20. Couwenberg AM, Burbach JPM, van Grevenstein WMU, Smits AB, Consten ECJ, Schiphorst AHW, et al. Effect of neoadjuvant therapy and rectal surgery on health-related quality of life in patients with rectal cancer during the first 2 years after diagnosis. Clin Colorectal Cancer 2018; 17: 499-512. doi: 10.1016/j.clcc.2018.03.009 21. Kim NK, Ba ik SH, Seong JS, Kim H, Roh JK, Lee KY, et al. Oncologic outcomes after neoadjuvant chemoradiation followed by curative resection with tumor-specific mesorectal excision for fixed locally advanced - impact of postirradiated pathologic downstaging on local reccurrence and survival. Ann Surg 2006; 244: 1024-30. doi: 10.1097/01.sla.0000225360.99257.73 22. Hernandez JM, Clark W, Weber J, Fulp WJ, Lange L, Shibata D. The impact of pathologic nodal status on survival following neoadjuvant chemoradia­tion for locally advanced rectal cancer. Int J Color Dis 2014; 29: 1061-8. doi: 10.1007/s00384-014-1917-8 23. Berger MD, Yang D, Sunakawa Y, Zhang W, Ning Y, Matsusaka S, et al. Impact of sex, age and ethnicity/race on the survival of patients with rectal cancer in the United States from 1988 to 2012. Oncotarget 2016; 7: 53668-78. doi: 10.18632/oncotarget.10696 24. Velenik V, Oblak I, Anderluh F. Long-term results from a randomized phase II trial of neoadjuvant combined-modality therapy for locally advanced rectal cancer. Radiat Oncol 2010; 5: 88. doi: 10.1186/1748-717x-5-88 25. Badakhshi H, Ismail M, Boskos C, Zhao K, Kaul D. The role of concomitant ra­diation boost in neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Anticancer Res 2017; 37: 3201-5. doi: 10.21873/anticanres.11681 26. O’Hare T, McDermott R, Hannon R. Late anastomotic breakdown with beva­cizumab in colorectal cancers, a case-based review. Ir J Med Sci 2018; 187: 333-6. doi: 10.1007/s11845-017-1676-y 27. Borzomati D, Nappo G. Infusion of bevacizumab increases the risk of intes­tinal perforation: results on a series of 143 patients consecutively treated. Updat Surg 2013; 65: 121-4. doi: 10.1007/s13304-013-0207-2 Velenik V et al. / Long term results of adding bevacizumab to radiochemotherapy in rectal cancer 469 28. Machida E, Miyakura Y, Takahashi J, Tamaki S, Ishikawa H, Hasegawa F, et al. Bevacizumab is associated with delayed anastomotic leak after low anterior resection with preoperative radiotherapy for rectal cancer: a case report. Surg Case Rep 2019; 5: 14. doi: 10.1186/s40792-019-0573-1 29. Floodeen H, Hallbk O, Rutegĺrd J, Sjahl R, Matthiessen P. Early and late symptomatic anastomotic leakage following low anterior resection of the rectum for cancer: are they different entities? Color Dis 2013; 15: 334-40. doi: 10.1111/j.1463-1318.2012.03195.x 30. Hyman N, Manchester TL, Osler T, Burns B, Cataldo PA. Anastomotic leaks after intestinal anastomosis: it’s later than you think. Ann Surg 2007; 245: 254-8. doi: 10.1097/01.sla.0000225083.27182.85 31. Iwamoto M, Kawada K, Hida K, Hasegawa S, Sakai Y. Delayed anastomotic leakage following laparoscopic intersphincteric resection for lower rectal cancer: report of four cases and literature review. World J Surg Oncol 2017; 15: 143. doi: 10.1186/s12957-017-1208-2 32. Lim S, Yu CS, Kim CW, Yoon YS, Park IJ, Kim JC. Late anastomotic leakage after low anterior resection in rectal cancer patients: clinical characteristics and predisposing factors. Color Dis 2016; 18: 135-40. doi: 10.1111/codi.13300 33. Matthiessen P, Lindgren R, Hallbk O, Rutegĺrd J, Sjahl R, Study GRCT on DS. Symptomatic anastomotic leakage diagnosed after hospital discharge following low anterior resection for rectal cancer. Color Dis 2010; 12: 82-7. doi: 10.1111/j.1463-1318.2009.01938.x 34. Morks AN, Ploeg RJ, Sijbrand Hofker H, Wiggers T, Havenga K. Late anasto­motic leakage in colorectal surgery : a significant problem. Color Dis 2013; 15: 271-5. doi: 10.1111/codi.12167 35. Shin US, Kim CW, Yu CS, Kim JC. Delayed anastomotic leakage following sphincter-preserving surgery for rectal cancer. Int J Color Dis 2010; 25: 843­9. doi: 10.1007/s00384-010-0938-1 36. Watanabe J, Ota M, Kawaguchi D, Shima H, Kaida S, Osada S, et al. Incidence and risk factors for rectovaginal fistula after low anterior resection for rectal cancer. Int J Color Dis 2015; 30: 1659-66. doi: 10.1007/s00384-015-2340-5 37. Matthiessen P, Hansson L, Sjah l, Rutegĺrd J. Anastomotic-vaginal fistula ( AVF) after anterior resection of the rectum for cancer - occurrence and risk factors. Color Dis 2010; 12: 351-7. doi: 10.1111/j.1463-1318.2009.01798.x 38. Velenik V, Šecerov-Ermenc A, But-Hadžic J, Zadnik V. Health-related qual­ity of life assessed by the EORTC QLQ-C30 questionnaire in the gen­eral slovenian population. Radiol Oncol 2017; 51: 342-50. doi: 10.1515/ raon-2017-0021 39. Grosek J, Novak J, Kitek K, Bajric A, Majdic A, Košir JA. Health-related qual­ity of life in Slovenian patients with colorectal cancer: a single tertiary care center study. Radiol Oncol 2019; 53: 231-7. doi: 10.2478/raon-2019-0015 40. Nagpal K, Bennett N. Colorectal surgery and its impact on male sexual func­tion. Curr Urol Rep 2013; 14: 279-84. doi: 10.1007/s11934-013-0341-x 41. Chew M, Yeh Y, Lim E, Seow-Choen F. Pelvic autonomic nerve preserva­tion in radical rectal cancer surgery: changes in the past 3 decades. Gastroenterol Rep 2016; 4: 173-85. doi: 10.1093/gastro/gow023 470 research article Breast size and dose to cardiac substructures in adjuvant three-dimensional conformal radiotherapy compared to tangential intensity modulated radiotherapy Ivica Ratosa1,2, Aljasa Jenko1, Zeljko Sljivic1,3, Maja Pirnat4, Irena Oblak1,2 1 Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 4 Department of Radiology, University Medical Centre Maribor, Maribor, Slovenia Radiol Oncol 2020; 54(4): 470-479. Received 18 March 2020 Accepted 10 May 2020 Correspondence to: Assist. Ivica Ratoša, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: iratosa@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to quantify planned doses to the heart and specific cardiac substructures in free-breathing adjuvant three-dimensional radiation therapy (3D-CRT) and tangential intensity modulated radio­therapy (t-IMRT) for left-sided node-negative breast cancer, and to assess the differences in planned doses to organs at risk according to patients’ individual anatomy, including breast volume. Patients and methods. In the study, the whole heart and cardiac substructures were delineated for 60 patients using cardiac atlas. For each patient, 3D-CRT and t-IMRT plans were generated. The prescribed dose was 42.72 Gy in 16 fractions. Patients were divided into groups with small, medium, and large clinical target volume (CTV). Calculated dose distributions were compared amongst the two techniques and the three different groups of CTV. Results. Mean absorbed dose to the whole heart (MWHD) (1.9 vs. 2.1 Gy, P < 0.005), left anterior descending coro­nary artery mean dose (8.2 vs. 8.4 Gy, P < 0.005) and left ventricle (LV) mean dose (3.0 vs. 3.2, P < 0.005) were all significantly lower with 3D-CRT technique compared to t-IMRT. Apical (8.5 vs. 9.0, P < 0.005) and anterior LV walls (5.0 vs. 5.4 Gy, P < 0.005) received the highest mean dose (D). MWHD and LV-D increased with increasing CTV size meanmean regardless of the technique. Low MWHD values (< 2.5 Gy) were achieved in 44 (73.3%) and 41 (68.3%) patients for 3D-CRT and t-IMRT techniques, correspondingly. Conclusions. Our study confirms a considerable range of the planned doses within the heart for adjuvant 3D-CRT or t-IMRT in node-negative breast cancer. We observed differences in heart dosimetric metrics between the three groups of CTV size, regardless of the radiotherapy planning technique. Key words: breast cancer; breast size; 3D-CRT; IMRT; heart dose; left anterior descending coronary artery Introduction Cardiovascular diseases are becoming the most critical competing mortality risk in women with early breast cancer treated with present-day ra­diotherapy (RT).1,2 The relative risk of radiation-induced heart failure increases with rising cardiac radiation exposure, typically reported as mean ab­sorbed dose to the whole heart (MWHD).3-5 MWHD values reflect local radiation therapy practices, and with the help of modern RT approaches, now ranging from 1.7–5.4 Gy6-8 and 1.22–1.65 Gy9, for mean and median values, respectively. However, even very low cardiac exposure does not eliminate the risk of radiotherapy-mediated cardiotoxicity, which has been demonstrated in recent studies.3,5,10 471 In many recent publications, authors favor the use of intensity modulated techniques over three-dimensional conformal radiotherapy (3D-CRT) in node negative breast cancer adjuvant RT, argu­ing for lower MWHD, decreased skin toxicity and more homogeneous dose distribution in the target volume.11-13 Besides the RT technique used, MWHD depends on the position of the patient’s heart rela­tive to the irradiated breast and the shape of their chest wall.14 Different simple anatomical measures were evaluated to predict increased MWHD and subsequently for the need to use one of the heart-sparing techniques, namely deep inspiration breath hold technique (DIBH). Useful anatomical meas­ures are increased chest wall separation (CWS)9, maximum heart distance (the distance between the anterior cardiac contour crossing over the posterior edge of the tangential fields)15, multidimensional assessment of the presence of the heart in contact with the chest wall14 and linear heart contact dis­tance from the left sternal to the beginning of the lung parenchyma edges at the 4th costal arch in the axial axis.16 It has also been shown that the shape and size of the clinical target volume (CTV) result in increased mean and/or maximum point heart doses.9,17,18 If a cohort of breast cancer patients with similar breast volume is defined, specific problems and resolutions can be proposed, because breast contours according to breast size and shape may be associated with the variations in the target vol­ume coverage and calculated dose to organs at risk.19 Three-dimensional treatment planning al­lows target volume to be measured and CTVs of = 500–975 cm3, 975–1.600 cm3 and = 1.600 cm3 have been typically, but not consistently, defined as small, medium and large breasts, respectively.20,21 Additionally, quite a few clinical studies have re­ported a comparison of the clinical adverse events in regard to the three groups of breast sizes.22 Although observed average MWHD in a popu­lation of breast cancer survivors is low, smaller fragments of the heart might have received doses exceeding 25–40 Gy.4,10,23,24 Subclinical cardiac dysfunction was observed early after adjuvant ra­diotherapy for breast cancer with molecular bio­markers25,26, radionuclide myocardial perfusion imaging27–29, echocardiography30–32, and functional magnetic resonance imaging.31 Limited data exist regarding the range of doses received by individu­al heart substructures with adjuvant free-breathing 3D-CRT or tangential intensity modulated radio­therapy (t-IMRT) for left-sided breast cancer. It has been shown that MWHD does not necessarily cor­relate to mean radiation doses, absorbed by cardiac chambers or coronary arteries in adjuvant breast cancer radiotherapy.33–36 Lately, detailed studies of the specific cardiac structures’ absorbed radiation dose in thoracic radiation therapy24,36,37, and the ef­forts to understand the specific radiation dose-vol­ume effects in the heart have emerged. 4,38–41 With expanding knowledge in this field, German Society of Radiation Oncology (DEGRO) recommends new stringent dose constraints for the heart and its substructures: MWHD < 2.5 Gy, left ventricle (LV) Dmean < 3 Gy (LV mean dose), LV V5 < 17% (volume of LV receiving = 5 Gy), LV V23 < 5% (volume of LV receiving = 23 Gy), left anterior descending coro­nary (LADCA) Dmean < 10 Gy (LADCA mean dose), LADCA V30 < 2% (volume of LADCA receiving = 30 Gy), and LADCA V40 < 1% (volume of LADCA receiving = 40 Gy).42 To standardize the reporting of cardiac imag­ing regardless of diagnostic modality, both The American Society of Echocardiography and the European Association of Cardiovascular Imaging recommend using a segmentation model of the LV to assess regional LV function.42,44 The LV segmen­tation model reflects coronary arteries’ territories and permits to compare echocardiography with other imaging modalities.43 Five main LV seg­ments, defined in a cardiac atlas by Duane et al.24 are based on a previously described 17-segmenta­tion model.44 In this work, we hypothesized that in the set­ting of the node-negative left-sided breast cancer adjuvant radiotherapy, the lowest median MWHD and doses to the cardiac substructures would be achieved with the t-IMRT, compared to 3D-CRT. In addition, we assumed that individual patient characteristics, which include chest wall separation and breast volume, would contribute to the differ­ences in absorbed doses to the heart and cardiac substructures, regardless of the treatment plan­ning technique. To test our hypothesis, we aimed to quantify doses to the heart and cardiac sub­structures in present-day free-breathing adjuvant 3D-CRT and t-IMRT and to analyze the differences in dosimetric metrics to organs at risk between three different groups of CTV according to breast size and other individual anatomical information. Patients and methods Patient selection and CT simulation The study was approved by the ethics review board committee (approval number KME 78/07/15). Based on the size of the CTV, we randomly selected 472 TABLE 1. Target goals used in the planning process PTV D2% < 108% PTVeval % > 95% V95 Whole Body Contour Global Dmax < V110% Dmean < 3.2 Gy Heart Gy < 10% V17V35 Gy < 5% Dmean < 10 Gy Ipsilateral Lung Gy < 25% V17V26 Gy < 20% = maximum dose; D = mean dose; Dx % = absorbed dose, received by x % of the PTV; PTV = planning target volume; PTVeval = planning target volume for evaluation; Vx % = fractional volume, receiving x % of the prescribed dose; Vx Gy = fractional volume receiving x Gy Dmaxmean patients with early left-sided node-negative breast cancer. The definitions of the small, medium, and large breast volumes were like those made avail­able elsewhere.22 The patients were referred to ad-juvant radiotherapy between the years 2014 and 2015. All patients underwent a free-breathing non-enhanced simulation computed tomography (CT) scan with a 3 mm slice thickness. The treatment position for all women was supine, on an inclined simulation table using a breast board, with both arms positioned above the head. Delineation, treatment planning, and data collection Whole heart, LV with its anterior, apical, inferior, lateral, and septal walls, right ventricle (RV), left atrium (LA), right atrium (RA), LADCA with proxi­mal, middle and distal segments, right coronary ar­tery (RCA), left circumflex coronary artery (LCX), and left main coronary artery (LMCA) were de­lineated by one radiation oncologist. We followed identification of the individual structure segments by the instructions proposed by Duane et al.24 in a recently published heart atlas. We used a 6 mm di­ameter for all coronary arteries’ segments, as previ­ously proposed.23 The thickness of the LV wall was set to 10 mm. An experienced cardiac radiologist reviewed the contoured cardiac segments. We de­lineated CTV to include total glandular breast tis­sue according to published guidelines.45 Planning target volume (PTV) was generated by adding a 5 mm uniform margin to the CTV, and the planning target volume for evaluation (PTVeval) was created similarly, with a modification that excludes 5 mm below the skin surface. Additionally, we collected anatomically based distance metrics, such as chest wall separation (CWS) and a previously described “4th arch” metric.16 We used Monaco (Elekta AB®, Stockholm, Sweden) as a contouring and treatment planning platform. The prescribed dose was 42.72 Gy in 16 fractions, 5 days per week. For the 3D-CRT treat­ment planning, we used 6MV photon tangential beam arrangement with wedge filters and addition­al 6MV or 15MV small beams in tangential or non-tangential beam direction where needed to achieve a homogeneous dose distribution. The “Collapsed Cone” algorithm was used to calculate the dose. For t-IMRT plans we used the same isocenter posi­tion as with 3D-CRT planning and two tangential 6 MV photon beams positioned in the same direction as for 3D-CRT plans. The plans were calculated us­ing inverse dose optimization with “Monte-Carlo” algorithm. Dynamic Multileaf Collimator (dMLC) technique was used with minimum segment size 1 cm and 30 control points, which generated 25–30 segments per beam. Although “the dose-to-water” reporting is typically used in clinical routine for the inverse optimization treatment plans and since “Collapsed Cone” algorithm does not have that option for calculation, we used “the dose-to-medi­um” reporting in our study for both 3D-CRT and t-IMRT planning in order to improve treatment plan comparability. In the planning optimization procedure, we used institutional target goals for both treatment plans (Table 1). Dose constraints for the specific cardiac substructures were not incorporated into the optimization process but we strived to keep the dose to the whole heart as low as possible without compromising the target coverage for both tech­niques. Each plan was thoroughly evaluated for target coverage and OAR. We reported nominal median absolute doses, without EQD2 (equivalent dose in 2 Gy per fraction) conversion. All treatment plans were created by one dosimetrist and one medical physicist. Statistical analyses Calculated dose distributions were compared amongst the two techniques and the three different groups of CTV. Due to mostly non-parametrically distributed data, dose distributions data between the groups were compared using the Kruskal-Wallis and Mann-Whitney tests. Friedman ANOVA and Wilcoxon signed-rank test were also used to compare values between the two techniques. All 473 TABLE 2. Target volumes’ and organs at risk’s metrics CTV [cm3] 800.6 (124.8–2970.9) 425.7 (124.8–545.5) 867.0 (652.1–1295.1) 1586.8 (1348.9–2970.9) 0.021 PTVeval [cm3] 990.7 (233.5–3336.1) 583.0 (233.5–711.1) 1035.9 (834.3–1576.5) 1874.3 (1605.8–3336.1) < 0.005 PTV [cm3] 1163.3 (340.1–3792.2) 730.7 (340.1–856.6) 1212.3 (985.1–1805) 2134.8 (1826.4–3792.2) < 0.005 CWS [cm] 23.1 (17.9–33.2) 19.5 (17.9–23.2) 24.0 (19.9–28.5) 27.5 (22.9–33.2) < 0.005 4th arch metrics [cm] 4.4 (0–11.6) 1.6 (0–9.6) 5.5 (0–11.6) 7.1 (0–10.7) 0.008 Heart [cm3] 677.7 (432.9–1192.7) 625.2 (432.9–912.8) 671.1 (563.5–872.4) 817.9 (620.1–1192.7) < 0.005 Left Ventricle [cm3] 173.8 (116–277.4) 161.3 (116–251.7) 173.8 (120.8–229.8) 188.7 (147.4–277.4) 0.018 Left Lung [cm3] 1245.1 (809.3–2127.9) 1458.9 (824.5–2127.9) 1123.8 (944.2–1619.2) 1230.6 (809.3–1541.7) 0.003 Right Lung [cm3] 1563.4 (855–2560.1) 1721.9 (992.9–2560.1) 1466.4 (855.1–1838.2) 1493.2 (1089.6–1925.6) 0.002 Lungs [cm3] 2879.7 (1504.6–4789.2) 3241.3 (1877.5–4789.2) 2634.4 (1504.6–3513.6) 2799.8 (1960.2–3479.2) 0.001 CTV = clinical target volume; CWS = chest wall separation distance at isocenter; PTV = planning target volume; PTVeval = planning target volume for evaluation numbers are presented as median values with a range. Statistical analyses were performed with IBM® SPSS® version 24.0 (SPSS Inc., Armonk: NY, IBM corporation). We considered a p-value = 0.05 as statistically significant. Results Patient population and treatment plans Sixty patients with left-sided breast cancer were included in this analysis, divided into groups of small (N = 22, 36.6%), medium (N = 21, 35.0%) and large (N = 17, 28.4%) CTV size. Target volumes’ and OAR’s metrics are presented in Table 2. There was a statistically significant difference between the three groups for all measured target volumes, OAR volumes, and anatomically based simple distance metrics. Regarding target cover­age, all except two dosimetric parameters (PTVeval %, PTVeval D2%), were superior in the 3D-CRT V107 group (Tables 3 and 4). Nevertheless, the t-IM­RT approach resulted in lower high-dose areas (PTVeval V105%) across all three CTV groups. Whole heart For the whole group of evaluated patients, 3D-CRT technique showed significant lower MWHD com­pared to t-IMRT (Table 5) with an absolute differ­ence of 0.2 Gy. Absolute difference in MWHD between the two techniques ranged from 0.06, 0.46 and 0.7 for the groups of medium-, large- and small-sized CTVs, respectively (Table 6). CTV size had an impact on MWHD regardless of the RT technique, while oth­er parameters were not statistically different except for heart-V5 Gy in 3D-CRT technique. In 3D-CRT, MWHD correlated with increased CWS relative to 18.0 cm (0.09 Gy/1 cm, p = 0.0022) and with CTV size (0.06 Gy/100 cm3, p = 0.0015). Low MWHD values (< 2.5 Gy) were achieved in 44 (73.3%) and 41 (68.3%) patients for 3D-CRT and t-IMRT tech­niques, correspondingly (Figure 1). Heart chambers Selected dose-volume parameters for the LV are presented in Table 5 and 6. For the whole group, 3D-CRT showed lower dosimetric metrics for the TABLE 3. Target volume dosimetric metrics PTVeval D98% [Gy] 40.6 (39.8–41.4) 40.3 (38.7–41.6) 0.002 PTVeval D2% [Gy] 44.7 (44.4–45.5) 43.8 (43.8–47.1) NS PTVeval D50% [Gy] 43.3 (42.7–43.7) 42.9 (42.2–43.9) < 0.005 PTVeval V95% [%] 98.1 (95.3–99.6) 96.8 (79.9–99.9) 0.001 PTVeval V105% [%] 1.3 (0.1–10.3) 4.3 (0.01–85.9) < 0.005 PTVeval V105% [cm3] 11.7 (0.08–656.7) 5.4 (0.06–68.0) 0.014 PTVeval V107% [%] 0 (0–1.4) 0.1 (0–9.6) < 0.005 PTVeval V107% [cm3] 0 (0–321.4) 0 (0–7.2) NS PTVeval V110% [%] 0 (0–0) 0 (0–0) NS Dmax [Gy] 45.7 (45.1–46.9) 46.9 (45.3–51) < 0.005 3D-CRT = three-dimensional conformal radiotherapy; D2% = near maximum dose, D50% = median dose; D98% = near minumum dose, Dmax = maximal absorbed dose, NS = not significant; PTVeval = planning target volume for evaluation; t-IMRT = tangential intensity modulated radiation therapy; Vx% = fractional volume, receiving x % of the prescribed dose TABLE 4. Target volume dosimetric metrics and CTV size 3D-CRT PTVeval V95% [%] 97.7 (95.3–99.6) 98.3 (96.2–99.5) 98.8 (97.5–99.4) 0.022 (S vs. M, S vs. L) t-IMRT PTVeval V95% [%] 97.9 (96.3–99.2) 97.3 (95.3–99.0) 96.8 (79.9–99.9) NS p value (3D-CRT vs. T-IMRT) NS p = 0.003 p = 0.013 3D-CRT PTVeval V105% [cm3] 8.1 (0.5–17.5) 12.5 (0.08–91.3) 87.3 (9.5–656.6) < 0.005 (S vs. M, S vs. L, M vs. L) t-IMRT PTVeval V105% [cm3] 7.4 (0.1–61.5) 5.9 (0.09–54) 4.2 (0.06–68.2) NS p value (3D-CRT vs. T-IMRT) NS NS p = 0.012 3D-CRT = three-dimensional conformal radiotherapy; L = large; M = medium; NS = not significant; PTVeval = planning target volume for evaluation; S = small; t-IMRT = tangential intensity modulated radiation therapy; Vx% = fractional volume, receiving x % of the prescribed dose Small CTV Medium CTV Large CTV LV contour, except for LV apical-Dmean and LV-V23 20 Gy. The lowest Dmean values of the dosimetric met­ 18 16 rics for LV, including anterior, lateral, septal, and 14 inferior LV wall, were obtained in the small CTV 12 group, regardless of treatment technique. 10 8 In 3D-CRT, apical and anterior LV walls received 6 the highest Dmean (Table 5), while lateral, septal, and 4 2 inferior regions, received 1.9, 1.6, and only 0.6 Gy, 0 < 2.5 Gy .5 Gy < 2.5 Gy .5 Gy 3D-CRT t-IMRT FIGURE 1. Mean whole heart dose and number of plans within each CTV groups, concerning optimal mean dose value. 3D-CRT = three-dimensional conformal radiotherapy; CTV = clinical target volume; Gy = Gray; t-IMRT = tangential intensity modulated radiation therapy TABLE 5. Radiotherapy technique and selected dose-volume parameters for the whole heart and selected cardiac substructures MWHD [Gy] 1.90 (0.61–4.14) 2.13 (1.06–4.4) < 0.005 LV-Dmean [Gy] 2.98 (0.78–8.03) 3.22 (1.31–7.25) < 0.005 LV-V5 Gy [%] 8.67 (0–26.3) 9.21 (0–26.02) 0.455 LV-V23 Gy [%] 2.46 (0–14.32) 1.86 (0–10.58) 0.003 LV anterior-Dmean [Gy] 5.00 (1.27–20.17) 5.42 (1.94–19.18) < 0.005 LV apical-Dmean [Gy] 8.97 (1.22–24.89) 8.47 (1.64–22.16) < 0.005 LADCA-Dmean [Gy] 8.20 (1.23–27.92) 8.39 (1.8–27.62) < 0.005 LADCA-V30 Gy [%] 5.39 (0–66.34) 2.01 (0–84.20) < 0.005 LADCA-V40 Gy [%] 0 (0–37.8) 0 (0–43.09) < 0.005 LADCA-prox-Dmean [Gy] 2.17 (0.62–8.68) 2.66 (1.22–12.43) < 0.005 LADCA-mid-Dmean [Gy] 9.63 (1.67–40.07) 11.05 (2.26–39.63) 0.956 LADCA-dist-Dmean [Gy] 13.73 (1.44–41.11) 15.93 (2.03–3.89) 0.132 *Wilcoxon signed-rank test; 3D-CRT = three-dimensional conformal radiotherapy; dist = distal; Dmean = mean dose; Gy = Gray; LADCA = left anterior descending artery; LV = left ventricle; mid = middle; MWHD = whole heart mean dose; prox = proximal; t-IMRT = tangential intensity modulated radiation therapy; Vx Gy = fractional volume receiving x Gy respectively. The Dmean of RV, RA, and LA were 1.41 Gy (range, 0.5–4.8), 0.5 Gy (0.3–1.2), and 0.6 Gy (0.4–1.5), respectively and were not statistically sig­nificantly different among different groups of the CTV size. Likewise, with IMRT, apical and anterior LV walls received similarly high mean radiation doses. The Dmean varied from 8.5 Gy (range, 1.64– 22.16), 5.4 Gy (1.94–19.18), 2.33 Gy (1.18–7.59), 2.18 Gy (1.01–4.46), and 1.11 Gy (0.77–1.98) for apical, anterior, lateral, septal and inferior LV walls, corre­spondingly. Seventeen-segmental LV models, rep­resented as a Bull’s eye diagram, with respective Dmean dose distributions, are presented in Figure 2. Low LV-Dmean ( < 3 Gy), LV-V5 (< 17%), and LV-V23 (< 5%) values were achieved in 51.6%, 88.3%, and 73.3% of treatment plans in 3D-CRT and in 41.6%, 88.3%, and 85.0% of treatment plans in t-IMRT, re­spectively. Coronary arteries Planned median Dmean values for LADCA and its segments are presented in Table 5. Median mean doses to other coronary arteries, namely RCA, LCX, and LMCA were 0.7 Gy (range, 0.3–4.7), 0.7 Gy (0.3–2.0), and 0.8 Gy (0.5–2.0), in the 3D-CRT group and 1.14 Gy (0.77–1.86), 1.10 Gy (0.79–2.18) and 1.31 Gy (0.96–2.17) in the t-IMRT group, re­spectively. For the entire group, only parameter LADCA-V30 Gy was found to be lower with t-IMRT compared to 3D-CRT technique, but the reduction 475 was seen only in the medium and large CTV-size groups. Compared to t-IMRT, 3D-CRT technique showed advantages in terms of lower planned Dmean values of proximal, middle and distal LADCA segments (Table 5). However, dose to the proximal LADCA segment increased with the CTV size, regardless of the planning method. The highest Dmean values of the middle and distal LADCA segments were achieved in patients with the medium or large tar­get volumes. Low LADCA-Dmean (< 10 Gy), LADCA-V30 Gy (< 2%), and LADCA-V30 Gy (< 1%) values were achieved in 55.0%, 48.3%, and 71.6% of treatment plans in 3D-CRT and in 56.6%, 51.6%, and 86.6% of treatment plans in t-IMRT, respectively. Figure 2 represents Bull’s eye diagrams of the LV and seg­ment models of the coronary arteries with reported median Dmean distributions for 3D-CRT technique. Discussion By tradition and its contouring pragmatism, MWHD is the most frequently reported surrogate for the assessment of the potential subsequent car-diotoxic effects after radiation therapy for breast cancer. In the present study, we aimed to compare doses to the individual cardiac structures in the circumstances that represent everyday practice in FIGURE 2. Bull’s eye diagrams of the left ventricle and segment models of the free-breathing node-negative left-sided breast can-coronary arteries with reported median D distributions in three-dimensional mean conformal radiotherapy plans, divided in groups according to clinical target volume cer adjuvant 3D-CRT or t-IMRT. Herein, we report size. Contouring segments of left ventricle consisted of anterior (segments 1 and 7), reasonably low median MWHD values achieved apical (segments 13–17), inferior (segments 4 and 10), lateral (segments 5, 6, 11, 12) with both techniques, 1.9 Gy with 3D-CRT and and septal regions (segments 2, 3, 8, 9). 2.1 Gy with t-IMRT. In the contemporary series, CTV = clinical target volume; Gy = Gray; LADCA = left anterior descending artery; LCX = left measured mean or median MWHD values in free-circumflex artery; LMCA = left main coronary artery; RCA = right coronary artery breathing node-negative left-sided breast cancer adjuvant RT are in the range of 2.6–3.6 Gy for 3D-CRT6,33,35,36 and 1.8–4.8 for the intensity modu­lated techniques.11,46,47 lated heart dose and increasing breast size, espe-In both evalu ated techniques, we observed cially when PTV exceeds 1500 cm3.17,18 Compared to statistically significant differences between the small-sized CTV, MWHD increased with medium- groups of small, medium, and large CTV sizes for and large- sized CTVs in our study, although the the following dose-volume parameters: MWHD, absolute differences between the groups were rela-mean doses for proximal LADCA segment, ante-tively small, ranging from 0.73 Gy and 0.97 Gy for rior, lateral, inferior, and septal LV walls. In me-the t-IMRT and 3D-CRT, respectively. Our results dium and large-sized CTV, we observed reduction imply that patients’ anatomy, including CWS and/ of LADCA-Dmean with t-IMRT technique, which or CTV/PTV volume, should be also considered was not statistically different. Our results are con-when choosing the appropriate radiotherapy tech-sistent with previously published studies showing nique (3D-CRT vs. modulated approaches), patient increased CWS, relative to 22 cm, to be one of the setup (prone or lateral vs. supine), and breathing predictors for a higher MWHD, in both normo- adaptation techniques. As previously mentioned, and hypofractionation.9 Other studies have also breast size grouping could be useful in this context, demonstrated the correlation between the calcu-helping to tailor whole breast irradiation.19 476 TABLE 6. Breast size and selected dose-volume parameters for the whole heart and cardiac substructures MWHD [Gy] 1.29 (0.61–3.75) 1.99 (1.06–3.98) 2.05 (1.06–3.84) 2.11 (1.62–3.54) 2.26 (1.04–4.14) 2.72 (1.46–4.4) < 0.005*; 0.047† Heart-V5 Gy [%] 2.56 (0.02–10.84) 3.77 (0.1–11.01) 4.99 (0.59–10.87) 4.34 (1.19–9.58) 5.29 (0–12.81) 6.19 (0.04–12.83) 0.043* Heart-V10 Gy [%] 1.28 (0–7.91) 2.01 (0–7.59) 2.71 (0.01–7.73) 2.24 (0.12–68.14) 3.09 (0–8.24) 3.17 (0–8.54) NS Heart-V17 Gy [%] 0.76 (0–6.61) 1.22 (0–6.08) 2.03 (0–6.52) 1.36 (0–4.79) 2.37 (0–6.92) 3.36 (0–6.42) NS Heart-V20 Gy [%] 0.62 (0–6.22) 1 (0–5.63) 1.83 (0–6.16) 1.17 (0–4.38) 2.15 (0–6.51) 2.01 (0–5.81) NS Heart-V35 Gy [%] 0.15 (0–4.12) 0.2 (0–3.4) 0.93 (0–4.15) 0.31 (0–2.3) 1.06 (0–4.32) 0.63 (0–2.91) NS Heart-V40 Gy [%] 0.02 (0–1.41) 0.01 (0–1.65) 0.24 (0–1.45) 0.03 (0–0.42) 0.03 (0–1.93) 0.04 (0–1.69) NS LV-Dmean [Gy] 2.3 (0.7–5.7) 2.9 (1.31–5.84) 3.2 (1.1–6.9) 3.15 (1.78–5.97) 3.5 (1.3–8.0) 3.92 (1.83–7.25) 0.019* LV-V5 Gy [%] 6.8 (0–17.4) 8.27 (0–17.39) 9.7 (0–22.0) 8.47 (0.46–19.87) 10.8 (0–26.3) 12 (0–26.02) 0.052* LV-V23 Gy [%] 1.4 (0–9.5) 1.73 (0–8.47) 2.8 (0–12.0) 1.81 (0–8.18) 3.3 (0–14.3) 3.1 (0–10.58) NS LV anterior-Dmean [Gy] 3.6 (1.2–12.8) 4.86 (1.94–12.35) 6.8 (2.0–15.9) 5.71 (2.69–14.48) 6.8 (1.9–20.1) 6.94 (2.58–19.18) 0.017* LV lateral-Dmean [Gy] 1.6 (0.7–2.8) 2.16 (1.18–3.38) 1.8 (0.9–6.3) 2.24 (1.55–5.12) 2.5 (1.2–8.7) 2.98 (1.73–7.59) < 0.001*, < 0.001† LV inferior-Dmean [Gy] 0.5 (0.3–3.3) 0.96 (0.77–1.17) 0.6 (0.5–0.9) 1.07 (0.9–1.32) 0.8 (0.6–2.0) 1.33 (0.96–1.98) < 0.005*, < 0.005† LV septal-Dmean [Gy] 1.2 (0.4–3.4) 1.8 (1.01–3.71) 1.6 (1.1–3.4) 2.19 (1.77–3.74) 1.9 (1.2–3.9) 2.56 (1.72–4.46) < 0.005*, < 0.005† LV apical-Dmean [Gy] 6.9 (1.2–19.6) 8.54 (1.64–19.79) 9.0 (1.7–21.9) 8.42 (2.46–19.68) 9.5 (1.2–24.8) 8.91 (1.76–22.16) NS LADCA-Dmean [Gy] 5.2 (1.2–27.9) 6.84 (1.8–27.62) 13.8 (2.6–25.2) 10.76 (3.01–20.73) 11.1 (2.2–21.2) 8.24 (2.84–21.22) NS LADCA-V30 Gy [%] 0.2 (0–66.3) 0.36 (0–63.48) 17.8 (0–59.0) 7.39 (0–84.2) 8.9 (0–43.3) 2.13 (0–46.34) NS LADCA-V40 Gy [%] 0 (0–37.8) 0 (0–43.09) 0.5 (0–32.9) 0 (0–3.22) 0 (0–19.2) 0 (0–7.26) NS LADCA-prox-Dmean [Gy] 1.6 (0.6–8.6) 2.22 (1.22–7.95) 2.9 (0.6–7.2) 2.96 (1.96–5.19) 2.5 (1.4–7.2) 2.84 (2.07–12.43) < 0.001*, 0.002† LADCA-mid-Dmean [Gy] 7.9 (1.6–40.0) 9.12 (2.26–39.63) 17.9 (2.0–38.7) 13.81 (4.22–30.95) 10.4 (2.5–29.8) 11.14 (3.23–36.01) NS LADCA-dist-Dmean [Gy] 5.5 (1.4–41.1) 8.58 (2.03–40.65) 26.9 (3.5–39.4) 17.46 (3.98–35.39) 14.0 (2.4–39.7) 16.32 (2.87–34.95) NS *intergroup comparison within 3D-CRT technique, using Kruskal-Wallis test; † intergroup comparison within t-IMRT technique using Kruskal-Wallis test; 3D-CRT = three-dimensional conformal radiotherapy; CTV = clinical target volume; Dmean = mean dose; dist = distal; Gy = Gray; LADCA = left anterior descending artery; LV = left ventricle; mid = middle; MWHD = whole heart mean dose; NS = not significant; prox = proximal; t-IMRT = tangential intensity modulated radiation therapy; Vx Gy = fractional volume receiving x Gy Despite the low MWHD for the whole group, our study confirms that apical and anterior parts of LV and mid or distal LADCA segments in both 3D-CRT and t-IMRT techniques receive dispropor­tionately higher Dmean radiation doses. Likewise, in the study by Tang et al., segments corresponding to anterior and apical LV wall absorbed the high­est doses, 9.2 Gy and 14.9 Gy, respectively. Patients were treated with tangential breast RT, with or without regional nodal irradiation and with or without DIBH.36 Corresponding values in our study were lower in both evaluated techniques, 3D-CRT vs. t-IMRT for anterior and apical LV walls were 5.0 vs. 5.4 Gy and 8.5 vs. 8.9 Gy, respectively. Lower numbers might reflect a difference in con­toured thickness of the LV wall, 6-9 mm in the study of Tang et al. compared to 10 mm used in our study, as suggested by Duane et al.24 In our work, the LADCA-Dmean was 8.2 Gy (range, 1.2–27.9) in 3D-CRT and 8.4 Gy (range, 1.8– 27.6) in t-IMRT, respectively. Drost et al. in their systematic review of heart doses reported vary­ing dose-volume measurements for the LADCA. The LADCA-Dmean ranged from 1.9–40.8 Gy (aver­age 12.4 Gy)6, which is similar to our data. In our series of treatment plans, we have demonstrated the highest Dmean for the middle LADCA segment in the group of women with medium-sized CTVs (17.9 Gy) but was not significantly different com­pared to the smallest or the largest CTV groups. With t-IMRT, it was possible to lower LADCA high-dose areas (V30 Gy), but not low-dose areas or mean doses to the coronary arteries. Carosi et al. observed no difference in MWHD when t-IM­RT was compared to 3D-CRT (2.0 vs. 1.9 Gy) in 24 patients with a median breast volume of 645 cm3. 477 However, the authors showed a statistically mean­ingful difference in LADCA Dmean (10.3 vs. 11.9 Gy, p=0.0003), LADCA-Dmax and LADCA-V17 Gy pa­rameters using t-IMRT compared to 3D-CRT.48 There are many possible explanations for the dissimilar reported heart and heart substructures’ absorbed doses in free-breathing left-breast only RT. The differences may arise from the discrep­ancy in the total dose prescription and the size of the radiation field, CTV definition and size, OAR contouring, including diameter of the coronary ar­teries and LV thickness, the lack of detailed heart contouring atlases, individual coronary topology, heart size, body mass index, CWS distance, and finally radiotherapy technique used.9,33,49–52 The use of contrast agent53 or automatic substructures’ segmentation without54 or with cardiac magnetic resonance imaging55 could improve contouring consistency, but these technical solutions are un­likely to be widely adopted in the near future. Non-automatic contouring is feasible as showed in a study by Francolini et al. Authors made multiple comparisons of delineated cardiac chambers and 5 left LV wall segments according to aforementioned cardiac atlas24 and confirmed high interobserver delineation consistency.56 Spatial variation in contouring has been shown to result in less than 1 Gy dose variation for most segments and in most regimens in adjuvant breast cancer RT, but higher dose variations up to 21.8 Gy were seen for segments close to the radiation field edge.24 Substantial variation in the estimated dose was observed for LADCA, regardless of which par­ticular delineation guidelines were used.57 Except for proximal LADCA (2.6 Gy vs. 2.5 Gy), absorbed mean Dmean values of LADCA segments and LV were lower in our study compared to the partially wide tangential technique used in Duane and co­workers’ research; 15.1 Gy vs. 25.1 Gy for middle LADCA segment, 17.6 Gy vs. 35.8 Gy for distal LADCA segment, and 3.2 vs. 6.7 Gy for LV. In the study of Wennstig et al., three radiation oncolo­gists, using the heart atlas of Feng et al., achieved substantial spatial agreement in delineating coro­nary arteries on 32 CT study sets. The agreement was the highest for LMCA and LADCA, and less for RCA.23,58 In our study, the coronary vessel di­ameter was set to 6 mm considering both cardiac and respiratory motion, similar to Wennstig and colleagues’ work.23 Based on recent clinical reports, the DEGRO group proposed stringent dose constraints for the heart and its substructures in adjuvant breast can­cer radiation treatment.42 We surpassed at least one of the proposed optimal dose constraints for LV (Dmean < 3 Gy, V5 < 17%, and V23 < 5%) or LADCA (Dmean < 10 Gy, V30 < 2%, and V40 < 1%) in 11.7–51.7% of all evaluated plans. In our plan optimization process, we did not use specific dose-volume con­straints for cardiac substructures. However, it has been shown that additional LADCA or LV con­straints in breast cancer adjuvant 3D-CRT or IMRT treatment planning might help to optimize heart dosimetric metrics further.23,59 In our study, the evaluation of the planned dose to the heart and specific cardiac substructures was performed in a free-breathing simulation CT scan and in the supine position. Ideally, the dose to car­diac substructures should also be evaluated for pa­tients treated using alternative treatment positions (lateral decubitus or prone) or with DIBH. Due to various reasons, most patients are still treated in the conventional free-breathing supine position, whereas prone positioning or DIBH is in the best-case scenario offered to only 28–83% of breast can­cer patients.15,60 All delineations were performed on a non-enhanced CT scan, an approach that may impact the visibility of the small cardiac segments. Additional drawback of our study is not including patients receiving peri-clavicular regional nodal irradiation with or without internal mammary lymph chain irradiation. Strengths of this study include careful contouring of individual cardiac substructures and using a cardiac atlas based on individual anatomy. An experienced cardiac radi­ologist thoroughly evaluated the contours. Conclusions This is the first study to evaluate the cardiac con­touring atlas for radiotherapy by Duane et al.24 si­multaneously considering different CTV size. We confirmed that regardless of very low Dmean val­ues for the whole heart achieved using a 3D-CRT or t-IMRT free-breathing adjuvant RT technique for breast cancer, a small volume of the heart may receive disproportionate D or D values ex- meanmax ceeding 40 Gy. We observed differences in heart dosimetric metrics between the small, medium, and large CTV sizes for both evaluated techniques, which may disappear with DIBH technique. With t-IMRT technique, only few dosimetric metrics were improved compared to 3D-CRT. The ob­served results in our study suggest that anatomic differences, especially breast volume and CWS, should be considered in clinical practice as well as in the dosimetric studies of various treatment plan- 478 ning techniques. Subdividing breast target volume into similar cohorts could be helpful in this context and further research is warranted. The quantifica­tion of the radiation dose variability of individual cardiac substructures is an important first step to understand the unique cardiac structures’ dose-volume predictors for cardiotoxicity in adjuvant, free-breathing breast cancer radiation therapy. In the future, reported absorbed doses may be paired with cardiac imaging and help to choose patients for whom more intense cardiac function monitor­ing is warranted. References 1. Abdel-Qadir H, Austin PC, Lee DS, Amir E, Tu J V, Thavendiranathan P, et al. 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Predictors of cardiac sparing in deep inspiration breath-hold for patients with left sided breast cancer. Front Oncol 2018; 8: 564. doi: 10.3389/ fonc.2018.00564 51. Wollschläger D, Karle H, Stockinger M, Bartkowiak D, Brdel S, Merzenich H, et al. Radiation dose distribution in functional heart regions from tan­gential breast cancer radiotherapy. Radiother Oncol 2016; 119: 65-70. doi: 10.1016/j.radonc.2016.01.020 52. Appelt AL, Vogelius IR, Bentzen SM. Modern hypofractionation schedules for tangential whole breast irradiation decrease the fraction size-corrected dose to the heart. Clin Oncol (R Coll Radiol) 2013; 25: 147-52. doi: 10.1016/j. clon.2012.07.012 53. Lee J, Hua K-L, Hsu S-M, Lin J-B, Lee C-H, Lu K-W, et al. Development of delineation for the left anterior descending coronary artery region in left breast cancer radiotherapy: an optimized organ at risk. Radiother Oncol 2017; 122: 423-30. doi: 10.1016/j.radonc.2016.12.029 54. 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Lorenzen EL, Taylor CW, Maraldo M, Nielsen MH, Offersen B V, Andersen MR, et al. Inter-observer variation in delineation of the heart and left anterior descending coronary artery in radiotherapy for breast cancer: a multi-centre study from Denmark and the UK. Radiother Oncol 2013; 108: 254-8. doi: 10.1016/j.radonc.2013.06.025 58. Feng M, Moran JM, Koelling T, Chughtai A, Chan JL, Freedman L, et al. Development and validation of a heart atlas to study cardiac exposure to radiation following treatment for breast cancer. Int J Radiat Oncol Biol Phys 2011; 79: 10-8. doi: 10.1016/j.ijrobp.2009.10.058 59. Tan W, Wang X, Qiu D, Liu D, Jia S, Zeng F, et al. Dosimetric comparison of intensity-modulated radiotherapy plans, with or without anterior myocar­dial territory and left ventricle as organs at risk, in early-stage left-sided breast cancer patients. Int J Radiat Oncol Biol Phys 2011; 81: 1544-51. doi: 10.1016/j.ijrobp.2010.09.028 60. Park HJ, Oh DH, Shin KH, Kim JH, Choi DH, Park W, et al. Patterns of practice in radiotherapy for breast cancer in Korea. J Breast Cancer 2018; 21: 244-50. doi: 10.4048/jbc.2018.21.e37 480 research article Long-term toxicity and survival outcomes after stereotactic ablative radiotherapy for patients with centrally located thoracic tumors Banu Atalar1,2, Teuta Zoto Mustafayev2, Terence T. Sio3, Bilgehan Sahin1, Gorkem Gungor2, Gokhan Aydn2, Bulent Yapici2, Enis Ozyar1,2 1 Department of Radiation Oncology, Acibadem MAA University, Istanbul, Turkey 2 Department of Radiation Oncology, Acibadem Maslak Hospital, Istanbul, Turkey 3 Department of Radiation Oncology, Mayo Clinic Hospital, Phoenix, Arizona Radiol Oncol 2020; 54(4): 480-487. Received 2 April 2020 Accepted 30 May 2020 Correspondence to: Banu Atalar, M.D., Department of Radiation Oncology, Acibadem MAA University, Istanbul, Turkey. E-mail: banu.atalar@acibadem.edu.tr Disclosure: Terence T. Sio, M.D., M.Sc., provides strategic and scientific recommendations as a member of the Advisory Board and speaker for Novocure, Inc. This position has no relation to this manuscript. All other authors have no additional conflict of interest to disclose. Background. Stereotactic ablative radiotherapy (SABR) is effective for thoracic cancer and metastases; however, adverse effects are greater for central tumors. We evaluated factors affecting outcomes and toxicities after SABR for patients with primary lung and oligometastatic tumors. Patients and methods. We retrospectively identified consecutive patients with centrally located lung tumors that were treated at our hospital from 2009-2016. The effects of patient, disease, and treatment-related parameters on local control (LC), overall survival (OS), and toxicity-free survival (TFS) were evaluated with multivariate analyses. Results. Among 65 consecutive patients identified with 70 centrally located tumors, 20 tumors (28%) were reirradiat-ed. Median (range) total dose for all tumors was 55 (30–60) Gy in 5 (3–10) fractions. Radiographic complete response was obtained in 43 lesions (61%). None of the analyzed factors were correlated with complete response. After a me­dian follow-up of 57 (95% CI, 48–65) months, 10 tumors (14%) relapsed and 37 patients (57%) died; the actuarial 2- and 5-year OS rates were 52% and 28%, respectively. Median OS was significantly lower in patients with grade 3 or higher toxicity vs. lower toxicity (5 vs. 39 months; P < 0.001). Among 17 severe toxicities, 5 were grade 5, and 3 of them were reirradiated to the same field. Grade 3 to 5 TFS was lower with vs. without reirradiation (2-year TFS, 63% vs. 96%; P = 0.02). Conclusions. Our study showed that modern SABR is effective for central lung tumors, and toxicities are accept­able. SABR for reirradiated central lung lesions and possibly for lesions abutting the tracheobronchial tree may result in higher risk of serious toxicities. Key words: lung cancer; radiation; stereotactic ablative radiotherapy; stereotactic body radiation therapy; survival outcomes; toxicity Introduction Because local control (LC) and survival have shown limited improvement after conventionally fractionated radiotherapy for early inoperable lung tumors, interest in alternative, hypofractionated treatment schedules has increased. Stereotactic ablative radiotherapy (SABR) has been effective for primary lung tumors, as well as pulmonary metastases that are associated with other primary organs.1,2 In early studies, biological effective doses (BEDs) to the tumor with an alpha/beta ratio of 10 (BED10) greater than 100 Gy given in 3 or 4 fractions resulted in better LC and improved overall survival (OS) compared with conventional radiotherapy.3-5 However, this potential therapeutic gain can come with a risk of increased toxicities including fatal events, although they are usually rare.6 Proximity 481 to the trachea or main bronchi, within 1–2 cm of the tracheobronchial tree (TBT), is directly related to increased toxicities observed clinically.6-8 As a re­sult, highly fractionated ablative schedules such as 54 Gy in 3 fractions should not be used for centrally located thoracic tumors with such proximity. Recently, the highly anticipated NRG Oncology/ Radiation Therapy Oncology Group (RTOG) 0813 trial was published.9 The maximally tolerated dose of 12 Gy per fraction over 5 fractions was reached in the study; however, the dose-limiting toxicity rate of 7.2% still gives certain clinicians pause for using a 5-fraction regimen, especially for “ultra-central” lesions.10-13 A more fractionated dosing scheme and strict adherence to the organs-at-risk constraints may still need to be defined, especial­ly for tumors that directly invade critical struc­tures. A phase II prospective study (LungTech) by the European Organisation for Research and Treatment of Cancer using 60 Gy in 8 fractions for central lung tumors is ongoing; another Canadian study, SUNSET, mainly focuses on ultracentral le­sions using SABR techniques.14,15 With the full results of these prospective trials still unavailable, we aimed to clarify the effects of current treatment regimens and predisposing fac­tors for increased toxicities in central lung cancers. In the current study, we identified patients treated in our center and reviewed their long-term out­comes regarding LC, OS, and toxicities after SABR for centrally located primary lung and oligometa-static tumors. Patients and methods Patient selection and grouping After approval by our institutional review board, we retrospectively searched our patient database for the records of all consecutive patients treated with their first SABR course to one or more cen­trally located lung lesions between October 2009 and April 2016 at our hospital. Primary stage I or II non-small cell lung cancers (NSCLCs), recurrent tumors after previous irradiation (regularly frac­tionated treatments), and oligometastatic tumors from other primary organs were included. Lesions were grouped according to distance from the tra­cheobronchial tree and mediastinum: 1) tumors with gross tumor volume (GTV) and/or planning target volume (PTV) very close to or abutting the tracheobronchial tree (= 1 cm); 2) tumors with GTV and/or PTV 1 to 2 cm away from the tracheobron­chial tree; 3) tumors intersecting the mediastinum; and 4) tumors abutting the aorta. Patients with at least 3 months of follow-up, or patients who died within 3 months after SABR completion, were in­cluded in all of the analyses. SABR treatments All patients were simulated in the supine position using a wing board. Patients had 1 of 3 motion management methods: 4-dimensional computed tomography (CT) using a Respiratory Gating System (Anzai Medical) or a Real-time Position Management System (Varian Medical Systems), CT performed during 3 phases (free breathing, end-ex­piratory phase, and inspiratory phase), or planning CT during free-breathing or during breath-hold. CT slice thickness was set at 1 to 1.5 mm. Positron emission tomography (PET)/CT fusion was used to assist delineation for some tumors. The target tumor (as GTV) was delineated on the maximum intensity projection when applicable or by using volumes from all 3 phases of breathing, which were united to form the internal target volume. No additional expansion was given to form the clinical target volume (i.e., clinical target volume equaled GTV). PTV margin was given as a 0.5 cm isotrop­ic expansion to the internal target volume for all cases. All patients were treated using a linear accel­erator (Trilogy or TrueBeam STx; Varian Medical Systems). One patient had a tumor treated by CyberKnife (Accuray, Inc). Organs-at-risk dose constraints and PTV cover­age were done according to the RTOG study pro­tocols. Kilovoltage portal imaging and cone beam CT were used in every fraction for every patient’s treatments during the daily setup. For the patient treated by CyberKnife, the Xsight lung tracking and Synchrony systems (Accuray, Inc) were used. Treatment dose and fractionation were deter­mined at the discretion of the treating physician, but lower doses or more protracted schedules, in general, were used for patients undergoing reirra­diation and for tumors abutting the tracheobron­chial tree. BED calculations, based on alpha/beta ratios of 10 (acute) and 3 (late) evaluations, were performed conventionally on the basis of classic ra­diobiology principles in radiation oncology. Statistical methods and outcomes Toxicity-free survival (TFS) and local relapse-free survival (LRFS) were calculated as time since the end of SABR to event occurrence (death or a 482 TABLE 1. Patient, tumor, and treatment characteristics for 65 patients (70 tumors) receiving stereotactic ablative radiotherapy (SABR) Age, year 64 (22–95) Men 50 (77) Primary cancer Lung 49 (70) Colorectal 10 (14) Other (breast, gastric, melanoma, germ cell, RCC) 11 (16) Treatment indication Primary lung (medically inoperable T1–T2) 12 (17) Relapse (primary lung and oligometastatic) 24 (34) Oligometastatic 34 (49) Previous radiation to chest 20 (29) Tumor location = 1 cm from tracheobronchial tree 24 (34) > 1 cm but = 2 cm from tracheobronchial tree 12 (17) Lesions intersecting mediastinum 22 (31) = 1 cm from thoracic aorta 12 (17) Left laterality 37 (53) Lesion size (PTV), cc 33.4 (7.3–461.5) Total dose, Gy 55 (30–60) Dose per fraction, Gy 9.75 (4–18) Fractions 5 (3–10) BED10, Gy 110 (48–151.2) BED10 < 100 Gy 16 (23) = 100 Gy 54 (77) BED3, Gy 228 (90–378) Treatment time, days 10 (5–19) Treatment time < 10 days 30 (43) = 10 days 40 (57) Treatment on consecutive days 6 (9) BED = biological effective dose; PTV = planned tumor volume; RCC = renal cell carcinoma; a Values are median (range) or No. of patients/tumors (%). grade 2 or higher toxicity for TFS and death or lo-coregional relapse for LRFS, whichever occurred earlier). OS for patients with multiple SABR treat­ments was calculated as time since the end of the last SABR to death. Toxicity was graded according to the Common Terminology Criteria for Adverse Events, 4th edition. OS, TFS, and LRFS were calculated using the Kaplan-Meier method, and log-rank tests were used for comparison between groups. Complete response was defined as shrinkage or radiographic disappearance of the tumor on 3-month follow-up scans, with decreasing maximum standardized up­take values (SUV). Partial response was defined as minimal decrease in tumor size or maximum SUV. Progression was defined as an increase in tumor size and also maximum SUV, concerning for resid­ual tumor or recurrence. Multivariate hazard ratios (HRs) and corresponding 95% CIs were calculated by Cox regression analysis. Statistical analysis was performed with IBM SPSS Statistics software ver­sion 23 (IBM SPSS Statistics). All P values were 2-sided, and P < 0.05 was considered statistically significant. Results Our search identified 65 patients (70 lesions) with at least 3 months of follow-up or who died within 3 months after SABR completion. The type of tu­mor was primary lung in 49 (70%) and oligometa-static in 21 (30%). The patient, tumor, and treat­ment characteristics are summarized in Table 1. The treatment planning was 4-dimensional CT in 15 patients (23%), CT during 3 phases in 43 (66%), and CT during free-breathing or during breath-hold in 7 (11%). PET/CT fusion was used to assist delineation for 50 patients (77%). Volumetric mod­ulated arc therapy was the most commonly used technique (34, 52%), followed by 3-dimensional conformal (29, 45%) and dynamic conformal arc (2, 3%) radiotherapies. Median (range) total dose was 55 Gy (30–60 Gy), fraction dose was 9.75 Gy (4–18 Gy), BED10 was 110 Gy (41–151 Gy), and BED3 was 228 Gy (90–378 Gy). The median (range) number of fractions was 5 (3–10). Reirradiation was performed for 20 tumors (28%) (Table 1). The median dose given as reirra­diation was lower than for other tumors (reirradia­tion BED10 dose: 94.4 Gy reirradiation vs. 110 Gy non-reirradiation; P = 0.009). After a median follow-up of 57 months (95% CI, 48–65 months), 43 (61%) of the tumors achieved complete response (Table 2). On univariate analy­sis, BED10 (> 100 vs. = 100 Gy), PTV size (> 33.4 vs. = 33.4 cc), and type of tumor (colorectal metasta­ses vs. other tumors) were not related to complete response radiographically by PET/CT at 3 months after the end of SABR treatments (all P > 0.05). 483 Locoregional control and survival LRFS was lower in patients with colorectal cancer as a primary tumor (2-year LRFS: colorectal me­tastases, 59% vs. other primary tumors, 89%; P = 0.02) (Figure 1A). LRFS also was lower in tumors that did not have a complete response 3 months after the end of SABR (2-year LRFS: no complete response, 51% vs. complete response, 100%; P < 0.001) (Figure 1B). On multivariate analyses, tu­mors with less than complete response had lower LRFS (HR, 18.2; 95% CI, 2.3–145.9; P = 0.006). Other factors, including previous radiotherapy, BED10 greater than 100 Gy, PTV size, or tumor location in relation to the tracheobronchial tree, had no effect on local relapse (all P > 0.05). Overall survival During follow-up, 10 tumors (14%) relapsed (2- and 5-year LC were 84% and 70%, respectively), and 37 patients (57%) died (2- and 5-year OS were 52% and 28%, respectively). Median OS was signif­icantly lower in patients who had toxicity of grade 3 or higher (5 months, grade = 3 toxicity vs. 39 months grade < 3 toxicity) (Figure 2A). Grade 3 or higher toxicity conferred a significantly increased risk of death (HR, 4.7, 95% CI, 2.0–11.2; P < 0.001). Median OS was slightly lower in patients with pri­mary lung cancer than in patients with other pri­mary cancer origins (19 months, lung cancer vs. 49 months, other cancers) (Figure 2B), but the risk of death was not significantly increased (HR, 2.3; 95% CI, 1.0–5.6; P = 0.06). Factors including previous radiotherapy, BED10 higher than 100 Gy, PTV size, or position of the lesions in relation to the tracheo-bronchial tree had no effect on OS (all P >0.05). SABR-related toxicities Seventeen toxicities of grade 2 or higher were ob­served in 13 patients, some patients have more than 1 toxicity (Table 2). Imaging examples of patients with tracheal rupture and vocal cord paralysis are shown in Figure 3. The most common toxicity was radiation-induced pneumonia. Less common tox­icities, including brachial plexus injury (giving rise to Lhermitte sign) and vocal cord paralysis (due to vagus or recurrent laryngeal nerve injury), were observed in 3 patients; radiation-related esophagi-tis occurred in 2 patients. Seven of the 10 toxicities of grade 3 to 5 were observed in reirradiation patients, which conferred an HR of 5.8 (95% CI, 1.7–20.3). Also, 7 of 10 grade TABLE 2. Tumor and patient outcomes after stereotactic ablative radiotherapy (SABR) for central lung tumors Response on 3-month PET/CT after SABR Complete response 43 (61) Partial response 19 (27) Progression 2 (3) Unknown (patient died before 3 months or imaging not 6 (9) performed) Locoregional control 2-year 84% 5-year 70% Median Not reached Overall survival 2-year 52% 5-year 28% Median 28 months 2-Year toxicity-free survival 81% All Toxicities (grade 2 or higher) 17 (26.2%) RT-induced pneumonitis 9 (13.8%) Brachial and recurrent laryngeal nerve injury 3 (4.6%) Esophagitis 2 (3%) Tracheal perforation 1 (1.5%) Fatal hemoptysis 1 (1.5%) Possible RT-related death 1 (1.5%) Toxicity, grade 5 (fatal) 5 (7.7%) RT-induced pneumonitis 2 (3%) Tracheal perforation 1 (1.5%) Fatal hemoptysis 1 (1.5%) Possible RT-related death 1 (1.5%) PET/CT = positron emission tomography/computed tomography; RT = radiotherapy; a Values are No. patients/tumors (%) or No. patients unless otherwise stated. 484 3 to 5 toxicities were observed in lesions abutting the tracheobronchial tree, for an HR of 4.5 (95% CI, 1.3–15.8). Among the 17 toxicities, 5 were grade 5 (fatal). Three out of 5 fatal toxicity patients were reirradiated to the same RT field, and one of them was irradiated to a neighboring field. The prior and reirradiation doses of each patients were 66Gy/33 fractions and 30 Gy/5 fractions; 40 Gy/10 fractions and 59.5Gy/7 fractions; 66 Gy/33 fractions and 30 Gy/5 fractions; and 45 Gy/15 fractions with the neighboring field dose and 50 Gy/5 fractions, re­spectively. We were able to get the medical reports and the thoracic CT for 3 of the patients and con­firmed the grade 5 toxicity; in regard to patient #4, which was reported as “possible RT-related death,” this was due to the fact that his death was unex­pected, and happened only a few weeks shortly after his SABR course; this information was given to us by his relatives. To be estimating this toxicity rate conservatively, we believe that it is reasonable to account for this in the statistics (so it did not ap­pear that we were biased), as the death did happen within one month after SABR. The last patient who had grade 5 toxicity after 1st SABR was treated to a totaldose of 59.5Gy in 7 fractions and notably he had a lesion encasing bronchus with a size of 55 mm which was considered to be a larger lesion for SABR. After a reasonable amount of effort, we could not locate his radiological images; however, the emergency medical notes noted symptoms and signs of him developing an acute pneumonia. As a result, we considered the possibility that it could be a RT-related pneumonia due to the proximity of timing to his SABR course. Survival free of grade 3 to 5 toxicity was lower after reirradiation than in patients without reirra­diation (2-year TFS: 63% after reirradiation vs. 96% without reirradiation) (Figure 4A); the HR was 5.1 (95% CI, 1.3–20.3; P = 0.02). TFS also was lower in tumors abutting the tracheobronchial tree (2-year TFS: 69%, tumors abutting the tracheobronchial tree vs. 93%, other cases) (Figure 4B), but the as­sociated risk did not reach statistical significance (HR, 3.5; 95% CI, 0.9–13.9; P = 0.08). Discussion Grade 3 or higher complications of SABR for cen­trally located lung tumors are still a substantial concern, as reported by multiple studies, includ­ing the most recently published NRG Oncology/ RTOG 0813 trial.5,6,8,9,12 Therefore, more studies are required to evaluate whether these findings are similar in the general population. To our knowl­edge, the current retrospective study is one of the FIGURE 3. Computed tomographic imaging examples of patients with a grade 3 or higher toxicity. (A) Patient with a tracheal rupture after reirradiation. (B) Patient with vocal cord paralysis after reirradiation (previous chest wall radiotherapy). The circled portion indicates the planning target volume. 485 largest series to date for centrally located and ultra-central lung tumors. Favorable outcome and toxic­ity profiles were achieved, which supports the use of 5-fraction and also moderately hypofractionated regimens in this population. The LC rates in our series are comparable to those of other published series which showed ex­cellent tumor control. Although we saw no cor­relation of BED10 doses higher than 100 Gy with better LC, previous studies indicated that BED10 of 100 Gy or higher led to better local progression-free survival and OS.3,4 The reason for the lack of correlation in our study may be the high number of reirradiation lesions, which were prescribed lower radiotherapy doses (mean reirradiation BED10 dose, 94.4 Gy). reirradiation lesions also had shorter follow-up, so their local recurrence rates may appear lower at the time of data analysis. The difference also may relate to the heterogeneity of these tumors, including colorectal oligometastatic, lung cancers with epidermal growth factor recep­tor or anaplastic large-cell lymphoma kinase–gene mutations, and other confounding factors such as chemotherapy before or after SABR. If only non- reirradiation primary lung lesions are considered, the LC rates in our study (2-year LC, 71%) are simi­lar to those in the literature.2 Metastatic tumors with a separate primary seemed to have higher LC rates (2-year LC, 81%) than those reported in the literature (51%–96%, with various radiotherapy doses).1 At this time, there is no clear correlation between LC and radiotherapy doses, although LC was found to be positively correlated with favora­ble response radiographically 3 months after SABR by PET/CT in our study (the use of PET/CT for follow-up is a routine practice at our institution). In our series, 2- and 5-year OS were 48% and 20%, respectively, for patients with primary lung cancer and were 60% and 44%, respectively, for patients with oligometastatic tumors. The 2-year OS rates in the literature range from 33% to 84% depending on primary tumor type, size and num­ber of lesions, disease-free survival from primary tumor treatment to onset of metastasis, and other treatment-related factors.1 Similarly, survival af­ter SABR for patients with NSCLC has also varied among studies, with 2-year OS ranging from 43% to 90% depending on radiotherapy dose, tumor size, clinical performance status, and tumor loca­tion (central vs. not).2 With 29% of our tumor cases being reirradiation and 16% of tumors being larger than 5 cm, our results are comparable to the histori­cal controls as a result. The higher rates of toxicities (including grade 5 cases) also contributed to the lower OS rates in our study. Compared with rates reported in the literature, a slightly higher rate of possible grade 5 toxicities was noted in our cohort; 5 patients who died had treatment complications that may have been causa­tive, including pneumonitis, tracheal perforation, and hemoptysis. OS in patients with grade 3 to 5 toxicity was short, with a median of only 5 months after SABR. Reirradiation carried significant risks in these cases because it resulted in a high cumu­lative dose in the mediastinum. More guidance and research in the future are required for making SABR safer in these clinical scenarios, in which pa­tients often have no other choice but reirradiation, along with proper counseling regarding potential treatment outcomes and adverse effects. For centrally located lung tumors or nodal re­currences after previous irradiation, some authors have discouraged the use of SABR because of the perceived high risks of toxicity.16,17 In other studies that included central lesions without prior radio­therapy, a higher rate of grade 5 toxicities was of­ten reported.16-18 In an analysis of 32 lesions (11 cen­tral) that were previously irradiated, Peulen et al. reported that treatment of central lung lesions and lesions with larger volumes resulted in higher tox­icity; 9 of 29 patients had grade 3 or higher toxic­ity, including 3 cases of fatal hemoptysis.17 Another prospective trial studying salvage SABR in NSCLC did not include any central lesions in their reirra­diation series.18 The GTV or PTV was within 1 cm of the tracheo-bronchial tree (ultracentral) in 24 (34%) of our pa­tients. Four of these patients had grade 5 toxicity. Because 3 of those patients also had reirradiation, 486 we do not know conclusively whether the death was related to reirradiation, tumor proximity to the tracheobronchial tree, or both. The literature re­ports conflicting results regarding the importance of proximity to the tracheobronchial tree (lesions abutting the tracheobronchial tree vs. other cen­tral lesions), with some studies considering these lesions as harboring similar risk as other central tumors and other studies advocating for more cau­tion in their treatment planning.7,8,10,13 Vocal cord paralysis is a rarely recognized com­plication of SABR. To our knowledge, only 2 stud­ies have reported its occurrence.19,20 Shultz et al. concluded that reirradiation to the vagal or recur­rent laryngeal nerve in 1 case and connective tissue disorders in another case led to nerve injury and paralysis of the vocal cord.20 Two of our patients had vocal cord paralysis, which was confirmed by laryngoscopy. In both patients, PET/CT was per­formed at the onset of voice hoarseness to exclude local recurrence or as part of follow-up: None of the patients had lesions that would otherwise ex­plain their symptoms. One of the patients had had SABR to the same lesion previously, and the other patient had previous ipsilateral breast irradiation (the contribution from the previous breast radio­therapy was estimated to be about 15 Gy to the new GTV [by SABR]). Both lesions were located adjacent to the aortic arch and invaded the vagus nerve; they were also in close proximity to the re­current laryngeal nerve (Figure 3). Our study has several limitations. The study was retrospective, and the patient population was more heterogeneous than in other reported series on this topic (in terms of radiotherapy dose and also inclu­sion of primary lung vs. oligometastatic tumors). Because our institution is a tertiary referral center, some patients’ follow-up was not completed in our department. The circumstances related to patients’ death were derived from interviews with relatives instead of medical records, which led us to recat­egorize 1 of the grade 5 toxicities as SABR related instead of “unknown cause.” Heterogeneity and lower patient numbers in different subgroups also may have limited our study power. Conclusions SABR is an effective treatment modality for cen­trally located lung cancers. SABR to reirradiation lesions, and possibly lesions abutting the tracheo-bronchial tree, appeared to carry a higher risk of higher grade toxicities developing in the long term. More research is needed to define the optimal dose and fractionation schedule for both centrally and ultracentrally located lung tumors. We are waiting for completion of more prospective trials, which will hopefully give more information regarding suitable treatment regimens and clearer factors that may predispose patients to increased toxicities after SABR for central lung cancers. Acknowledgment Portions of this manuscript were presented at the International Association for the Study of Lung Cancer 19th World Conference on Lung Cancer, Toronto, Canada, September 23–26, 2018. References 1. Shultz DB, Filippi AR, Thariat J, Mornex F, Loo BW, Jr., Ricardi U. Stereotactic ablative radiotherapy for pulmonary oligometastases and oligometastatic lung cancer. J Thorac Oncol 2014; 9: 1426-33. doi:10.1097/ JTO.0000000000000317 2. Shah JL, Loo BW, Jr. Stereotactic ablative radiotherapy for early-stage lung cancer. Semin Radiat Oncol 2017; 27: 218-28. doi: 10.1016/j.semra­donc.2017.03.001 3. Lagerwaard FJ, Haasbeek CJ, Smit EF, Slotman BJ, Senan S. Outcomes of risk-adapted fractionated stereotactic radiotherapy for stage I non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2008; 70: 685-92. doi: 10.1016/j. ijrobp.2007.10.053 4. Timmerman R, Paulus R, Galvin J, Michalski J, Straube W, Bradley J, et al. Stereotactic body radiation therapy for inoperable early stage lung cancer. JAMA 2010; 303: 1070-6. doi: 10.1001/jama.2010.261 5. Onishi H, Araki T, Shirato H, Nagata Y, Hiraoka M, Gomi K, et al. Stereotactic hypofractionated high-dose irradiation for stage I nonsmall cell lung car­cinoma: clinical outcomes in 245 subjects in a Japanese multiinstitutional study. Cancer 2004; 101: 1623-31. doi: 10.1002/cncr.20539 6. Timmerman R, McGarry R, Yiannoutsos C, Papiez L, Tudor K, DeLuca J, et al. Excessive toxicity when treating central tumors in a phase II study of stereotactic body radiation therapy for medically inoperable early-stage lung cancer. J Clin Oncol 2006; 24: 4833-9. doi: 10.1200/JCO.2006.07.5937 7. Tekatli H, Haasbeek N, Dahele M, De Haan, Verbakel PW, Bongers E, et al. Outcomes of hypofractionated high-dose radiotherapy in poor-risk patients with “ultracentral” non-small cell lung cancer. J Thorac Oncol 2016; 11: 1081-9. doi: 10.1016/j.jtho.2016.03.008 8. Haseltine JM, Rimner A, Gelblum DY, Modh A, Rosenzweig KE, Jackson A, et al. Fatal complications after stereotactic body radiation therapy for central lung tumors abutting the proximal bronchial tree. Pract Radiat Oncol 2016; 6: e27-33. doi: 10.1016/j.prro.2015.09.012 9. Bezjak A, Paulus R, Gaspar LE, Timmerman RD, Straube WL, Ryanet WF, al. Safety and efficacy of a five-fraction stereotactic body radiotherapy sched­ule for centrally located non-small-cell lung cancer: NRG Oncology/RTOG 0813 Trial. J Clin Oncol 2019; 37: 1316-25. doi: 10.1200/JCO.18.00622 10. Chaudhuri AA, Tang C, Binkley MS, Jin M, Wynne JF, von Eyben R, et al. Stereotactic ablative radiotherapy (SABR) for treatment of central and ultra-central lung tumors. Lung Cancer 2015; 89: 50-6. doi: 10.1016/j.lung­can.2015.04.014 11. Chang JY, Li QQ, Xu QY, Allen PK, Rebueno N, Gomez DR, et al. Stereotactic ablative radiation therapy for centrally located early stage or isolated parenchymal recurrences of non-small cell lung cancer: how to fly in a “no fly zone”. Int J Radiat Oncol Biol Phys 2014; 88: 1120-8. doi: 10.1016/j. ijrobp.2014.01.022 487 12. Haasbeek CJ, Lagerwaard FJ, Slotman BJ, Senan S. Outcomes of stereotactic ablative radiotherapy for centrally located early-stage lung cancer. J Thorac Oncol 2011; 6: 2036-43. 12. doi: 10.1097/JTO.0b013e31822e71d8 13. Raman S, Yau V, Pineda S, Le LW, Lau A, Bezjak A, et al. Ultracentral tumors treated with stereotactic body radiotherapy: single-institution experience. Clin Lung Cancer 2018; 19: e803-e810. doi: 10.1016/j.cllc.2018.06.001 14. Adebahr S, Collette S, Shash E, Lambrecht M, Le Pechoux C, Faivre-Finn C, et al. LungTech, an EORTC Phase II trial of stereotactic body radiotherapy for centrally located lung tumours: a clinical perspective. Br J Radiol 2015; 88: 20150036. doi: 10.1259/bjr.20150036 15. Giuliani M, Mathew AS, Bahig H, Bratman SV, Filion E, Glicket D, et al. SUNSET: stereotactic radiation for ultracentral non-small-cell lung cancer - a safety and efficacy trial. Clin Lung Cancer 2018; 19: e529-32. doi: 10.1016/j. cllc.2018.04.001 16. Matsuo Y. A systematic literature review on salvage radiotherapy for local or regional recurrence after previous stereotactic body radiotherapy for lung cancer. Technol Cancer Res Treat 2018; 17: 1533033818798633. doi: 10.1177/1533033818798633 17. Peulen H, Karlsson K, Lindberg K, Tullgren O, Baumann P, Lax I, et al. Toxicity after reirradiation of pulmonary tumours with stereotactic body radiother­apy. Radiother Oncol 2011; 101: 260-6. doi: 10.1016/j.radonc.2011.09.012 18. Sun B, Brooks ED, Komaki R, Liao Z, Jeter M, McAleer M, et al. Long-term outcomes of salvage stereotactic ablative radiotherapy for isolated lung recurrence of non-small cell lung cancer: a phase II clinical trial. J Thorac Oncol 2017; 12: 983-92. doi: 10.1016/j.jtho.2017.02.018 19. Binkley MS, Hiniker SM, Chaudhuri A, Maxim PG, Diehn M, Loo BW Jr, et al. Dosimetric factors and toxicity in highly conformal thoracic reirradiation. Int J Radiat Oncol Biol Phys 2016; 94: 808-15. doi: 10.1016/j.ijrobp.2015.12.007 20. Shultz DB, Trakul N, Maxim PG, Diehn M, Loo BW, Jr. Vagal and recurrent laryngeal neuropathy following stereotactic ablative radiation therapy in the chest. Pract Radiat Oncol 2014; 4: 272-8. doi: 10.1016/j.prro.2013.08.005 488 research article Does regular quality control improve the quality of surgery in Slovenian breast cancer screening program? Andraz Perhavec1,2, Sara Milicevic1, Barbara Peric1,2, Janez Zgajnar1,2 1 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(4): 488-494. Received 4 April 2020 Accepted 26 April 2020 Correspondence to: Prof. Janez Žgajnar, M.D., Ph.D., Department of Surgical Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. E-mail: e-mail: jzgajnar@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of our study was to evaluate the quality of surgery of Slovenian breast cancer screening pro­gram (DORA) using the requested EU standards. Furthermore, we investigated whether regular quality control over the 3-year period improved the quality of surgical management. Patients and methods. Patients who required surgical management within DORA between January 1st, 2016 and December 31st, 2018 were included in the retrospective study. Quality indicators (QIs) were adjusted mainly ac­cording to European Society of Breast Cancer Specialists (EUSOMA) and European Breast Cancer Network (EBCN) recommendations. Five QIs for therapeutic and two for diagnostic surgeries were selected. Additionally, variability in achieving the requested QIs among surgeons was analysed. Results. Between 2016 and 2018, 14 surgeons performed 1421 breast procedures in 1398 women. There were 1197 therapeutical (for proven breast cancer) and 224 diagnostic surgical interventions respectively. Overall, the minimal standard was met in two QIs for therapeutic and none for diagnostic procedures. A statistically significant improve­ment in three QIs for therapeutic and in one QI for diagnostic procedures was observed however, indicating that regular quality control improves the quality of surgery. A high variability in achieving the requested QIs was observed among surgeons, which remained high throughout the study period. Conclusions. Adherence to all selected surgical QIs in patients from screening program is difficult to achieve, especially to those specifically defined for screen-detected lesions. Regular quality control may improve results over time. Reducing the number of surgeons dedicated to breast pathology may reduce variability of management inside the institution. Key words: breast surgery; mammography screening program; quality control Introduction Breast cancer is the most common female can­cer with an estimated incidence of 523,000 cases in Europe in 2018 and the third leading common cause of death from cancer (138,000 cases per year).1 A 5-year survival rate in women with breast cancer ranges from 25.1% to 95.7% and depends mainly on the stage of the disease at the time of di­agnosis.2 Screening programs are efficient in early detection of cancer and lead to a better prognosis and less intensive treatment.3,4 As the management of early breast cancer is complex, the optimal outcomes are ensured in the specialized multidisciplinary breast cancer centres.5 Comprehensive quality assurance is of great im­portance for maintaining the appropriate balance between benefits and harms.3 European Society of Breast Cancer Specialists (EUSOMA) and European Breast Cancer Network (EBCN) provided a set of quality indicators (QIs) in order to establish mini­mum standard of care and to improve the quality of care, patient satisfaction and outcome. QIs also allow standardised quality of care evaluation.5-7 489 Slovenian breast cancer screening program, called DORA, is a national population-based or­ganized screening program inviting women aged 50–69 to biannual mammography and it is aimed to detect breast cancer in asymptomatic women in early stages. Quality of surgery and the impact of regular quality control on improvement of QIs within screening program is not known. In 2016, we defined and regularly monitored a set of sur­gical QIs mainly from EUSOMA and EBCN for women that undergo surgical procedure for a sus­picious or malignant lesion detected in Slovenian Breast Cancer screening Program. The aim of our study was to evaluate the quality of surgical treatment of patients from the DORA program and to investigate whether the surgi­cal approach fulfils the requested EU standards. Furthermore, we investigated whether regular quality control over the 3-year period improved the quality of surgical management. Patients and methods A retrospective study of women who required sur­gical management within Slovenian Breast Cancer screening Programme between January 1st, 2016 and December 31st, 2018 was performed. The data were prospectively collected from the National screening programme registry and missing data supplemented by reviewing patient’s records. Slovenian National screening program DORA, with centrally organized invitation system, pro­vides screening mammography every two years for women aged from 50 to 69 with residence in Slovenia. The programme was initiated in April 2008. The average participation rate in the years between 2008 and 2018 was 73%. Between 2016 and 2018, 216,717 women were screened in the DORA programme. A total of 1352 (0.6%) breast cancers were detected during that time.8 For the purpose of this study, patients were di­vided into two groups: the group in which breast cancer was preoperatively histologically or cytologi­cally confirmed (B5 or C5 lesions) and the group in which diagnostic surgical procedure was performed due to lesions of uncertain malignant potential. A decision on management of screen-detected breast cancer was always made within multidisci­plinary tumour board. Thus, this QI was not moni­tored, as it was not expected to change over time. Since all patients were operated at the same insti­tution (Institute of oncology Ljubljana), the same rationale approaches were used. All but one QIs have been chosen according to EUSOMA recommendations from 2010 and EBCN recommendations from 2006.6,7 We did not in­clude all QIs as we do not routinely collect all the required data. Furthermore, we tried to avoid be­coming overwhelmed with too many indicators. QIs that have been defined and regularly moni­tored in patients in which breast cancer was preop­eratively histologically or cytologically confirmed are: (1) median waiting time (in days) from multidisci­plinary tumour board to surgery and propor­tion of patients who waited less than 15 work­ing days from multidisciplinary tumour board to surgery: both EUSOMA and EBCN consider waiting time as a QI; since in our study only patients with screen detected lesions were in­cluded, we decided to choose more strict EBCN recommendation; (2) proportion of patients (invasive and noninva­sive cancers) who received a single (breast) operation for the primary tumour (excluding reconstruction): EUSOMA recommendation considers invasive and noninvasive cancers separately and the recommendation is stricter for invasive cancers (80%) compared to nonin­vasive cancers (70%); we decided to combine invasive and noninvasive cancers into one QI with stricter criteria to provide more robust numbers and to avoid becoming overwhelmed with too many rather similar QIs; (3) proportion of patients with invasive breast cancer not greater than 3 cm (total pathologi­cal size, including DCIS component) who un­derwent breast conserving treatment (BCT): EUSOMA QI; (4) proportion of patients with invasive cancer and axillary clearance performed with at least 10 lymph nodes (LN) examined: EUSOMA QI; (5) mean weight of the excised specimen and the pro­portion of specimens from the breast with weight less than 80 g when breast conserving surgery was performed: this is neither the EUSOMA nor the EBCN QI. We decided to include it as a sur­rogate of expected cosmetic result. The average specimen from breast conserving surgery should weigh between 20 and 40 g and as a general rule, 80 g of breast tissue is the maximum weight that can be removed from a medium-sized breast without resulting in deformity.9 QIs that have been regularly monitored in pa­ tients in which diagnostic surgical procedure was performed due to lesions of uncertain malignant potential are: 490 (1) median waiting time (in days) from multidisci­plinary tumour board to surgery and propor­tion of patients who waited less than 15 work­ing days from multidisciplinary tumour board to surgery: EBCN QI; (2) mean weight of the excised specimen and the proportion of specimens with weight less than 30 g: EBCN QI. Patients who received neoadjuvant chemother­apy were excluded from calculations of median waiting time. All selected QIs have been regularly monitored each year, starting in 2016. All statistical analyses were performed using SPSS for Windows, version 22.0. Data were sum­marized using frequencies and percentages for cat­egorical variables and median or mean for continu­ous variables. Chi square test was used to compare categorical variables and Mann-Whitney test or ANOVA for continuous variables. P-values = 0.05 were considered statistically significant. This study was conducted according to the rules of the Ethical Committee of the Institute of oncol­ogy Ljubljana. Results Between 2016 and 2018, 14 surgeons performed 1421 breast procedures in 1398 women from breast cancer screening program DORA. There were 1197 procedures therapeutic because of histologically or cytologically proven invasive or in situ breast can­cer and 224 procedures were diagnostic because of lesions of uncertain malignant potential. To determine whether our surgical approach reaches the requested EU standards, quality of care was evaluated by predefined QIs. Pathologic characteristics of patients that under­went therapeutic surgical procedures split by the year of treatment are shown in Table 1. Quality in­dicators of therapeutic surgical procedures split by the year of surgery are shown in Table 2. Pathologic characteristics of patients that under­went diagnostic surgical procedures split by the year of surgery are shown in Table 3. Quality in­dicators of diagnostic surgical procedures split by the year of surgery are shown in Table 4. Nineteen out of 224 (8.5%) patients needed reop­eration after diagnostic breast surgical procedure; 5/224 (2.2%) re-excision, 4/224 (1.8%) mastectomy, 2/224 (0.9%) re-excision followed by mastectomy and 8/224 (3.6%) only sentinel lymph node biopsy. There were 1421 breast procedures performed by 14 surgeons. The variability among surgeons in 3 years period is shown in Table 5. Discussion Regularly reporting and analysing outcome data is important in order to allow centres to ensure patients with breast cancer the optimal manage­ment and to recognise the particular areas, where the improvements are required. Namely, focusing on QIs shows higher compliance to recommended treatment and better outcome.10-14 Furthermore, by providing outcome data, the new scientific knowl­edge is incorporated and minimum standards are upgraded.5 TABLE 1. Characteristics of therapeutic procedures performed between 2016 and 2018 Procedure type in breast BCT mastectomies Procedure type in axilla: none SNB ALND SNB + ALND Histologic tumour type Benign In situ Microinvasive carcinoma Invasive carcinoma Other 927 (77.4%) 218 (73.6%) 312 (74.8%) 397 (82.0%) 270 (22.6%) 78 (26.4%) 105 (25.2%) 87 (18.0%) 202 (16.9%) 34 (11.5%) 72 (17.3%) 96 (19.8%) 858 (71.7%) 211 (71.3%) 304 (72.9%) 343 (70.9%) 87 (7.3%) 35 (11.8%) 25 (6.0%) 27 (5.6%) 50 (4.2) 16 (5.4%) 16 (3.8%) 18 (3.7%) 64 (5.3%) 6 (2.0%) 19 (4.6%) 39 (8.1%) 214 (17.9%) 50 (16.9%) 84 (20.1%) 80 (16.5%) 19 (1.6%) 2 (0.7%) 12 (2.9%) 5 (1.0%) 899 (75.1%) 238 (80.4%) 302 (72.4%) 359 (74.2%) 1 (0.1%) 0 (0.0%) 0 (0.0%) 1 (0.2%) 0.007 0.002 0.004 Mean tumour diameter ± 17.8 ± 13.3 18.9 ± 14.6 18.2 ± 13.6 16.8 ± 12.0 0.127 S.D. (mm) ALND = axillary lymph node dissection; BCT = breast conserving treatment; S.D. = standard deviation; SNB = sentinel node biopsy 491 TABLE 2. Quality indicators of therapeutic surgical procedures split by the year of treatment 1. QI: waiting time Median (days) Proportion of patients with waiting time less than 15 working days 2. QI: proportion of patients (invasive and noninvasive cancers) who received a single (breast) operation for the primary tumour (excluding reconstruction) 3. QI: proportion of patients with invasive breast cancer not greater than 3 cm (total size, including DCIS component) who underwent BCT 4. QI: proportion of patients with invasive cancer and axillary clearance performed with at least 10 LN examined 5. QI: weight of the excised specimen from the breast Mean ± S.D. (g) Proportion of specimens with weight = 80g (%) Not more than 15 working days = 90% Minimum standard: 80% Target: 90% Minimum standard: 70% Target: 80% Minimum standard: 95% Target: 98% Proportion of specimen after BCT with weight less than 80g = 90% 33 4.2% 1088/1197 (90.9%) 669/801 (83.5%) 124/137 (90.5%) 69.0 ± 42.5 70.0% 38 1.7% 266/296 (89.9%) 165/211 (78.2%) 45/51 (88.2%) 75.3 ± 50.3 61.8% 34 2.9% 379/417 (90.9%) 220/265 (83.0%) 40/41 (97.6%) 63.8 ± 35.9 75.6% 28 < 0.001 6.9% 443/484 0.735 (91.5%) 284/325 0.019 (87.4%) 39/45 (86.7%) 0.014 69.7 ± 42.3 0.009 70.0% BCT = breast conserving treatment; DCIS = ductal carcinoma in situ; LN = lymph nodes; QI = quality indicator; S.D.= standard deviation In 2016, we defined a series of five surgical QIs including DCIS component) who underwent BCT) for therapeutic and two surgical QIs for diagnostic and none for diagnostic procedures. Furthermore, procedures for women diagnosed with breast le-we observed a statistically significant improve-sion within national screening program. Overall, ment in three QIs for therapeutic (waiting time, the minimal standard was met in two QIs for ther-proportion of patients with invasive breast cancer apeutic (proportion of patients (invasive and non-not greater than 3 cm (total size, including DCIS invasive cancers) who received a single (breast) component) who underwent BCT and weight of operation for the primary tumour (excluding re-the excised specimen) and in one QI for diagnostic construction) and proportion of patients with inva-procedures (waiting time), indicating that regular sive breast cancer not greater than 3 cm (total size, quality control may improve the quality of sur- TABLE 3. Results of the diagnostic surgical procedures split by the year of treatment Procedure type in axilla: - none 208 (92.9%) 75 (96.2%) 59 (89.4%) 74 (92.5%) - SNB 16 (7.1%) 3 (3.8%) 7 (10.6%) 6 (7.5%) - ALND 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) - SNB + ALND 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) Histologic tumour type - Benign 177 (79.0%) 63 (79.5%) 52 (78.8%) 62 (77.5%) - In situ 28 (12.5%) 11 (14.1%) 6 (9.1%) 11 (13.8%) - Microinvasive carcinoma 1 (0.4%) 1 (1.3%) 0 (0%) 0 (0.0%) - Invasive carcinoma 14 (6.3%) 2 (2.6%) 6 (9.1%) 6 (7.5%) - Other malignat 4 (1.8%) 1 (1.3%) 2 (3%) 1 (1.3%) Mean tumour diameter ± S.D. (mm) 15.5 ± 14.1 15.0 ± 14.1 19.0 ± 18.7 12.2 ± 5.7 0.289 0.610 0.701 ALND = axillary lymph node dissection; S.D. = standard deviation; SNB, sentinel node biopsy 492 TABLE 4. Quality indicators of diagnostic surgical procedures split by the year of surgery 1 QI: waiting time - Median (days) - Proportion of patients with waiting time less than 15 working days Not more than 15 working days = 90% 41 10.3% 44.5 2.6% 42 6.2% 34 21.5% < 0.001 2 QI: weight of the excised specimen from the breast - Mean ± S.D. (g) - Proportion of specimens with weight = 30g Proportion of specimen with weigh less than 30g = 90% 37.3 ± 23.4 47.8% 40.3 ± 25.8 46.2% 32.6 ± 22.1 60.6% 38.2 ± 22.1 38.8% 0.131 QI = quality indicator; S.D. = standard deviation gery. However, the number of QIs meeting the minimum standard was the same through all three studying years. Timely treatment is an important requisite for the quality of surgery as it maximizes the benefit of early detection and reduces anxiety of patients and their families.5 This is especially important in screen-detected lesions as participation rate is critically dependent on patient’s satisfaction. Thus, we set a recommendation for waiting time as de­fined by EBCN (15 working days), which is much stricter compared to EUSOMA (6 weeks). In the first year of our quality control monitoring, only 1.7% of patients with therapeutic operation and 2.6% of patients with diagnostic operation met the EBCN recommendations on waiting time for sur­gery, which is far below the minimum standard (= 90%). Several reasons contributed to a long wait­ing time. First, our institution has been faced with increasing number of surgical oncology patients and the screening patients had to be scheduled for surgical intervention regarding the waiting time and indications of all patients. Second, during the analysed period our institution was the only hospi­tal in Slovenia performing the surgery of screening patients. Recently another institution, after meet­ing all necessary quality checks, started to operate on these patients, which will contribute to lower the waiting times. Finally, in many patients longer preoperative preparation including examinations by other physicians (i.e. cardiologist, diabetologist etc.) were required and thus the surgical interven­tion was postponed until it could be safely per­formed. Several measures have been taken to shorten waiting time. First, patients were referred to the TABLE 5. The variability of QIs within the institution for each studying year and altogether Therapeutic procedures Diagnostic procedures Number of procedures: least active surgeon - most active surgeon Proportion of mastectomies: surgeon with lowest proportion - surgeon with highest proportion Median waiting time (in days): surgeon with lowest - surgeon with highest waiting time Single (breast) operation for the primary tumour (excluding reconstruction): surgeon with lowest - surgeon with highest proportion Invasive breast cancer not greater than 3 cm who underwent BCT: surgeon with lowest - surgeon with highest proportion Weight (g) of the excised specimen from the breast: surgeon with lowest mean - surgeon with highest mean Patients with invasive cancer and axillary clearance performed with at least 10 LN examined: surgeon with lowest - surgeon with highest proportion Number of procedures: least active surgeon - most active surgeon Median waiting time (in days): surgeon with lowest - surgeon with highest waiting time Weight (g) of the excised specimen from the breast: surgeon with lowest mean - surgeon with highest mean 1–166 1–45 6–62 1–67 17.8%–100.0% 10.0%–100.0% 12.5%–44.4% 0.0%–100.0% 28.0–45.0 24.0–45.0 27.0–41.0 23.0–136.0 83.1%–100.0% 73.3%–100.0% 71.4%–100.0% 75.0%–100.0% 0.0%–92.6% 0.0%–94.4% 40.0%–100.0% 77.8%–100.0% 35.5–89.0 33.5–104.5 28.0–94.0 37.0–81.5 71.4%–100.0% 50.0%–100.0% 80.0%–100.0% 50.0%–100.0% 3–30 1–12 1–11 1 -12 25.5–45.0 24.0–59.0 28.5–76.0 20.0–71.0 18.0–70.0 22.0–73.0 15.0–55.0 18.0–94.0 BCT = breast conserving treatment; LN = lymph nodes 493 surgeon in maximum 2 weeks after the decision on operation was made within multidisciplinary meeting. Second, all other investigations needed before surgery were made on the day of appoint­ment with surgeon. Finally, we provided addition­al time in operating theatre dedicated for women from screening program. Because of described measures, the waiting time had significantly im­proved over the next two years. Nevertheless, ac­tivities to shorten waiting time should be intensi­fied to reach the minimum standard. Three QIs from EUSOMA working group had been chosen to monitor the quality of surgery in women with proven breast cancer: proportion of patients (invasive and noninvasive cancers) who received a single (breast) operation for the primary tumour (excluding reconstruction), proportion of patients with invasive breast cancer not greater than 3 cm (total size, including DCIS component) who underwent BCT and proportion of patients with invasive cancer and axillary clearance performed with at least 10 LN examined. The recommended standard was met for the first two QIs every single year. Furthermore, QI proportion of patients with invasive breast cancer not greater than 3 cm (total size, including DCIS component) who underwent BCT significantly improved over the study period. On the other hand, we had not reached the mini­mal proportion of patients with invasive cancer and axillary clearance performed with at least 10 LN examined. However, in the majority of patients low number of examined lymph nodes was not the result of low technical skills of the surgeons, but rather the decision of the surgeons that complete axillary lymph node dissection might not be neces­sary, following the trends towards minimizing the axillary surgery as reviewed in an article by Henke et al..15 Overall, the number of these patients is very low. As more data become available that less ag­gressive axillary surgery does not affect survival in breast cancer patients, the inclusion of this QI in monitoring surgical quality control should be re­considered. Indeed, in the last version of EUSOMA QIs from 2017, this QI is no longer included and was replaced by QI monitoring the ability to avoid axillary overtreatment (proportion of patients with no more than 5 SLN excised).5 As the breast is aesthetically sensitive organ and important for woman’s self-esteem, the cosmetic results are of utmost importance in breast surgery. However, measurement of cosmetic results are dif­ficult and subjective assessments could not be ruled out. Weight of the excised specimen is a proxy for expected final cosmetic result and preserved shape and symmetry with the contralateral breast.16 Although far from ideal as it does not take into account the effect of oncoplastic reconstruction, it represents an objective measurement of the sur­geon’s ability to balance between aggressiveness and clear margins on the one hand and cosmetic is­sues on the other. The weight of the specimen was significantly reduced in the second and third stud­ying year compared to the first one for therapeutic procedures and a trend towards weight reduction was observed for diagnostic procedures. However, the goal was still not met and efforts to reduce the weight of the specimens should be continued. Besides monitoring surgical QIs at the institu­tional level, we also analysed the variability of man­agement among surgeons. As all surgeons were informed about their own results each year, we would expect that the variability among surgeons becomes less pronounced over time. However, the variability remained high throughout the studying years, which is most probably the reflection of the number of surgeons involved in breast surgery at our department. As recommended by EUSOMA, any breast surgeon at the breast centre must carry out primary surgery as first operator on at least 50 newly diagnosed breast cancers a year. If the centre has surgeons in training, those responsible for su­pervising trainees might perform fewer than 50 pri­mary cases as first operator. In this case documen­tation on their role as second operator supervising trainees must be available.17 Although surgeons involved in breast surgery at our department oper­ate other breast lesions besides those detected in the screening program, many surgeons still do not meet the requested volume standards. To further improve the compliance with QIs and to reduce the variability of surgical management inside the insti­tution, the importance of concentrating the breast pathology to a reduced number of surgeons meet­ing the requested standards could not be overem­phasized. It seems that this measure may provide important step towards improved quality of breast surgery. Although many studies addressed the compli­ance to QIs as defined by EUSOMA, our study is the first considering only screen-detected breast le­sions and including both, diagnostic and therapeu­tic procedures. Since women with screen-detected lesions represent a specific population and the quality of management of those women, includ­ing the quality of surgery, is important not only for the woman in question but also for the appropriate participation rate and the operation of the screen­ing program as a whole, we included QIs specifi- 494 cally recommended for screen-detected lesions as well. As a result, our results are not fully compa­rable to other studies considering EUSOMA QIs only. Nevertheless, the results from other studies show, similar to ours, that complete adherence to guidelines is difficult to achieve. However, contin­uous monitoring is of paramount importance as it results in better performance of QIs over time.18-20 Besides being the first study addressing the topic of monitoring surgical QIs within breast cancer screening program, other strengths of our study are large number of included cases, com­prehensive data collection from a prospective da­tabase and thus low number of missing data and the recent nature of the data. Furthermore, in the study period all patients with screen-detected le­sions detected in the national screening program, underwent surgery at our institution, minimizing the selection bias. The limitations of our study are limited number of QIs that were monitored and inclusion of old EUSOMA QIs defined in 2010. Looking ahead, a set of our QIs should be updated according to the latest EUSOMA recommenda­tions. Furthermore, all potential cofounders were not taken into account in our analysis. First, the re­sults of most EUSOMA indicators improves over time independent of quality control as demon­strated by van Dam et al.18; these time trends were not considered in our study. Second, without mul­tivariate analysis adjusting for differences in case mix firm conclusions are difficult to draw. In con­clusion, our results showed that adherence to all surgical QIs in patients from screening program is difficult to achieve, especially to those specifically defined for screen-detected lesions. Nevertheless, regular quality control may improve results over time. Reducing the number of surgeons dedicated to breast pathology may reduce variability of man­agement inside the institution. Acknowledgment The study was supported by the research program of the Slovenian research agency P3-0352. References 1. 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Quality in­dicators in breast cancer care: an update from the EUSOMA working group. Eur J Cancer 2017; 86: 59-81. doi: 10.1016/j.ejca.2017.08.017 6. Rosselli Del Turco M, Ponti A, Bick U, Biganzoli L, Cserni G, Cutuli B, et al. Quality indicators in breast cancer care. Eur J Cancer 2010; 46: 2344-56. doi: 10.1016/j.ejca.2010.06.119 7. Perry N, Broeders M, de Wolf C, Tornberg S, Holland R, von Karsa L. European guidelines for quality assurance in breast cancer screening and diagnosis [Internet]. Eur Guidel 2006 [cited 2020 Mar 30]. Available from: http://screening.iarc.fr/doc/ND7306954ENC_002.pdf 8. DORA programme colleagues. [Annual Report on the DORA Breast Cancer National Screening Program 2018]. [Slovenian]. [Internet]. Institute of Oncology Ljubljana - DORA National Breast Cancer Screening Programme 2019 [cited 2020 Mar 30]. Available from: https://dora.onko-i.si/fileadmin/ user_upload/Dokumenti/DORA_letno_porocilo_2018_knjiznica.pdf 9. Cicero U, Rietjens M, Mahmoud ET, Virgilio S. Oncoplastic and reconstructive breast surgery. Cham: Springer; 2019. 10. Rizzo M, Bumpers H, Okoli J, Senior-Crosby D, O’Regan R, Zelnak A, et al. Improving on national quality indicators of breast cancer care in a large pub­lic hospital as a means to decrease disparities for african american women. Ann Surg Oncol 2011; 18: 34-9. doi: 10.1245/s10434-010-1204-z 11. Wilcox N, McNeil JJ. Clinical quality registries have the potential to drive improvements in the appropriateness of care. Med J Aust 2016; 205: S27-9. doi: 10.5694/mja15.00921 12. Cheng SH, Wang CJ, Lin JL, Horng CF, Lu MC, Asch SM, et al. Adherence to quality indicators and survival in patients with breast cancer. Med Care 2009; 47: 217-25. doi: 10.1097/MLR.0b013e3181893c4a. 13. Van Dam PA, Verheyden G, Sugihara A, Trinh XB, Van Der Mussele H, Wuyts H, et al. A dynamic clinical pathway for the treatment of patients with early breast cancer is a tool for better cancer care: implementation and prospec­tive analysis between 2002-2010. World J Surg Oncol 2013; 11: 70. doi: 10.1186/1477-7819-11-70 14. Plavc G, Ratoša I, Žagar T, Zadnik V. Explaining variation in quality of breast cancer care and its impact: a nationwide population-based study from Slovenia. Breast Cancer Res Treat 2019; 175: 585-94. doi: 10.1007/s10549­019-05186-z 15. Henke G, Knauer M, Ribi K, Hayoz S, Gerard MA. Tailored axillary surgery with or without axillary lymph node dissection followed by radiotherapy in patients with clinically node-positive breast cancer (TAXIS): study protocol for a multicenter, randomized phase-III trial. Trials 2018; 19: 667. doi: 10.1186/s13063-018-3021-9 16. Ojala K, Meretoja TJ, Leidenius MHK. Aesthetic and functional outcome after breast conserving surgery - Comparison between conventional and oncoplastic resection. Eur J Surg Oncol 2017; 43: 658-64. doi:10.1016/j. ejso.2016.11.019 17. Biganzoli L, Cardoso F, Beishon M, Cameron D, Cataliotti L, Coles CE, et al. The requirements of a specialist breast centre. Breast 2020; 51: 65-84. doi: 10.1016/j.breast.2020.02.003 18. Van Dam PA, Tomatis M, Marotti L, Heil J, Mansel RE, Rosselli del Turco M, et al. Time trends (2006–2015) of quality indicators in EUSOMA-certified breast centres. Eur J Cancer 2017; 85: 15-22. doi: 10.1016/j. ejca.2017.07.040 19. Van Dam PA, Tomatis M, Marotti L, Heil J, Wilson R, Rosselli Del Turco M, et al. The effect of EUSOMA certification on quality of breast cancer care. Eur J Surg Oncol 2015; 41: 1423-9. doi: 10.1016/j.ejso.2015.06.006 20. Hartmann-Johnsen OJ, Kĺresen R, Schlichting E, Naume B, Nygĺrd JF. Using clinical cancer registry data for estimation of quality indicators: Results from the Norwegian breast cancer registry. Int J Med Inform 2019; 125: 102-9. doi: 10.1016/j.ijmedinf.2019.03.004 495 research article Experimental validation of Monte Carlo based treatment planning system in bone density equivalent media Djeni Smilovic Radojcic1,3, Bozidar Casar2,4, David Rajlic1, Manda Svabic Kolacio1, Ignasi Mendez2, Nevena Obajdin1, Dea Dundara Debeljuh1,5, Slaven Jurkovic1,3 1 Medical Physics Department, University Hospital Rijeka, Rijeka, Croatia 2 Department for Dosimetry and Quality of Radiological procedures, Institute of Oncology Ljubljana, Ljubljana, Slovenia 3 Department of Medical Physics and Biophysics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia 4 Faculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia 5 General Hospital Pula, Radiology Department, Pula, Croatia Radiol Oncol 2020; 54(4): 495-504. Received 10 June 2020 Accepted 9 July 2020 Correspondence to: Assist. prof. Slaven Jurkovic, Ph.D., Medical Physics Department, University Hospital Rijeka, Kresimirova 42, HR-51000 Rijeka, Croatia. E-mail: slaven.jurkovic@medri.uniri.hr Disclosure: No potential conflicts of interest were disclosed. Djeni Smilovic Radojcic and Bozidar Casar have equally contributed to this work. Introduction. Advanced, Monte Carlo (MC) based dose calculation algorithms, determine absorbed dose as dose to medium-in-medium (D) or dose to water-in-medium (D). Some earlier studies identified the differences in the m,mw,m absorbed doses related to the calculation mode, especially in the bone density equivalent (BDE) media. Since the calculation algorithms built in the treatment planning systems (TPS) should be dosimetrically verified before their use, we analyzed dose differences between two calculation modes for the Elekta Monaco TPS. We compared them with experimentally determined values, aiming to define a supplement to the existing TPS verification methodology. Materials and methods. In our study, we used a 6 MV photon beam from a linear accelerator. To evaluate the accuracy of the TPS calculation approaches, measurements with a Farmer type chamber in a semi-anthropomorphic phantom were compared to those obtained by two calculation options. The comparison was made for three parts of the phantom having different densities, with a focus on the BDE part. Results. Measured and calculated doses were in agreement for water and lung equivalent density materials, re­gardless of the calculation mode. However, in the BDE part of the phantom, mean dose differences between the calculation options ranged from 5.7 to 8.3%, depending on the method used. In the BDE part of the phantom, neither of the two calculation options were consistent with experimentally determined absorbed doses. Conclusions. Based on our findings, we proposed a supplement to the current methodology for the verification of commercial MC based TPS by performing additional measurements in BDE material. Key words: treatment planning system; dose-to-medium; dose-to-water; experimental validation of dose calcula­tion; Monte Carlo Introduction Implementation of advanced radiation therapy techniques into clinical practice has set high de­mands on the quality and accuracy of various devices used for radiation treatment planning, treatment delivery, and dose verification. Besides the required high performance of medical linear accelerators and their ancillary systems, there are also strict requirements on dose calculation and optimization using treatment planning systems (TPS). Precise dose calculation is one of the most critical steps in the radiation therapy process since it is the basis for accurate and safe treatment deliv­ery using high-energy photon beams. To provide necessary dosimetric accuracy, the verification of the calculated doses should be performed using a reproducible and reliable methodology. To ensure acceptable reliability of the verification results, an appropriate methodology for dose verification 496 should be carefully selected, while the limitations of the specific method must be fully understood. The latter is essential for an adequate interpreta­tion of the verification results. Comprehensive verification methodology for the evaluation of calculation algorithms built in the TPSs has been proposed by the International Atomic Energy Agency (IAEA).1,2 However, the rapid development of treatment delivery devices and, consequently, the utilization of the advanced radiation therapy techniques call for further devel­opment of the verification methods. In some pub­lished studies and documents3-5, methodologies for the verification of dosimetry parameters for the im­plementation of Intensity Modulated Radiotherapy (IMRT) have been proposed. However, neither the means of verification nor the methods were explic­itly spelled out. Presently, Monte Carlo based dose calculation algorithms built in the TPS are assumed to be the most accurate computational systems for the ap­propriate simulation of particle transport and dose calculation.6,7 Those algorithms offer two alterna­tive options for the calculation and reporting of the absorbed dose: dose-to-medium as calculated by Monte Carlo algorithms, referred as dose to medi­um-in-medium, , , and dose-to-water convert­ed from dose-to-medium using stopping power ratios water-to-medium, referred as dose to water-in-medium, , , or sometimes “biological dose to water”.8-10 The first approach calculates absorbed energy in a medium voxel divided by the mass of the medium element, while the second calculates the absorbed energy in a small cavity of water di­vided by the mass of that cavity. For brevity, and , calculation options will be denoted as (dose-to-medium) and (dose-to-water) respec­tively in the rest of the paper. Since it is a matter of debate whether to use or calculation approach for dose planning9-13, the American Association of Physicists in Medicine (AAPM) Task Group 10510 recommended that the material to which the dose is computed should be explicitly indicated and conversion between dose-to-medium and dose-to-water calculation modes should be available. Several previously published studies9,12,14-16 were dedicated to comparisons of the two mentioned calculation modes built in the con­temporary TPSs. Those studies have shown that differences between dose-to-medium and dose-to-water calculation modes can be expected in bone density equivalent (BDE) material. While is the quantity inherently computed by MC dose algorithms, calculation approach is still indis­pensable in clinical radiation therapy due to some practical and historical experience of prescribers.10 Because there is still no agreement regarding the calculation approach that should be used as a clini­cal standard and due to the absence of the appro­priate verification methodology, the present work aimed to propose a supplement to the existing ver­ification methodology to establish the validity of both approaches. For that purpose, calculated ab­sorbed doses using and options were com­pared to those determined experimentally in the semi-anthropomorphic phantom focusing on the dose differences in the part of the phantom having density close to the bone density. The ultimate goal of the study was to define and propose an additional verification procedure as a supplement to the set of existing preclinical com­missioning tests provided in the IAEA TECDOC 15832, for the specific case where TPS uses Monte Carlo based calculation algorithms. Such addition­al test may well eliminate potential misinterpreta­tions of the commissioning results for bone density material, where and calculation approaches lead to different conclusions.9,12,14-16 We have to note that the proposed addendum to the verification methodology has no intention to be an answer to which reporting mode, or , should be used for radiotherapy treatment prescription or dose calculation, neither to discuss possible limitations of the conversion methodology from to , which is based on stopping power ratios water-to-medium.8 Materials and methods In this work we used 6 MV photon beam gener­ated by Siemens Oncor Expression (Siemens Healthineers, Erlangen, Germany) linear accel­erator, Siemens Somatom Open Computerized Tomography (CT) simulator (Siemens Helthineers, Erlangen, Germany) and Elekta Monaco treatment planning system version 5.11 (Elekta, Stockholm, Sweden). Monaco TPS is a Monte Carlo based sys­tem which calculates absorbed dose using the approach that can be converted to mode using water-to-medium stopping power ratios to ac­count for different energy absorption in both me­dia.17 Linear accelerator and Elekta Monaco ver. 5.11 TPS were commissioned and prepared for the clinical implementation of Intensity Modulated Radiotherapy according to the international rec­ommendations.1,2,4,18-21 All dosimetric measure- Smilovic Radojcic D et al. / Experimental validation of Monte Carlo based treatment planning system 497 FIGURE 1. Photo of the semi-anthropomorphic CIRS Thorax phantom with interchangeable rod inserts (left) and its CT image (right). Positions of 10 interchangeable rod inserts are marked with numbers from 1 to 10. Five measuring points are in the water equivalent part of the phantom (grey area), four points are in the lung density equivalent material (black area), and one point is in the bone density equivalent part of the phantom (white area). ments were performed using a PTW 30013 Farmer type ionization chamber and PTW UNIDOS elec­trometer (PTW, Freiburg, Germany). Standard measurements in the CIRS Thorax phantom Accuracy of the TPS Monaco ver. 5.11 calcula­tion algorithm was experimentally verified using a semi-anthropomorphic CIRS Thorax phantom (CIRS Inc., Norfolk, VA, USA) consisting of a body made of water equivalent material (. = 1.003 g/cm3), lung equivalent parts (. = 0.207 g/cm3), and bone equivalent part (. = 1.506 g/cm3) with cylindrical holes for placement of ionization chamber into interchangeable rod inserts having three different densities.2 The phantom was scanned using the Somatom Open CT simulator. Acquired CT images were used for the delineation of volumes of interest and subsequent dose calculations. Measurements of absorbed dose were performed at ten measur­ing positions within the phantom (Figure 1) for 15 different irradiation set-ups (Table 1), using a PTW Farmer-type ionization chamber. All meas­urements were carried out at the central part of the selected radiation fields, excluding the regions of high dose gradients. Measured doses were compared to the cor­responding doses obtained by both calculation options, and . Dose differences and between measured and calculated values for dose-to-medium and dose-to-water calculation approach, were calculated according to the IAEA methodology1,2 as: [1] [2] where denotes measured absorbed dose at the selected measuring point, while stands for the absorbed dose measured at the refer­ence point, which was chosen on the central axis of the beam at the isocenter (Table 1). Dose differences and between calcu­lated and measured doses were analysed for both calculation options through the comparison of the respective average values and [3] [4] The index i stands for a particular dose differ­ence for i-th dose measurement and corresponding calculated dose for two different calculation modes in the selected part of the CIRS Thorax phantom (water equivalent part, lung equivalent part, or bone density equivalent part). 498 TABLE 1. Irradiation set-ups for measurements in 6 MV photon beam used for experimental verification of the Monaco ver. 5.11 treatment planning systems (TPS) calculation algorithm in the semi-anthropomorphic CIRS Thorax phantom. Reference and measuring points (I1 to I10) are shown in the last two columns; subscripts 1 to 10 correspond to the labelling in Figure 1 1 10×10 SSD 0 I5 I1, I3, I5-10 2 3 Single square fields 10×10 4×4 SAD SAD 0 0 I5 I5 I1, I3, I5-10 I1-9 4 10×10 SAD 90 I3 I2-10 5 Rectangular field 10× 15 SAD 300 I1 I1, I4, I6-8, I10 6 (6+8)×15 SAD 0 I5 I1-10 7 8 Single asymmetric fields (3+8)×15 (4+10)×15 SAD SAD 90 180 I5 I5 I1, I5-10 I1-3, I5-10 9 (3+7)×15 SAD 300 I5 I2-10 12×10 SAD 0 12×10 SAD 180 10 4 fields (box) 12×8 SAD 90 I5 I2-5 12×8 SAD 270 4×4 SAD 30 11 3 fields 16×4 SAD 90 I5 I2, I5-9 16×4 SAD 270 12 Diamond-shaped field 14×14 SAD 0 I3 I1, I3, I5-10 13 Irregular L shaped field / SAD 45 I1 I1-2, I4-6, I8-10 14 MLC cylinder shaped field / SAD 0 I2 I1,2, I5, I8,9, I10 16×4 SAD 90 15 3 non-coplanar fields 16×4 SAD 270 I5 I1, I5-6, I8, I10 4×4a SAD 30 a Couch angle = 270° SAD = source to axis distance; SSD = source to surface distance Throughout the study, all calculations within Monaco TPS were performed on a 0.2 cm calcula­tion grid, with 0.5% statistical uncertainty per con­trol point. Differences between Dm and Dw calculation modes in the bone density equivalent part of the CIRS Thorax phantom In the second part of the study, we were aiming to determine differences between and calcula­tion approaches in the Monaco ver. 5.11 TPS in the bone equivalent part of the CIRS Thorax phantom, following the same methodology as described in the preceding section. Three irradiation geometries (single asymmet­ ric rectangular fields having different gantry an­ gles: 0°, 90°, and 180°) were selected for this part of the study (Table 1, set-ups 6, 7, and 8). For each of those irradiation geometries, two phantom assem­blies were used to analyze differences between the two calculation approaches with respect to the measurements performed by PTW 30013 Farmer type ionization chamber in the bone density equiv­alent (BDE) part of the CIRS Thorax phantom. In the first assembly, referred to as non-standard, the water equivalent insert with the ionization cham­ber was placed into the BDE part of the phantom (Figure 2A). In this way, the measuring point in the phantom was surrounded by water equivalent material of sufficient thickness to fulfill conditions required by the Bragg-Gray cavity theory for the determination of absorbed dose in terms of dose to water. In the second assembly, referred to as stand­ard, the BDE insert was placed in the BDE part of the phantom (Figure 2B). Smilovic Radojcic D et al. / Experimental validation of Monte Carlo based treatment planning system 499 FIGURE 2. CT image of the CIRS Thorax phantom: water equivalent insert inside BDE part of the phantom (A); a BDE insert inside bone density equivalent (BDE) part of the phantom (B) and cross-section of small “water cylinders” of different dimensions delineated inside BDE part of the phantom to find limits for calculating geometry where cavity theory applies (top right). In the last part of the study, the phantom as­sembly was additionally virtually modified for the calculation purposes in the Monaco TPS: cylinders of various volumes (constant length and different diameters) were delineated inside the BDE insert on the CT scans (Figure 2, top right). This approach was utilized to obtain the limits above which the differences between and calculation ap­proaches become non-significant and in agreement with experimentally determined absorbed doses. The length of the cylinders was set equal to the length of the cavity volume of the PTW 30013 ioni­zation chamber, while the electron density of such cylinders was set to be equal to the electron density of the water. According to the IAEA TRS-398 Code of practice22, the charge measured by an ionization chamber calibrated in terms of absorbed dose to water is directly proportional to the absorbed dose in water at the point of measurement in the absence of the chamber. By delineating cylinders having the electron density of water inside the BDE part of the phantom, we have tried to simulate the mentioned theoretical situation to different degrees. To verify the accuracy of dose-to-medium and dose-to-water calculation modes, we have ana­lyzed differences and between calculated and measured absorbed doses for both calculation modes and different volumes of “water cylinders” smaller than the volume of the PTW 30013 ioni­zation chamber’s cavity volume (0.6 cm3), using Eqs. [1] to [4]. We were aiming to find the volume of “water cylinder,” above which there will exist an agreement between calculated and measured doses without a statistically significant difference between both calculation approaches. Our final challenge was to define an addendum to the ex­isting TPS verification methodology based on the described method and experimental findings from the present work. Evaluation of results and estimation of uncertainties The uncertainty of was estimated as the com­bination in quadrature of the statistical uncertainty of and the uncertainty of Monte Carlo calcula­tion of 0.5% (1 SD) for , using a coverage factor k = 2 (2 SD). The uncertainty of was calculated in the same manner. We considered that the and calculation modes differed significantly within 95% confi­dence limits (two standard deviations – 2 SD, i.e., coverage factor k = 2) if the relation [5] was satisfied. is a combined uncertainty which was determined as the combination in quadra­ture of the individual uncertainties of and . This estimation was considered conservative due to the fact that the uncertainties of the terms were included in the compute of the individual uncertainties and . Secondly, we considered that the dose calcula­tions within Monaco TPS were in agreement with the experimentally determined doses if the condi­tions 500Smilovic Radojcic D et al. / Experimental validation of Monte Carlo based treatment planning system [6] [7] were satisfied. At this point we note, that through­out the rest of the paper all combined uncertainties are stated within two standard deviations, i.e., us­ing a coverage factor k = 2. Results Standard measurements in the CIRS Thorax phantom Differences between calculated and measured ab­sorbed doses for two calculation modes, dose-to-medium and dose-to-water , were determined using Eqs. [1] and [2] for all 15 standard irradia­tion configurations and ten measurement points in the CIRS Thorax semi-anthropomorphic phantom (Table 1). Mean values of percentage dose differ­ences and calculated by Eqs. [3] and [4] are presented with corresponding uncertainties in terms of two standard deviations in Figure 3, separately for the water equivalent part (five measurement points), lung density equivalent part (four measurement points), and BDE part (one measurement point) of the phantom. Statistical significance of the obtained Smilovic Radojcic D et al. / Experimental validation of Monte Carlo based treatment planning system 501 TABLE 2. Differences and between two different calculation options in the Monaco ver. 5.11. treatment planning systems (TPS) and measured data obtained in the bone density equivalent (BDE) part of the CIRS Thorax phantom, according to Eqs. [1] and [2]. Two phantom assemblies and three simple beam set­ups were considered for this part of the study standarda - 2.9 2.9 (6+8) x 15 cm2 Gantry = 0° non-standardb - 0.7 - 0.2 standarda - 3.0 5.1 (3+8) x 15 cm2 Gantry = 90° non-standardb - 0.7 - 0.1 standarda - 5.7 5.4 (4+10) x 15 cm2 Gantry = 180° non-standardb 0.5 1.3 a BDE insert with the ionization chamber placed in the BDE part of the phantom b Water equivalent insert with the ionization chamber placed in the BDE part of the phantom TABLE 3. Mean differences, and , between calculated and measured doses in the bone density equivalent (BDE) part of the CIRS Thorax phantom for and calculation approaches, respectively. The absorbed doses were calculated using the Monaco ver. 5.11 treatment planning systems (TPS) in the center of delineated “water cylinders” of volume V, in the BDE part of the phantom. Corresponding combined uncertainties are denoted as and for dose-to-medium and dose-to-water calculation options, respectively 0 - 3.9 2.1 4.4 1.9 0.035 - 2.6 1.5 2.5 1.9 0.141 - 1.4 1.3 1.8 1.7 0.279 - 0.3 1.2 1.2 1.5 0.573 0.3 1.4 0.4 1.3 The differences between the two calculation approaches, dose-to-medium and dose-to-water, were, however, significant in BDE media (Table 2 and Figure 3). Andreo et al.9 have shown that a 10% difference in ICRP bone can be expected for Monaco ver. 5.0 TPS between two calculation modes after conversion of to . Results of the present study confirm those findings as well as the opposite signs of mean percentage dose differences for and reporting modes in the case when Monaco ver. 5.11 TPS has been used. Considerable differences between calculated dose distributions using and calculation approaches have also been reported in clinical studies.15,23 Differences between Dm and Dw calculation modes in the bone density equivalent part of the CIRS Thorax phantom In the BDE part of the CIRS Thorax semi-anthro­pomorphic phantom, mean percentage dose dif­ferences and were calculated by applying 502 Eqs. [1] and [2] for two phantom assemblies - stand­ard and non-standard and three selected irradiation geometries, as shown in Table 2. In the case of non­standard geometry, both and were within 1%, demonstrating that there is a negligible differ­ence between applied calculation modes. However, differences between the respective mean values and were statistically significant if standard geometry was utilized. The latter case was also assumed as our first result in the part of the study where we at­tempted to find the volume of “water cylinder” de­lineated in the Monaco ver. 5.11 TPS for which the difference between and would become non-significant. For further discussion, analysis, and graphical presentation, the exponential function was selected to fit the data from Table 3. The general form of the fitting function is given as [8] Smilovic Radojcic D et al. / Experimental validation of Monte Carlo based treatment planning system 503 2. Measured doses are compared to the calculat­ed ones using both calculation modes, and , applying Eqs. [1] to [4] for the additional four “water cylinders” delineated in the TPS. 3. Obtained mean values and of the per­centage dose differences are fitted by the ana­lytical function from Eq. [8]. Finally, the acceptability of the tested TPS algo­rithm is based on two conditions, which have to be fulfilled concurrently: i) Differences between dose-to-me­dium and dose-to-water calculation approach­es should fall within 1% for the “water cylin­der” of volume 0.6 cm3,i.e., [11] Fulfilment of this condition means that both calculation options yield to the same results within statistical uncertainty for large vol­umes, as expected. Since significant differ­ences do exist for small volumes of delineated “water cylinders,” we have to consider this fact as well. The maximal difference can be obtained from the corresponding fit­ting functions for V = 0 cm3 (in our study, the maximal difference between both calculation options was 7.6%). ii) Obtained values and have to fall be­low 1% (see Eqs [6] and [7]) for large volumes of delineated “water cylinders”. If this condition is fulfilled, one can conclude that TPS dose cal­culations are in agreement with experimentally determined doses for both calculation modes. It is important to note that our investigation was limited to the region of charged particle equilibri­um (CPE) and for 6 MV photon beam only. Conclusions In the present study, a Monte Carlo based calcula­tion algorithm built in the Elekta Monaco ver. 5.11 TPS was analyzed for 6 MV photon beam. It was confirmed that both calculation approaches, dose-to-medium and dose-to-water, yield to the simi­lar results in the water equivalent and lung den­sity equivalent parts of the semi-anthropomorphic phantom and are in agreement with experimental­ly determined absorbed doses. In the bone density equivalent part of the phan­tom, significant differences were observed when calculations were compared to the measured ab­sorbed doses. While the dose-to-medium approach yields to lower doses compared to the measured ones, calculations utilizing the dose-to-water com­puting approach revealed similar differences but of opposite sign. The observed differences can lead to ambiguity regarding the acceptability of the ver­ification results before the clinical implementation of a newly commissioned TPS Monaco. To overcome the ambiguity on the pertinence of the verification results in the bone density equiv­alent material, a supplement to the current TPS commissioning methodology has been proposed, having in mind inherent differences between the two calculation modes. This supplement relies on the findings from the present study. We consider it as a consistent and efficient method for the ex­perimental verification of the absorbed dose cal­culation in both calculation modes and . A proposed supplementary test to the present veri­fication methodology of the algorithm built in the Monaco TPS can assure higher accuracy and confi­dence compared to the current methodology. While the selection of beams in this study as­sumes conditions of charged particle equilibrium, it would be highly interesting and worthwhile to set-up the study where CPE is violated, e.g., for small fields where lateral CPE does not exist. However, an experimental determination of ab­sorbed doses in small fields is demanding. It re­quires determination of detector specific correction factors, which have to be utilized individually for the selected detector and are associated with addi­tional uncertainties.24-26 The latter can pose a prob­lem to conduct such a study with sufficient reliabil­ity and robustness. Acknowledgement Bozidar Casar acknowledges the financial support from the Slovenian Research Agency through the research Grant P1-0389. References 1. 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Code of practice for the quality assurance and control for intensity modulated radiotherapy. Delft, Netherlands; 2013. 5. Smilowitz JB, Das IJ, Feygelman V, Fraass BA, Kry SF, Marshall IR, et al. AAPM Medical Physics Practice Guideline 5.a.: Commissioning and QA of treat­ment planning dose calculations - megavoltage photon and electron beams. J Appl Clin Med Phys 2015; 16: 14-34. doi: 10.1120/jacmp.v16i5.5768 6. Van Dyk J, Battista J. Has the use of computers in radiation therapy im­proved the accuracy in radiation dose delivery? J Phys Conf Ser 2014; 489: 012098. doi: 10.1088/1742-6596/489/1/012098 7. Fogliata A, Cozzi L. Dose calculation algorithm accuracy for small fields in non-homogeneous media: the lung SBRT case. Phys Med 2017; 44: 157-62. doi: 10.1016/j.ejmp.2016.11.104 8. Reynaert N, Crop F, Sterpin E. On the conversion of dose to bone to dose to water in radiotherapy treatment planning systems. Phys Imaging Radiat Oncol 2018; 5: 26-30. 2018. doi: 10.1016/j.phro.2018.01.004 9. Andreo P. Dose to ‘water-like’ media or dose to tissue in MV photons radio­therapy treatment planning: still a matter of debate. Phys Med Biol 2015; 60: 309-37. doi: 10.1088/0031-9155/60/1/309 10. Chetty IJ, Curran B, Cygler JE, DeMarco JJ, Ezzell G, Faddegonet BA, et al. Report of the AAPM Task Group No. 105: issues associated with clinical implementation of Monte Carlo-based photon and electron external beam treatment planning. Med Phys 2007; 34: 4818-53. doi: 10.1118/1.2795842 11. Liu HH, Keal P. D m rather than D w should be used in Monte Carlo treat­ment planning. (Point/Counterpoint), Med Phys 2002; 29: 922-4. doi. 10.1118/1.1473137 12. Ma C-M, Li J, Dose specification for radiation therapy: dose to water or dose to medium? Phys Med Biol 2011; 56: 3073-89. doi: 10.1088/0031­9155/56/10/012 13. Reynaert N, Van der Marck S, Schaart D, Van der Zee W, Van VlietVroegindeweij C, Tomsej M, et al. Monte Carlo treatment planning for photon and electron beams. Radiat Phys Chem 2007; 76: 643-86. doi: 10.1016/j.radphyschem.2006.05.015 14. Dogan N, Siebers JV, Keall PJ. Clinical comparison of head and neck and pros­tate IMRT plans using absorbed dose to medium and absorbed dose to wa­ter Phys Med Biol 2006; 51: 4967-80. doi: 10.1088/0031-9155/51/19/015 15. Walters BRB, Kramer R, Kawrakow I. Dose to medium versus dose to water as an estimator of dose to sensitive skeletal tissue. Phys Med Biol 2010; 55: 4535-46. doi: 10.1088/0031-9155/55/16/S08 16. Sterpin E. Potential pitfalls of the PTV concept in dose-to-medium planning optimization. Phys Med 2016; 32: 1103-10. doi: 10.1016/j.ejmp.2016.08.009 17. Siebers JV, Keall PJ, Nahum AE. Converting absorbed dose to medium to absorbed dose to water for Monte Carlo based photon beam dose calcula­tions. Phys Med Biol 2000; 45: 983-95. doi: 10.1088/0031-9155/45/4/313 18. Abratt R, Aguirre F, Andreo P, Coffey M, Drew J, El Gueddari B, et al. IAEA Comprehensive audits of radiotherapy practices: a tool for quality improve­ment quality assurance team for radiation oncology-QUATRO. Vienna: International Atomic Energy Agency; 2007. 19. Ezzell GA, Galvin JM, Low D, Palta J, Rosen I, Sharpe MB, et al. Guidance document on delivery, treatment planning, and clinical implementation of IMRT: report of the IMRT Subcommittee of the AAPM radiation therapy committee. Med Phys 2003; 30: 2089-115. doi: 10.1118/1.1591194 20. Klein EE, Hanley J, Bayouth J, Yin FF, Simon W, Dresser S, et al. Task Group 142 report: quality assurance of medical accelerators. Med Phys 2009; 36: 4197-212. doi: 10.1118/1.3190392 21. Ezzell GA, Burmeister JW, Dogan N, LoSasso TJ, Mechalakos JG, Mihailidis D, IMRT commissioning: multiple institution planning and dosimetry compari­sons, a report from AAPM Task Group 119. Med Phys 2009; 36: 5359-73. doi: 10.1118/1.3238104 22. Andreo P, Burns DT, Hohlfeld K, Huq MS, Kanai T, Laitano F, et al. Absorbed dose determination in external beam radiotherapy: An international code of practice for dosimetry based on standards of absorbed dose to water. IAEA TRS-398. Vienna: International Atomic Energy Agency; 2006. 23. Smilovic RadojcicĐ, Švabic Kolacio M, Radojcic M, Rajlic D, Casar B, Faj D, et al. Comparison of calculated dose distributions reported as dose-to-water and dose-to-medium for intensity-modulated radiotherapy of nasopharyngeal cancer patients. Med Dos 2018; 43: 363-9. doi: 10.1016/j. meddos.2017.11.008 24. Palmans H, Andreo P, Huq MS, Christaki K, Alfonso R, Izewska J, et al. Dosimetry of small static fields used in external beam radiotherapy: An International Code of Practice for reference and relative dose determina­tion. Technical Report Series No. 483. IAEA TRS483. Vienna: International Atomic Energy Agency; 2017. doi: 10.1002/mp.13208 25. Casar B, Gershkevitsh E, Mendez I, Jurkovic S, Huq MS. A novel method for the determination of field output factors and output correction factors for small static fields for six diodes and a microdiamond detector in megavolt-age photon beams. Med Phys 2019; 46: 944-63. doi: 10.1002/mp.13318 26. Casar B, Gershkevitsh E, Mendez I, Jurkovic S, Huq MS. Output correction factors for small static fields in megavoltage photon beams for seven ioniza­tion chambers in two orientations - perpendicular and parallel. Med Phys 2020; 47: 242-59. doi: 10.1002/mp.13894 505 research article Comparison of three film analysis softwares using EBT2 and EBT3 films in radiotherapy Tamás Pza1,2, Zsuzsánna Zongor1, Barbara Melles-Bencsik1, Da Zita Tatai-Szab1, Tibor Major1,3, Csilla Pesznyák1,2 1 National Institute of Oncology, Centre of Radiotherapy, Budapest, Hungary 2 Budapest University of Technology and Economics, Institute of Nuclear Techniques, Budapest, Hungary 3 Department of Oncology, Semmelweis University, Budapest, Hungary Radiol Oncol 2020; 54(4): 505-512. Received 23 February 2020 Accepted 27 June 2020 Correspondence to: Tamás Pza, National Institute of Oncology, Centre of Radiotherapy, Ráth Gygy 7-9, 1122 Budapest, Hungary. E-mail: poczatamas87@gmail.com Disclosure: No potential conflicts of interest were disclosed. Introduction. The purpose of the study was to compare the results of gamma value based film analysis according to the used type of self-developer film and software product. Material and methods. The films were irradiated with different treatment techniques such as 3D conformal and intensity modulated radiotherapy with static and rotational delivery. Stereotactic plans with conformal and intensity modulated arc techniques, using coplanar and non-coplanar beam setup were also evaluated. The data of irradi­ated film were compared with the planned planar dose distribution exported from the treatment planning system. Three film analysis software programs were evaluated: PTW Mephysto (PTW), FilmQA Pro (FQP) and radiohromic.com (RC). Both EBT2 and EBT3 types of films were examined. The comparisons of dose distributions were performed with gamma analysis using 10% cut-off level. Results. The results of the gamma analysis for larger fields were between 78.3% and 98.3%, 75.7% and 100%, 80.2% and 98.8% with PTW, FQP and RC, respectively. The results of evaluation in case of stereotactic measurements were 76.8%–99.2% for PTW, 95.7%–100% for FQP and 91.2%–99.9% for RC. Conclusions. All the three software programs are suitable for calibrating and evaluating films, performing gamma analysis, and can be used for patient specific quality assurance measurements. There is no direct connection be­tween gamma passing rate and absolute accuracy or software quality, it is just a feature of the software. The inter­pretation of own results has to be defined on an institutional level according to given workflow and preliminary results. Key words: radiochromic; IMRT; gamma analysis; film analysis software Introduction Over the years, film dosimetry has been developed into a powerful tool for radiotherapy treatment verification and quality assurance as a two-dimen­sional radiation detector system. Radiochromic film technology is based on diacetylene-dye radi­ation-sensitive monomers, which polymerize and change colour due to radiation. These types of films are self-developers, their colour changes directly after irradiation and they do not require chemical processing or film developing equipment. The do-simetric analysis can be applied by using a photo scanner and a special software.1 The speed of po­lymerization depends on the environmental condi­tions, but it stabilizes after 24–48 h.2 The darkening of the film is increasing with the exposed dose, and their relation is generally approximated by poly­nomial or rational functions. Radiochromic films are nearly tissue-equivalent, with low energy- and dose rate dependency.3 For linear accelerators with more photon energies only one film calibration is necessary, but in kV photon energy range a new calibration is needed.4,5 They have a high-spatial resolution suitable for dose distribution measure­ment in radiation fields with high dose gradients, 506 for example, in stereotactic irradiation or brachy-therapy. Radiochromic films are water equivalent because the active layer is made up of low atomic number materials. The different types of films could have different layer arrangements, symmet­ric or asymmetric, and different material construc­tions containing C, H, O and Li. The contamination with high atomic number material (like Cl) is kept low, so the films are nearly water-equivalent.6,7 The radiation sensitive monomers are located between an adhesive and a polyester protection layer. Since the introduction of the EBT2 film type, a yellow marker dye has been built in the active layer of the film to provide information about the subtle differ­ences in the thickness of the active layer, thereby making the increment of the homogeneity possible, and reducing the sensitivity to artificial light.6-8 Materials and methods Films In this study we investigated the GafChromic EBT2 and EBT3 films (Ashland Inc., Wayne, New Jersey). The size of EBT2 film was 8” x 10”. The layer ar­rangement of the EBT2 film is not symmetric; con­sequently, film orientation is important. The sub­strate of the film is clear polyester (175 µm) coated with an active layer (28µm) which is covered by a 25 µm pressure-sensitive adhesive wrap and the top of the film also has a polyester layer (50 µm).9 The size of GafChromic EBT3 was 13” x 17”. The single active layer of the film is nominally 28 µm thick and contains the active component, a marker dye. The active layer is between two 125 µm trans­parent matte polyester subtracts.10 The film is sym­metric, and an anti-Newton ring feature is added by the manufacturer. Scanner To digitize the film EPSON Expression 10000XL (Epson, Nagano, Japan) flatbed scanner was used with A3 scanning surface. The applied scanning parameters were as follows: transmission mode, positive film, no colour correction, landscape ori­entation, 48-bit RGB, 72 dpi resolution and TIFF file format.10 Considerable warm up effect was not ob­served for our scanner, but before every scanning we waited at least fifteen minutes, and the first five scanned images were never used.11 Every pixel in a colour image has three-channel (RGB) image sig­nal. The scanned images can be evaluated by film analysis software.12 The film-scanner response may depend on thickness variations in the active layer coated on the film, electronic noise, scanner insta­bility, lateral artefact, local variations produced by systemic problems of the scanner, Newton rings, dust, scratches or other damage. The orientations of the films were noted during the irradiation, and they were positioned on the same way at the scanning, always at the same distance from the borders of the sensitive area of the scanner.13 The uniformity of the scanner bed was defined by plac­ing four, non-irradiated film pieces at the corners of the scanner to cover the whole scanning surface. The inhomogeneity map of our scanner was deter­mined to find the quasi-homogeneous part of the scanner. According to this map, only the homo­geneous middle part of the scanner was used for scanning. The films have also non-uniformity, but this effect was not examined or corrected. During the scanning, a glass layer was used as compress­ing media. The precision of the scanning method and the applied corrections affect the results of the gamma analysis, so the used methods always have to be reported.14-17 Calibration measurement During the calibration a CIRS Plastic Water sheet phantom (CIRS Inc., Norfolk, VA, USA) was ap­plied and the film was placed between the layers of the phantom (5 cm above and 10 cm under the film). The films were irradiated at the source sur­face distance (SSD) of 95 cm. The size of the calibra­tion films was 2×5 cm, and the number of calibra­tion points were 15 cGy, 30 cGy, 50 cGy, 100 cGy, 200 cGy, 300 cGy, 400 cGy, 550 cGy, 650 cGy, 750 cGy, 850 cGy, 950 cGy and the non-irradiated film (0 cGy). The waiting time between irradiation and scanning was always 24 hours. Always the same frame positions were used and lateral correction was not applied. The scanning of one calibration series was re­peated 18 and 60 months after the initial scan. The changes of the optical density values were evalu­ated for all three colour channels. The absolute and relative differences were also calculated. Treatment planning and irradiation The irradiation was performed with a Varian TrueBeam (Varian Medical Systems, Palo Alto, CA, USA) linear accelerator and Eclipse 13.6 (Varian Medical Systems, Palo Alto, CA, USA) treatment planning system with Analytical Anisotropic Algorithm (AAA) was used for dose calculation. Pocza T et al. / Film analysis software in radiotherapy 507 A pelvic case (prostate and nodal target) was planned with different treatment techniques, such as 3D conformal (3DCRT), intensity modulated radiation therapy with sliding-window (IMRT) and RapidArc (RA), and simultaneous integrated boost with arc therapy (RA-SIB). Small field, ste­reotactic radiotherapy plans were also created with conformal arc (CA) and RA techniques us­ing coplanar and non-coplanar (NC) beam setup. For the pelvic plan 10 MV, for the stereotactic CA plan 6 MV, for the stereotactic RA plan 6 MV-FFF energy was used. The original patient treatment plan was copied to the CT scan of the CIRS solid water phantom. The same phantom was also used for the calibration. The depth of the film was 5 cm with 10 cm backscatter, the isocenter of the plan was positioned at the middle of the film. After the recalculation, the 2D dose distribution at the slice position of the film was exported, and the plan was delivered to the film with the given setup. The planned dose distribution was compared with the results of the film dose distribution using a film analysis software. The gamma method The gamma map comparison as introduced by Low et al. is widely used to judge agreement be­tween treatment plan dose distribution and dose measurement.18 The gamma map function .(rtest) can be defined as minimum value of the following function, ac­cording to rreference: Where d(r) is the dosemap of the test distri­ testtest bution and dreference) is the reference distribu- (rreference tion. (.dose, .distance) also known as ‘gamma criterion’. The tolerance .dose is given in % and the distance in mm. .distance A point of the test distribution (rtest) passes the test, if .= 1. In our study the data were analysed using gamma evaluation with the following cri­teria: 2%, 2mm and 3%, 3mm, and normalization for local dose and global dose maximum with 10% threshold.18-20 During gamma comparison automatic matching with enabled rotation correction was used, and if it decided to be necessary, manual correction was applied. The planned distributions were the refer­ence, and the film measurements were evaluated and compared to them.21 Statistics of gamma analysis and comparison was applied for the three different softwares. The results were calculated with GraphPad 8.0.1 (GraphPad Software, San Diego, CA) using ANOVA and post-hoc Dunn’s test, based on all evaluated scans. Software Several different software programs can be ap­plied for dosimetric the evaluation of radiochromic films. Three software products were analysed for film evaluation: PTW Mephysto (PTW), FilmQA Pro (FQP) and Radiochromic.com (RC). Each of them is dedicated for film scanning, calibration, dose map creation, gamma evaluation, and for im­age processing.22-26 PTW Mephysto (PTW) The film analysis module of PTW Mephysto 3.0 software includes film scanning (FilmScan), cali­bration (FilmCal), film analysing (FilmAnalyse) options, and for gamma analysis PTW Mephysto VeriSoft 6.3 was used. This software works on the basis of single channel dosimetry which only takes into consideration the red channel from the RGB channels. One pixel value on the scanned film rep­resents one dose value. In case of a single-channel dosimetry all response artefacts convert directly to dose artefacts. Applying this method, important data can be lost. Unfortunately, we could not find more information about the mathematical method of the software. FilmQA Pro (FQP) The FilmQA Pro 4.0 software is based on the Micke-Mayer method.27 Different multichannel methods have been proposed in literature for film evalu­ation. This software is compact, tree structured with folders and files. Appropriate tutorials and training material on the handling of the software can be found on http://www.gafchromic.com.28 In multichannel approach, three pixel values (RGB) on the scanned image represent one dose value.27-29 Multichannel dosimetry makes it possible to re­duce the artefacts, for example; the thickness of ac­tive layer, fingerprints or dust from the dose image. Radiochromic films provide a different response in each of the three colour channels, that way the signals can be separated into a dose-dependent 508 and a dose-independent part. The latter one can be corrected, so we can use the only dose-related data for the evaluation of films. Choosing wrong multichannel model, errors of the different colour channels can be combined, because their errors correlate with each other, and the overall error can be increased. Therefore, multichannel dosimetry is can be worse than single-channel dosimetry in case of wrong model selection.30 As the dose range increases, the response of radiochromic film will be increasingly nonlinear. For this reason, the fit­ted polynomial function can oscillate between the data points at higher dose regions, therefore, if a polynomial calibration function is used, more cali­bration points are needed for fitting. The FilmQA Pro uses a special rational function for fitting the calibration curve on calibration data points be­cause the rational function is monotonic, does not oscillate between data values and appropriate to the dosimetric properties of the radiochromic film. In clinical practice, four or five calibration points are enough for a correct calibration. The calibration data have been fit using a func­tion: when the scanner response at dose D is X(D) and, a,b,c are constants. Radiochromic.com (RC) This is a cloud computing web application for cali­bration and dosimetry of radiochromic films. The version number was 2.7. The user interface has a clear layout, available in a browser. In can be used as a free software with some limitations, and its extended version is commercially available. The software also applies multichannel dosimetry as FilmQA Pro but uses another channel independ­ent perturbation model, the truncated normal distribution model. This model is considered as a metamodel which minimizes the uncertainty in the dose inherent in the method of channel independ­ent perturbation. This model applies the first order Taylor expansion to the dose due to small pertur­bation.31 D(r) represents the dose absorbed by the film at point r. is the absolute dose measured by the channel k (R,G,B), when no disturbance is present, and it is calculated directly from the calibration model is the first derivative of the dose, with respect to the NOD (net optical density), at point r. is an error term accounting for the difference between the dose absorbed by the film and the dose measured in the channel k after correction by the per­ turbation. The calculation algorithm of the program and the method of film analysis can be found on the website of the software: https://radiochromic.com. In order to perform calibration, dosimetry evalu­ation and gamma analysis, we uploaded the cali­bration films, the scanned film and the dose map exported from the treatment planning system to Radiochromic.com. It is also possible to make re­calibration during the film evaluation.32 From ver­sion 3.0 the application employs the Multigaussian model.26 Results Auxiliary results Scanner homogeneity The homogeneity map of the scanner can be seen in Figure 1. Based on these results, it can be observed that the top 8 cm and the bottom 7 cm borders of the scanner’s sensitive area are inhomogeneous. There are small inhomogeneities on the right and the left part of the scanner bed. For the film evalua­tion, we can use an approximately 15 cm wide ho­mogeneous area in the centre of the scanner. Inside the homogeneous area the optical density has less than 4% deviation, outside the area it reaches 13%. FIGURE 1. Inhomogeneity map of the full scanning surface (A) and the homogeneous area in the centre of the scanner glass (B). Pocza T et al. / Film analysis software in radiotherapy 509 Long-time darkening 18 and 60 months after the first scanning of the calibrating films, we scanned the same EBT2 films again. For films that received lower dose the rela­tive post-irradiation colouration was higher. The difference between the channels in terms of col-ouration is getting wider by time. In Figure 2 the relative change of the pixel values compared to the original scan at 18 months and 60 months can be observed, according to the exposure. Gamma analysis results The results of the gamma analysis from the type of films and the three software products can be found in Table 1. The parameters of the gamma analy­sis were: 3%, 3 mm, and 2%, 2 mm, the negligible threshold dose was 10%, and the normalization of gamma analysis was performed on global dose maximum. A sample result - the evaluation of the RA-SIB plan with three software programs can be found in Figure 3. The results of gamma analysis of stereotactic fields with EBT3 films can be found in Table 2. The negligible threshold dose was 10%. The gamma analysis was calculated in two ways; in the first case, the normalization was executed for global dose maximum and in the second case, we applied a harder limit, when the normalization was per­formed for local plan dose. According to statistical analysis, the passing rates for FilmQA Pro were significantly higher than PTW Mephysto and Radiochromic.com. TABLE 1. Pass rates of the gamma analysis using three software products; the negligible threshold dose was 10% and the normalization of gamma analysis was performed on global dose maximum. (RA: RapidArc, SIB: simultaneous integrated boost) PTW Mephysto 87.1 % 95.5 % 89.2 % 98.2 % 83.9 % 91.5 % 86.3 % 98.3 % EBT2 FilmQA Pro 98.9 % 100.0 % 75.7 % 93.4 % 99.9 % 100.0 % 87.3 % 92.8 % radiochromic.com 87.2 % 98.1 % 80.2 % 93.1 % 90.4 % 98.5 % 84.2 % 95.3 % PTW Mephysto 86.6 % 94.4 % 78.3 % 93.8 % 92.0 % 97.8 % 86.8 % 93.4 % TABLE 2. Pass rates of gamma analysis of small stereotactic fields; the negligible threshold dose was 10% for EBT 3 films (CA: Conformal Arc, RA: RapidArc, NC: non-coplanar) PTW Mephysto 97.0 % 99.2 % 87.9 % 90.2 % 95.1 % 98.6 % 89.2 % 93.5 % global FilmQA Pro 100.0 % 100.0 % 98.5 % 100.0 % 99.8 % 100.0 % 99.2 % 100.0 % radiochromic.com 98.2 % 99.9 % 97.2 % 99.8 % 95.0 % 99.7 % 96.4 % 99.6 % PTW Mephysto 95.2 % 97.2 % 87.4 % 89.9 % 93.1 % 97.9 % 76.8 % 83.1 % 510 FIGURE 3. Evaluation of the simultaneous integrated boost (SIB) plan with PTW Mephysto (A), FilmQAPro (B) and radiohromic.com (C). TABLE 3. Statistical evaluation and visualisation of the gamma passing rates for the Discussion three different softwares, according to all analysed cases During the preparation of the film data, the cali- Gamma passing rate statistics (%) bration and the scanning process have to be han- PTW Mephysto FilmQA Pro Radiochromic.com Minimum 76.8 75.7 80.2 Maximum 99.2 100.0 99.9 Median 92.6 99.5 95.9 Mean 91.4 96.6 94.2 Std. Deviation 5.9 5.8 5.5 Lower 95% CI 89.3 94.5 92.3 Upper 95% CI 93.5 98.8 96.2 Dunn’s multiple comparisons test Adjusted P Value Significant? PTW Mephysto vs. FilmQA Pro <0,0001 Yes PTW Mephysto vs. Radiochromic.com 0.1824 No FilmQA Pro vs. Radiochromic.com 0.0005 Yes dled very carefully. Based on the results showed in Figure 1, it is recommended to limit the scanner ar­ea to the homogeneous part, or corrections need to be applied at the border of film scanner. In case of large PTVs which cover the whole film surface, the gamma analysis showed higher deviations which were caused by the edge effects during our IMRT treatment plan evaluation. The quality of calibration curves and the time passed since the preparation of the calibration curve can also influence the results of dosimetry analysis. More accurate results can be received with a larger number of calibration points and shorter intervals between the calibration and the film evaluation. According to our results seen in Figure 2, in case of re-evaluation of older film scans the recalibration is crucial.33,34 The presented changes are summation of the film ageing and the scanner characteristics changes, and both effect have to be taken into con­sideration during long-time usage. Our gamma analysis results are in accordance with those found in the literature. The fact that in many cases the threshold dose and the normaliza­tion method (local dose or global maximum dose) are not published makes the comparison more dif­ficult. Agnew et al. showed, that the selection and the settings of the software has a crucial effect on the gamma passing rates.35 Cosumano et al. exam­ined stereotactic irradiation plans (small field, large fraction dose) with EBT3 films. For the gamma cri­teria of 5%, 1 mm they received 94.3%.36 For the ste­reotactic plan, Wen et al. applied a different criteria, for 3%, 1 mm and they found a 95±4.2% agreement during the evaluation of plans.37 Hanusová et al. Pocza T et al. / Film analysis software in radiotherapy 511 using PTW VeriSoft v3.1 found in average 97.03% for EBT3 and 85.81% for EBT2 films agreement with for static IMRT fields with 3%, 3 mm and 5% threshold level.38 Lewis et al. applied FQP software and achieved a correspondence ranged between 95% and 99% for all the treatment fields studied using the gamma test criterion of 2%, 2 mm to eval­uate the measurements.39 Also with FQP Marrazzo et al. found with single- and multichannel analysis for linac measurements the passing rates in aver­age with 2%, 2mm criteria are 91% and 80%, with 3%, 3mm criteria are 98% and 94%, respectively.40 According to Calvo-Ortega et al. with RC software the agreement is between 87.6% and 99.8% using fast protocol for IMRT plans with 3%, 3 mm criteria and 10% threshold.41 The software products have possibilities for au­tomatic dose map and film fusion, but these do not always work perfectly. Manual matching is pos­sible for all three software programs; in this case, results highly depend on the user’s skills and ex­perience. During the evaluation with FilmQA Pro and RC there is an opportunity for recalibration of sensitometric curves with the actual zero and dose maximum points. This option has a signifi­cant impact on the workflow of gamma analysis, it makes easier and faster the usage of the films from the same badge. Table 3 summarizes the statistical evaluation of our measurements. The FQP has sig­nificantly higher passing rate, than the other two softwares. As Table 1 shows, for EBT3 films the difference between the software programs is lower than for EBT2 films. In case of stereotactic plans, the agreement for the CA plans were better than for the RA plans, and for the coplanar cases were better than for the non-coplanars, as can be seen on Table 2. Using local instead of global normalization the number of passing points were decreased, but the differences between the plans were the same, independently from the used software. The ad­vantage of self-developing film as compared to the semi-conductor or ionchamber based detector ma­trix is that it has a better spatial resolution, which allows us to handle the high dose-gradients in case of state-of-art ultra-conformal (stereotactic) plans. The disadvantage of films as compared to other de­tectors is that the usage of film is time-consuming. The film has to be prepared before the measure­ment and they have to be handled very carefully. The results cannot be executed immediately after the measurement, the irradiated films have to be scanned and calibrated according to strictly de­fined methods after the irradiation. Based on our measurements, the EBT2 and EBT3 films are suitable for dose plan verification of 3DCRT and IMRT treatments combined with any of the 3 analysed software programs. All three evaluation programs are suitable for calibrating and evaluating films, and performing the gamma analysis. The deficiency of this paper is that some applications have been improved in the last few years, new models like Multigaussian are imple­mented. By using different softwares in the gamma analysis, the authors cannot exclude the influence of the implementation of the gamma calculation in the final result. Therefore, this paper is not testing which software provides more accurate film dose distributions, neither which dose distributions are more similar to the ones calculated with the treat­ment planning system. Based on the results, it is recommended to always use a new calibration curve during the film evaluation and the homoge­neous area of the scanner should be used for scan­ning. Both types of films and the three software products are very sensitive to calibration, the users must pay close attention to preparation, film han­dling and timing. We recommend using 2%, 2 mm agreement criteria with 10% threshold for evaluat­ing with gamma analysis. This way the results will be slightly lower, but it will be easier to identify the problematic points during the evaluation. Every institute has to define their own limit of acceptance level according to their own workflow and experi­ence. Acknowledgement This work was supported by the Higher Education Excellence Program of the Ministry of Human Capacities in the frame of Biotechnology research area of Budapest University of Technology and Economics (BME FIKP-BIO). References 1. Niroomand-Rad A, Blackwell CR, Coursey BM, Radiochromic film dosimetry: Recommendations of AAPM Radiation Therapy Committee Task Group 55. Med Phys 1998; 25: 2093-115. doi: 10.1118/1.598407 2. Girard F, Bouchard H, Lacroixa F. 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Phys Medica 2015; 31: 1035-42. doi: 10.1016/j.ejmp.2015.08.010 41. Calvo-Ortega JF, Pozo M, Moragues S, Casals J. Fast protocol for radiochromic film dosimetry using a cloud computing web application. Phys Medica 2017; 39: 1-8. doi: 10.1016/j.ejmp.2017.05.072 Radiol Oncol 2020; 54(4): 371-376. doi: 10.2478/raon-2020-0053 Sodobno zdravljenje raka zunanjega spolovila Merlo S Izhodišca. Rak zunanjega spolovila predstavlja 3–5 % rakavih obolenj ženskega spolovila. Slovenska incidenca je 5,5 / 100.000, kar predstavlja 57 novih primerov letno. Najpogostejši histološki tip (90 %) je plošcatocelicni rak. Etiološko razvršcamo rak zunanjega spolovila na dva tipa. Prvi tip je povezan z okužbo s humanim papiloma virusom in drugi tip, ki s to okužbo ni povezan. Najpogostejši in obicajno dolgotrajni simptom raka zunanjega spolovila je srbenje. Glavni in najprimernejši diagnosticni postopek za potrditev diagnoze raka zunanjega poslovila je punch oz. incizijska biopsija. Kirurgija v kombinaciji z radioterapijo predstavlja standardno zdravljenje. Biopsija varovalne bezgavke z limfoscintigrafijo pa je danes standardni del kirurškega zdravljenja, ki zmanjšuje kirurško obolevnost. Sistemskega zdravljenja se obicajno poslužujemo pri paliativnih bolnicah oz. razsejani obliki bolezni. Zakljucki. Rak zunanjega spolovila je redko obolenje. Zaradi patogeneze predstavljata kirurgija in radioterapija glavni izbiri zdravljenja. Biopsija varovalne bezgavke je sodoben nacin zdravljenja raka zunanjega spolovila in pomenljivo zmanjša obolevnost. Napredki pri zdravljenju raka zunanjega spolovila pa so doprinesli k zmanjšanju smrtnosti med slovenskimi bolnicami. Radiol Oncol 2020; 54(4): 377-393. doi: 10.2478/raon-2020-0060 Primarno zdravljenje plošcatocelicnega raka v podrocju glave in vratu s kombinacijo radioterapije in imunoterapije. Pregled trenutnih klinicnih raziskav Plavc G, Strojan P Izhodišca. Plošcatocelicni rak v podrocju glave in vratu je pri vecini bolnikov že ob ugotovitvi diagnoze lokoregionalno napredovala bolezen in se kljub agresivnemu zdravljenju pogosto ponovi. Odkar so zavi­ralci imunskih kontrolnih tock (angl. immune checkpoint inhibitors) pokazali klinicno dobrobit pri zdravlje­nju bolnikov z recidivnim ali razsejanim plošcatocelicnim rakom glave in vratu, je njihova vloga tudi v pri­marnem zdravljenju nemetastatske bolezni predmet številnih klinicnih raziskav. Predklinicni podatki kažejo na sinergisticno delovanje socasne radioterapije in imunoterapije, zato mnoge raziskave preucujejo to kombinacijo v kontekstu definitivne, neoadjuvantne ali adjuvantne terapije nemetastatske oblike bolezni pri bolnikih s tem rakom. Zaradi zapletene interakcije med imunskim sistemom, rakom, imunoterapijo in radioterapijo ima vsak od pristopov prednosti in slabosti. Predstavljamo biološko ozadje sinergisticnega delovanja socasne imunoradioterapije, prednosti in slabosti posameznih terapevtskih pristopov in posre­dujemo kriticen pregled zakljucenih in še potekajocih klinicnih raziskav. Zakljucki. Medtem ko je imunoterapija z zaviralci imunskih kontrolnih tock že postala standarden del zdravljenja bolnikov z recidivnim ali razsejanim plošcatocelicnim rakom glave in vratu, je ucinkovitost takšne imunoterapije pri nematatstaski obliki bolezni še vedno del intenzivnega klinicnega preizkušanja. Obsevanje lahko pripomore k premagovanju številnih mehanizmov, s katerimi se rakave celice izognejo imunskemu sistemu, kar vodi v sinergisticno delovanje z imunoterapijo. Glede na to, da na ucinkovitost kombinacije imunoradioterapije vplivajo številni dejavniki, so podrobnosti v zasnovi klinicnih raziskav, ki preucujejo ta pristop, izjemno pomembne. Radiol Oncol 2020; 54(4): 394-408. doi: 10.2478/raon-2020-0055 Korelacije med kazalci difuzijskega tenzorskega slikanja in magnetnoresonancno spektroskopijo v posameznih regijah glioblastomih. Pilotna raziskava Flores-Alvarez E, Rios-Piedra EA, Cruz-Priego GA, Durand-Muz C, Moreno-Jimenez S, Roldan-Valadez E Izhodišca. Korelacije med posameznimi kazalci difuzijskega tenzorskega slikanja in z magnetnoreso­ nancno spektroskopijo dolocenimi razmerji presnovkov v posameznih regijah glioblastomih še niso bili povsem raziskani. Bolniki in metode. Pri bolnikih z glioblastomom smo retrospektivno preverili korelacije med razmerji presnovkov v možganovini (holin/N-acetil aspartat [Cho/NAA], lipidi in laktat / kreatin [LL/Cr] in mio-ino­sitol/kreatin [mI/Cr]) in enajstimi kazalci difuzijskega tenzorskega slikanja: srednje difuzivnosti (angl. mean diffusivity; MD), frakcijske anizotropije (angl. fractional anisotropy, FA), ciste izotropne difuzije (angl. pure isotropic diffusion, p), cisto anizotropno difuzijo (angl. pure anisotropic diffusion, q), celokupno magnitu-do difuzijskega tenzorja (angl. total magnitude of the diffusion tensor, L), linearnim tenzorjem (angl. linear tensor, Cl), planarnim tenzorjem (angl. planar tensor, Cp), sfericnim tenzorjem (angl. spherical tensor, Cs), relativno anizotropijo (angl. relative anisotropy, RA), aksialno difuzivnostjo (angl. axial diffusivity, AD) in radialno difuzivnostjo (angl. radial diffusivity, RD) v istih predelih možganovine. Opazovali smo predel robnega postkontrastnega ojacanja, predel peritumorskega edema in predel bele možganovine nor-malnega izgleda. Za vrednotenje korelacij skupno 546 magnetnoresonancnih spektroskopij in meritev difuzijskega tenzorskega slikanja smo uporabili Spearmanov kolicnik. Rezultati. V predelu robnega postkontrastnega ojacanja smo našli štiri znacilne korelacije: FA . LL/Cr, Rs = -.364, p = .034; Cp . LL/Cr, Rs = .362, p = .035; q . LL/Cr, Rs = -.349, p = .035; RA . LL/Cr, Rs = -.357, p = .038. Deset dodatnih korelacij smo odkrili v predelu peritumorskega edema: AD . LL/Cr, AD . mI/Cr, MD . LL/Cr, MD . mI/Cr, p . LL/Cr, p . mI/Cr, RD . mI/Cr, RD . mI/Cr, L . LL/Cr, L . mI/Cr. Zakljucki. Raziskava je pokazala, da obstajajo med kazalci magnetnoresonancne spektroskopije in difuzijskega tenzorskega slikanja pri bolnikih z glioblastomom - predvsem v predelu peritumorskega ede­ ma - znacilne korelacije, kljub temu, da rezultati odsevajo razlicne biološke znacilnosti tumorjev. Da bi opisane povezave pojasnili, so potrebne nadaljne raziskave, tako pri bolnikih z glioblastomom, kot tudi pri drugih boleznih v možganovini. Radiol Oncol 2020; 54(4): 409-418. doi: 10.2478/raon-2020-0052 Vzorcenje nadledvicnih ven pri primarnem aldosteronizmu. 15 let izkušenj nacionalnega referencnega centra Kocjan T, Jensterle M, Vidmar G, Vrckovnik R, Berden P, Stankovic M Izhodišca. Vzorcenje nadledvicnih ven je kljucni del diagnosticne obravnave primarnega aldostero­nizma, ki loci med enostransko in obojestransko boleznijo ter doloca izbiro zdravljenja. Pregledati smo uspešnost prvih 15 let vzorcenja nadledvicnih ven pri primarnem aldosteronizmu v nacionalnem endo­krinološkem referencnem centru. Zacetno obdobje smo primerjali z obdobjem po vkljucitvi usmerjenega radiologa v letu 2012. Dodatno smo rezultate vzorcenja nadledvicnih ven primerjali z izvidi CT in ocenili delež operiranih biolnikov z dokazano enostransko boleznijo. Bolniki in metode. V retrospektivno presecno raziskavo smo vkljucili vse bolnike s primarnim aldoste­ronizmom, pri katerih smo naredili vzorcenje nadledvicnih ven po njegovi uvedbi v obdobju 2004 do konca 2018. Vzorcenje nadledvicnih ven smo opravili sekvencno med neprekinjenim spodbujanjem s Synacthenom. Ko je bilo razmerje koncentracije kortizola med nadledvicno veno in spodnjo votlo veno vsaj 5, smo vzorcenje nadledvicnih ven smatrali za uspešno. Rezultati. Pregledali smo podatke 235 bolnikov (168 moških; starost 32–73, mediana 56 let; indeks telesne mase 18–48, mediana 30,4 kg/m2). Povprecno število letno opravljenih postopkov vzorcenja nadledvicnih ven se je povecalo iz 7 v obdobju 2004–2011 na 29 v obdobju med 2012–2018 (p < 0,001). Vzorcenje nadledvicnih ven je bilo potrebno ponoviti v 10 % primerov; uspešnih je bilo 77 % vseh postop­kov in pri 86 % bolnikov. Delež bolnikov z uspešnim vzorcenjem nadledvicnih ven (92 % med 2012–2018 proti 66 % med 2004–2011, p < 0.001) in uspešnih postopkov vzorcenja nadledvicnih ven (82 % proti 61 %, p < 0.001) je bil v zadnjem obdobju statisticno znacilno višji. Zakljucki. Število postopkov vzorcenja nadledvicnih ven in njihova uspešnost sta se s casom povecala. Uvedba usmerjenega radiologa in tehnicni napredek sta razširila in izboljšala izvedbo vzorcenja nadled­vicnih ven. Radiol Oncol 2020; 54(4): 419-428. doi: 10.2478/raon-2020-0061 Pomen difuzijsko poudarjenih tehnik magnetnoresonancnega slikanja 3 Tesla pri adneksalnih tumorjih Dimova J, Zlatareva D, Bakalova R, Aoki I, Hadjidekov G Izhodišca. Namen raziskave je bil opredeliti razlicne vrste adneksalnih tumorjev in ugotoviti uporabnost difuzijsko poudarjenih slikovnih tehnik v primerjavi s standardnimi sekvencami ob uporabi magnetno re­ sonancnega slikanja (MRI) 3T. Bolniki in metode. V raziskavo smo vkljucili 174 žensk, starih od 13 do 87 let, pri katerih smo v obdobju treh let naredili magnetnoresonancno preiskavo medenice. Preiskave smo opravili na dveh radioloških oddelkih in dveh razlicnih aparatih: pri 135 bolnicah smo uporabili MRI 3 Tesla Siemens Verio in pri 39 bolnicah MRI 3 Tesla Philips Ingeina. V raziskavo smo vkljucili 98 preiskovank, pri katerih je bil odkrit vsaj en adneksalni tumor. Nekatere od preiskovank so bile obravnavane retrospektivno. Upoštevali smo tudi anamnesticne podatke, ugotovitve klinicnih pregledov in laboratorijske podatke. Rezultati. V skupini 98 preiskovank s povprecno starostjo 47,2 let smo odkrili 124 ovarijskih tumorjev. V skladu z magnetnoresonancnimi kriteriji je bilo 59,2 % benignih, 30,6 % malignih in 10,2 % mejnih. 58,1 % tumorjev je bilo cisticne, 12,9 % trdne in 29 % mešane konsistence. Histološka analiza je potrdila 74,4 % tumorjev kot benigne in 25,6 % kot maligne. Vsi maligni tumorji so na MRI kazali omejeno difuzijo. 64 prei­skovank je opravilo MRI s kontrastnim sredstvom, pri 34 je bila uporaba kontrastnega sredstva kontraindi­cirana. 39 % tumorjev (61 %) je kazalo ojacenje po dodatku kontrasta. Zakljucki. Opredelitev adneksalnih tumorjev je kljucna v predoperativni obravnavi bolnikov. MRI 3T, še posebno difuzijsko poudarjene slikovne tehnike, pomembno izboljšajo natancnost diagnosticne obrav-nave. Radiol Oncol 2020; 54(4): 429-436. doi: 10.2478/raon-2020-0057 Vpliv genetske variabilnosti v IL1B in MIR146A na tveganje za nastanek plevralnih plakov in malignega mezotelioma Piber P, Vavpetic N, Goricar K, Dolžan V, Kovac V, Franko A Izhodišca. Izpostavljenost azbestu je povezana s tveganjem za nastanek plevralnih plakov in maligne­ga mezotelioma, saj njegova vlakna aktivirajo makrofage, cemur sledi sprošcanje vnetnih mediatorjev, med drugim tudi interlevkina 1ß (IL1ß). Na izražanje IL-1ß vpliva genetska variabilnost IL1B in regulatorne mikroRNA (miRNA). Ta raziskava je preucevala vpliv polimorfizmov IL1B in MIR146A na tveganje za razvoj plevralnih plakov in malignega mezotelioma. Preiskovanci in metode. V raziskavo smo vkljucili 394 bolnikov s plevralnimi plaki, 277 bolnikov z mali­gnim mezoteliomom in 175 zdravih preiskovancev, pri katerih smo dolocili polimorfizme IL1B in MIR146A. Za statisticno analizo smo uporabili logisticno regresijo. Rezultati. Noben polimorfizem ni pokazal statisticno znacilnega vpliva na tveganje za razvoj plevralnih plakov. Polimorfizem MIR146A rs2910164 je statisticno znacilno zmanjšal tveganje za nastanek malignega mezotelioma (razmerje obetov [OR] = 0,31; 95 % interval zaupanja [CI] = 0,13–0,73; p = 0.008). Nosilci dveh polimorfnih alelov so imeli manjše tveganje za nastanek malignega mezotelioma tudi po prilago­ ditvi po starosti in spolu (OR= 0,34; 95 % CI = 0,14–0,85; p = 0,020). Nosilci vsaj enega polimorfnega alela IL1B rs1143623 v podskupini z znano izpostavljenostjo azbestu so imeli v multivariatni analizi manjše tve­ ganje za nastanek malignega mezotelioma (OR = 0,50; 95 % CI = 0,28–0,92; p = 0,025). Interakcija med polimorfizmoma IL1B rs1143623 in IL1B rs1071676 je imela statisticno znacilen vpliv na povecanje tveganja za maligni mezoteliom (p = 0,050). Zakljucki. V raziskavi smo pokazali, da bi genetska variabilnost vnetnega mediatorja IL-1ß lahko vpliva-la na tveganje za razvoj malignega mezotelioma, ne pa plevralnih plakov. Radiol Oncol 2020; 54(4): 437-446. doi: 10.2478/raon-2020-0054 Razmerje med nevtrofilci in limfociti lahko napoveduje izid zadravljenja pri razširjenem drobnocelicnem raku pljuc Drpa G, Šutic M, Baranašic J, Jakopovic M, Samaržija M, Kukulj S, Kneževic J Izhodišca. Razmerje med nevtrofilci in limfociti (NLR), razmerje med trombociti in limfociti (PLR) ter razmerje med limfociti in monociti (LMR) so analizirali pri razlicnih rakih in opredeljevali njihov pomen pri napovedovanja poteka bolezni. Cilj pricujoce raziskave je bil ugotoviti povezavo med temi parametri in preživetjem bolnikov z drobnocelicnim rakom pljuc, saj je bilo objavljenih zelo malo raziskav pri tej vrsti raka. Bolniki in metode. Retrospektivno smo analizirali 140 bolnikov, ki smo jim diagnosticirali drobnocelicni rak pljuc na Klinicnem oddelku Jordanovac med letoma 2012 in 2016. Razširjeno bolezen smo ugotovili pri 80 bolnikih, omejeno obliko bolezni pa pri 60 bolnikih. Analizirali smo potencialni napovedni pomen razlicnih laboratorijskih parametrov, vkljucno z NLR, PLR in LMR, dolocenih pred zacetkom zdravljenja. Rezultati. Razširjenost bolezni, odgovor na zdravljenje, obsevanje prsnega koša in profilakticno obseva­nje glave, pa tudi hemoglobin, število monocitov, C-reaktivni protein in laktatna dehidrogenaza (LDH) so bili napovedno dejavnik pri vseh bolnikih. Ko smo loceno analizirali bolnike z ozirom na razširjenosti bo­lezni, smo ugotovili, da imajo napovedni pomen poteka bolezni pri razširjeni bolezni le kožne metastaze in vrednosti LDH in NLR, ne glede na mejno vrednost. Napovedni pomen pri omejeni obliki bolezni pa so imeli stanje zmogljivosti, obsevanje prsnega koša, profilakticno obsevanje glave ter vrednosti hemoglo­bina in kreatinina. Zakljucki. NLR, izracunan pred zacetkom zdravljenja, je imel napovedni pomen poteka bolezni pri raz­širjeni obliki drobnocelicnega rakam pljuc, medtem ko PLR in LMR nista bila napovedno pomembna pri nobeni od analiziranih skupin bolnikov. Radiol Oncol 2020; 54(4): 447-454. doi: 10.2478/raon-2020-0058 Izsledki o nacinih zdravljenja III. stadija nedrobnocelicnega pljucnega raka v Srednji in Vzhodni Evropi Zemanová M, Jakopovic M, Stanic K, Lazar-Poniatowska M, Vrankar M, Rusu P, Ciuleanu T, Radosavljevic D, Bogos K, Nawrocki S Izhodišca. Namen raziskave je bil zbrati izsledke o nacinih zdravljenja III. stadija nedrobnocelicnega pljucnega raka v Srednji in Vzhodni Evropi. Na podlagi izsledkov o klinicnih praksah so avtorji oblikovali strokovno mnenje o pomanjkljivostih obravnave in o kazalnikih kakovosti. Bolniki in metode. Multidisciplinarna strokovna skupina 10 zdravnikov iz 7 držav je sistematicno pre­gledala literaturo ter uporabila modificiran postopek Delphi za dolocitev pomanjkljivosti obravnave in obravnavala kazalnike kakovosti pri bolnikih s III. stadijem nedrobnocelicnega pljucnega raka. Uporabili so podroben vprašalnik, s katerim so opredelili nacine zdravljenja teh bolnikov in prepoznavali vzorce nji-hove obravnave v Srednji in Vzhodni Evropi. Najprej je vprašalnik izpolnila skupina onkologov internistov, onkologov radioterapevtov in pulmologov. Nato se je strokovna skupina osebno srecala na sestanku in pregledala rezultate vprašalnika ter pripravila drugi krog postopka Delphi, v katerem je izdelala in dopol­ nila dodatno anketo. Raziskavo so koncali s koncnim pregledom in sintezo izsledkov. Rezultati. Strokovna skupina je dosegla popolno soglasje pri nizu klinicnih priporocil, ki temeljijo na medicinskih dokazih. Odgovori v vprašalniku so pokazali zelo razlicne vzorce zdravljenja v regiji. Seznam pomanjkljivosti obravnav in ovir za kakovostno oskrbo je bil pripravljen s skoraj popolnim soglasjem stro­kovne skupine. Zakljucki. Vzorci zdravljenja bolnikov s III. stadijem nedrobnocelicnega pljucnega raka kažejo na ve­liko raznolikost obravnave v Srednji in Vzhodni Evropi. Avtorji ugotavljajo, da je predvsem razpoložljivost slikovnodiagnosticnih preiskav pomanjkljiva in da je sorazmerno majhen delež bolnikov zdravljenih s ke­moradioterapijo in namenom ozdravitve pri neresektabilnih tumorjih. Radiol Oncol 2020; 54(4): 455-460. doi: 10.2478/raon-2020-0059 Zdravljenje otrok in mladostnikov z rabdomiosarkomom v štirih evropskih državah z nizkimi izdatki za zdravstvo Cesen Mazic M, Bonevski A, Mikeškova M, Mihut E, Bisogno G, Jazbec J Izhodišca. Preživetje otrok z rakom je v državah Vzhodne in Centralne Evrope 10–20 % nižje v primerjavi z evropskimi državami, ki za zdravstvo namenijo vec denarja. Ocenili smo preživetje otrok in mladostnikov z rabdomiosarkomom, ki so bili zdravljeni v štirih evropskih državah z nizkimi izdatki za zdravstvo (Slovenija, Hrvaška, Slovaška, Romunija). Bolniki in metode. V retrospektivni raziskavi smo ocenili preživetje za vse bolnike zdravljene v izbranem casovnem obdobju v Sloveniji in Hrvaški. Slovaška je vkljucila bolnike iz dveh otroških onkoloških bolni­šnic, kar predstavlja polovico vseh bolnikov z rabdomiosarkomom iz te države. Romunija je posredovala podatke za bolnike zdravljene v enem samem centru, kar ustreza desetini vseh pricakovanih bolnikov. Rezultati. Raziskava je zajela 178 otrok in mladostnikov z rabdomiosarkomom, ki so bili zdravljeni v ob- dobju od januarja 2000 do decembra 2015. Povprecna starost ob diagnozi je bila 7,7 let. Tretjina otrok je bila starejših od 10 let, cetrtina je imela tumor z alveolarno histologijo in 72 % neugodno lokacijo primar­nega tumorja. Delež bolnikov z razširjeno boleznijo, zasevki v regionalnih bezgavkah (24 %) ali oddaljenimi zasevki (27 %), je bil vecji od pricakovanega. Vsi bolniki so prejeli sistemsko kemoterapijo; 57 % bolnikov je bilo v sklopu lokalnega zdravljenja obsevanih in 63 % operiranih. Pet letno preživetje brez ponovitve in celokupno preživetje je znašalo 50,7 % oziroma 59,6 %. Pet letno preživetje bolnikov z lokalizirano boleznijo je bilo 72 %, tistih z oddaljenimi zasevki pa le 24 %. Zakljucki. Otroci in mladostniki z rabdomiosarkomom, ki so zdravljeni v državah Vzhodne in Centralne Evrope, imajo slabše preživetje kot vrstniki iz evropskih držav z velikimi izdatki za zdravstvo. Aktivno so-delovanje v mednarodnih klinicnih raziskavah na podrocju otroške onkologije bi lahko izboljšalo izhod bolnikov v državah z nižjimi izdatki za zdravstvo. Radiol Oncol 2020; 54(4): 461-469. doi: 10.2478/raon-2020-0043 Vpliv socasne kemoradioterapije s kapecitabinom in bevacizumabom na preživetje, pozno toksicnost in z zdravjem povezano kakovostjo življenja pri bolnikih z lokalno napredovalim rakom danke. (Prospektivna raziskava faze II »CRAB«) Velenik V, Zadnik V, Omejc M, Grosek J, Tuta M Izhodišca. Le malo raziskav je porocalo o zgodnjih rezultatih ucinkovitosti in toksicnosti kombiniranega zdravljenja lokalno napredovalega raka danke z dodajanjem bevacizumaba k predoperativni kemora­ dioterapiji. Dolgorocnih podatkov o preživetju in poznih zapletih pa ni. Poleg tega nobena raziskava ni porocala o oceni z zdravjem povezane kakovosti življenja. Bolniki in metode. Po vec kot 5 letih spremljanja smo posodobili rezultate klinicne raziskave II. faze pri 61 bolnikih z lokalno napredovalim rakom danke. Zdravili smo jih z neoadjuvantno s kapecitabinom, radi­oterapijo in bevacizumabom (raziskava CRAB) pred operacijo in adjuvantno s kemoterapijo. Sekundarni cilji posodobljene analize so bili lokalna kontrola, preživetje brez bolezni, celokupno preživetje, pozna to-ksicnost in kakovost življenja (pred zacetkom zdravljenja in eno leto po zdravljenju) z vprašalnikom EORTC QLQ-C30 in EORTC QLQ-CR38. Rezultati. Srednji cas spremljanja bolnikov je bil 67 mesecev. V tem obdobju je umrlo 16 bolnikov (26,7 %). 5-letna stopnja celokupnega preživetja, preživetja brez bolezni in lokalna kontrola so bila 72,2 %, 70 % in 92,4 %. Bolniki s patološkimi pozitivnimi podrocnimi bezgavkami ali patološkimi T3–4 tumorji so imeli znatno slabše preživetje kot bolniki s patološkimi negativnimi ali T0–2 tumorji. Devet bolnikov (14,8 %) je razvilo pozne zaplete stopnje 3 ali vec kombiniranega zdravljenja. Prvi dogodek smo beležili 12 mesecev in zadnji 87 mesecev po operaciji (srednji cas 48 mesecev). Na podlagi rezultatov vprašalnika EORTC QLQ-C30 eno leto po zdravljenju ni bilo bistvenih sprememb globalne kakovosti življenja in treh simp­tomov (bolecina, nespecnost in diareja), fizicno in socialno delovanje pa sta se znatno zmanjšala. Na podlagi rezultatov QLQ-CR38 se je telesna samopodoba bistveno izboljšala, težave z izgubo teže so se znatno zmanjšale, vendar ob povecanju spolne disfunkcije pri moških in povecanju neželenih ucinkov kemoterapije. Zakljucki. Za izboljšanje dolgorocnih rezultatov preživetja potrebujejo bolniki z lokalno napredovalim rakom danke in z visokimi dejavniki tveganja, kot so pozitivne patološke bezgavke in visoki patološki sta­ dij T, agresivnejše zdravljenje. Posebno skrb moramo nameniti uravnavanju njihovih posameznih vidikov kakovosti življenja ter pojavu in reševanju poznih sopojavov kombiniranega zdravljenja. Radiol Oncol 2020; 54(4): 470-479. doi: 10.2478/raon-2020-0050 Obsevalni volumni po operaciji zaradi raka leve dojke in nacrtovana absorbirana doza srcnih struktur pri tridimenzionalnem konformnem obsevanju ali tangencialni obliki intenzitetno modulirajocega obsevanja Ratoša I, Jenko A, Šljivic Ž, Pirnat M, Oblak I Izhodišca. Namen raziskave je bila primerjava nacrtovanih absorbiranih doz tridimenzionalnega kon­formnega obsevanja (3D-CRT) in tangencialne oblike intenzitetno modulirajocega obsevanja (t-IMRT) pri bolnicah, ki smo jih zdravili z obsevanjem po operaciji zaradi raka leve dojke. Z raziskavo smo želeli natancneje analizirati nacrtovano absorbirano dozo srca, srcnih votlin in koronarnih arterij ob socasnem upoštevanju razlicne anatomije oziroma velikosti obsevalnih volumnov. Bolniki in metode. V analizo smo vkljucili 60 posnetkov racunalniške tomografije (CT) bolnic. Na vsak set slik CT smo s pomocjo atlasa vrisali kriticne zdrave organe, vkljucno s posameznimi strukturami srca ter tarcne volumne. Za vsak set CT smo pripravili dva obsevalna nacrta: 3D-CRT in t-IMRT. Nacrtovana absorbirana doza je bila 16-krat 2,67 Gy. Posnetke CT smo razvrstili v skupino z majhnim, srednjim in velikim klinicnim tarcnim volumnom (CTV). Rezultati. Povprecne absorbirane doze za celotno srce (1,9 proti 2,1 Gy; P < 0,005), levo sprednjo ko­ronarno arterijo (8,2 proti 8,4 Gy; P < 0,005) in za levi prekat (3,0 proti 3,2 Gy; P < 0,005) so bile nižje ob uporabi obsevalne tehnike 3D-CRT. Posamezni srcni segmenti so prejeli najvišje povprecne doze, pred­vsem apikalni (8,5 proti 9,0 Gy; P < 0,005) in sprednji predel (5,0 proti 5,4 Gy; P < 0,005) levega prekata. Povprecni dozi za celotno srce in levi prekat sta bili višji ob vecanju CTV neodvisno od obsevalne tehnike. Nizke vrednosti povprecne obsevalne doze za srce (< 2,5 Gy) so bile dosežene pri 44 (73,3 %) bolnicah s tehniko 3D-CRT in pri 41 (68,3 %) bolnicah s tehniko t-IMRT. Zakljucki. Rezultati raziskave potrjujejo precejšnje razlike v povprecni absorbirani dozi srca ali levega prekata, pri bolnicah, ki se zdravijo z obsevanjem po operaciji zaradi raka leve dojke. Ugotovljene razlike so lahko posledica tako uporabljene tehnike obsevanja kakor tudi telesne konstitucije bolnice, kamor spada tudi velikost obsevalnega volumna. Radiol Oncol 2020; 54(4): 480-487. doi: 10.2478/raon-2020-0039 Dolgotrajni rezultati toksicnosti in preživetja po stereotakticni ablativni radioterapiji pri bolnikih s centralnimi pljucnimi tumorji Atalar B, Mustafayev TZ, Sio TT, Sahin B, Gungor G, Aydin G, Yapici B, Ozyar E Izhodišca. Stereotakticna ablativna radioterapija (SABR) je ucinkovita pri primarnih tumorjih prsnega ko­ša in metastazah; vendar so neželeni ucinki vecji pri centralnih tumorjih. Ocenili smo dejavnike, ki vplivajo na izid in toksicnost po SABR pri bolnikih s primarnimi pljucnimi in oligometastatskimi tumorji. Bolniki in metode. Retrospektivno smo pregledali zaporedne bolnike s centralno ležecimi pljucnimi tumorji, ki smo jih v naši bolnišnici zdravili od leta 2009 do 2016. Vpliv bolnikov, bolezni in parametrov po­ vezanih z zdravljenjem na lokalno kontrolo, celokupno preživetje in preživetje brez toksicnosti smo ocenili z multivariatno analizo. Rezultati. Med 65 zaporednimi bolniki s 70 centralno ležecimi tumorji je bilo ponovno obsevanih 20 tumorjev (28 %). Srednja skupna doza (razpon) za vse tumorje je bila 55 Gy (30–60) v 5 (3–10) frakci­jah. Radiološki popoln odgovor smo ugotovili pri 43 lezijah (61 %). Noben od analiziranih dejavnikov ni bil povezan s popolnim odgovorom. Po srednjem casu spremljanja 57 (95 % interval zaupanja [CI] 48–65) mesecev se je 10 tumorjev (14 %) znova pojavilo, 37 (57 %) bolnikov je umrlo; 2- in 5-letne sto­pnje celokupnega preživetja so bile 52 % oziroma 28 %. Srednje celokupno preživetje je bilo bistve-no nižje pri bolnikih s 3. ali višjo stopnjo toksicnostjo proti nižji toksicnosti (5 v primerjavi z 39 meseci; P < 0,001). Med 17 primeri resne toksicnosti jih je bilo pet 5. stopnje, med njimi so bili trije ponovno obseva­ni na isto polje. Preživetje bolnikov brez toksicnosti stopnje 3 do 5 je bila nižja pri bolnikih, ki so bili ponovno obsevani (2-letno preživetje brez toksicnosti 63 % v primerjavi z 96 %; P = 0,02). Zakljucki. Raziskava je pokazala, da je sodobna SABR ucinkovita pri centralnih pljucnih tumorjih, toksic­nost pa je sprejemljiva. SABR pri ponovno obsevanih centralnih pljucnih lezijah in verjetno lezijah, ki se naslanjajo na traheobronhialno drevo, lahko povzroci vecje tveganje za resno toksicnost. Radiol Oncol 2020; 54(4): 488-494. doi: 10.2478/raon-2020-0030 Ali redna kontrola kakovosti izboljšuje kirurško zdravljenje bolnic iz slovenskega presejalnega programa za raka dojk? Perhavec A, Milicevic S, Peric B, Žgajnar J Izhodišca. Namen pricujoce raziskave je bil analizirati kakovost kirurškega zdravljenja bolnic s presejal­nega programa za raka dojk (DORA) z uporabo uveljavljenih evropskih kazalnikov kakovosti. Dodatno smo analizirali ali se kakovost kirurškega zdravljenja z leti izboljšuje. Bolnice in metode. V retrospektivno raziskavo smo vkljucili bolnice s programa DORA, ki so v obdobju od 1. januarja 2016 do 31. decembra 2018 potrebovale kirurški poseg. Za analizo smo uporabili kazalni­ ke kakovosti Evropskega združenje specialistov za rak dojke (angl. European Society of Breast Cancer Specialists; EUSOMA) in Evropske mreže raka dojke (angl. European Breast Cancer Network; EBCN), pet kazalnikov za terapevtske in dva za diagnosticne operacije. Dodatno smo primerjali rezultate med kirurgi. Rezultati. V triletnem obdobju 2016–2018 je 14 kirurgov opravilo 1421 operacij dojk pri 1398 bolnicah; 1197 terapevtskih (za potrjene rake dojk) in 224 diagnosticnih. Od izbranih zahtevanih kazalcev kakovosti smo dosegali dva za terapevtske in nobenega za diagnosticne operacije. Ugotovili pa smo statisticno znacilen napredek pri treh kazalnikih za terapevtske in enem za diagnosticne operacije, kar nakazuje, da redno spremljanje kakovosti vodi k izboljšanju kirurškega zdravljenja. Med kirurgi smo opazili visoko variabilnost pri doseganju kazalcev kakovosti, ki je ostala visoka preko celotnega analiziranega obdobja. Zakljucki. Dosegati merila kazalnikov kakovosti je zahtevno, zlasti ko uporabljamo specificne kazalnike za presejalne programe. Redna kontrola kakovosti vodi k izboljševanju rezultatov. Omejitev kirurškega zdravljenja na manjše število bolj izkušenih kirurgov bi lahko vodila do manjših razlik v kakovosti kirurškega zdravljenja. Radiol Oncol 2020; 54(4): 494-504. doi: 10.2478/raon-2020-0051 Eksperimentalno preverjanje racunskega algoritma Monte Carlo nacrtovalnega sistema v snovi z gostoto kosti Smilovic Radojcic Đ, Casar B, Rajlic D, Švabic Kolacio M, Mendez I, Obajdin N, Dundara Debeljuh D, Jurkovic S Izhodišca. Novejši racunalniški nacrtovalni sistemi, ki uporabljajo simulacijo Monte Carlo, lahko absor­birano dozo izracunavajo na dva nacina: kot dozo v snovi in kot dozo v vodi. Predhodne raziskave so pokazale, da obstajajo pomembne razlike med obema nacinoma, posebej v primeru, ko izracunavamo dozo v gostejših snoveh z gostoto podobno gostoti kosti. Ker moramo pred klinicno uporabo racunske algoritme nacrtovalnih sistemov eksperimentalno preveriti, smo opravili analizo dveh nacinov izracuna­vanja doze nacrtovalnega sistema Elekta Monaco. Izracunane doze smo primerjali z eksperimentalno dobljenimi vrednostmi z namenom, da definiramo dodatni postopek k že obstojeci metodologiji prever­janja racunalniških nacrtovalnih sistemov. Materiali in metode. V raziskavi smo uporabili fotonske žarke 6 MV iz linearnega pospeševalnika. Izvedli smo meritve s Farmerjevo ionizacijsko celico v semi-antropomorfnem fantomu in dobljene eksperimen­ talne rezultate primerjali z izracunanimi vrednostmi. Primerjali smo absorbirane doze pri treh razlicnih delih fantoma, ki imajo razlicne gostote. Osredotocili smo se na podrocje, kjer je gostota podobna gostoti kosti. Rezultati. Izmerjene in izracunane doze so bile skladne za vse dele fantoma, kjer je gostota podobna gostoti vode ali gostoti pljuc. V podrocju, kjer je gostota podobna gostoti kosti, smo našli statisticno po­membne razlike med eksperimentalnimi in izracunanimi vrednostmi absorbirane doze. Pomembne razlike smo našli tudi med obema nacinoma izracunavanja doze – te so bile v razponu od 5,7 do 8,3 %, odvisno od metode, ki smo jo uporabili. Zakljucki. Na osnovi naših izsledkov smo predlagali dodatek k trenutni metodologiji za preverjanje komercialnih nacrtovalnih sistemov Monte Carlo z izvedbo dodatnih meritev v snovi, ki ima podobno gostoto, kot jo imajo kosti. Radiol Oncol 2020; 54(4): 505-512. doi: 10.2478/raon-2020-0049 Primerjava treh programskih paketov za analizo filmov v radioterapiji ob uporabi filmov EBT2 in EBT3 Pza T, Zongor Z, Melles-Bencsik B, Tatai-SzabDZ, Major T, Pesznyák C Izhodišca. Namen raziskave je bila primerjava programskih paketov za filmsko dozimetrijo z analizo vre­ dnosti gama ob uporabi razlicnih tipov filmov. Materiali in metode. Filme smo obsevali na vec nacinov in uporabili razlicne nacrtovalne oziroma ob-sevalne tehnike. Uporabili smo tehniko 3D konformnih obsevalnih polj in intenzitetno modulirano tehniko s staticnimi in dinamicnimi obsevalnimi polji. Dodatno smo analizirali tudi obsevalne nacrte pripravljene za stereotakticno locno tehniko s konformnimi in intenzitetno moduliranimi obsevalnimi polji za koplanar­na in nekoplanarna polja. Dozno porazdelitev na obsevanih filmih smo primerjali s tisto, ki smo jo dobili v racunalniškem nacrtovalnem sistemu. Pri analizi smo uporabili tri razlicne pakete programske opreme, ki jih uporabljamo za filmsko dozimetrijo, PTW Mephysto (PTW), FilmQA Pro (FQP) in Radiochromic.com (RC) ter dva tipa filmov, EBT2 in EBT3. Primerjavo doznih porazdelitev smo naredili z analizo gama kjer smo privzeli 10 % mejno stopnjo. Rezultati. Pri vecjih obsevalni poljih so bile vrednosti gama med 78,3 % in 98,3 %, 75,7 % in 100 % ter 80,2 % in 98,8 % po vrsti za programske pakete PTW, FQP in RC. V primeru, ko smo uporabili stereotakticne obsevalne tehnike, so bile vrednosti gama od 76,8 % do 99,2 % za PTW, od 95,7 % do 100 % za FQP in od 91,2 % do 99,9 % za programski paket RC. Zakljucki. Analiza vrednosti gama je pokazala, da so vsi trije testirani programski paketi ustrezni za film-sko dozimetrijo in jih lahko uporabljamo za individualno dozimetrijo pri bolnikih. Raziskava je pokazala, da ni neposredne povezave med rezultati analize gama in absolutno natancnosti ali kakovostjo programske opreme; razlicni rezultati so pretežno povezani z individualnimi lastnostmi posameznega programskega paketa. Izsledki pricujoce raziskave dovoljujejo vkljucitev dozimetricnih postopkov z radiokromskimi filmi v posamezne procese klinicnega dela. Fundacija "Docent dr. J. Cholewa" je neprofitno, neinstitucionalno in nestrankarsko združenje posameznikov, ustanov in organizacij, ki želijo materialno spodbujati in poglabljati raziskovalno dejavnost v onkologiji. Dunajska 106 1000 Ljubljana IBAN: SI56 0203 3001 7879 431 Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education – a report for the fourth quarter of 2020 Doc. Dr. Josip Cholewa Foundation for cancer research and education continues with its planned activi­ties in the fourth quarter of 2020. Its primary focus remains the provision of grants and scholarships and other forms of financial assistance for basic, clinical and public health research in the field of on­cology. In parallel, it also makes efforts to provide financial and other support for the organisation of congresses, symposia and other forms of meetings to spread the knowledge about prevention and treat­ment of cancer, and finally about rehabilitation for cancer patients. In Foundation's strategy, the spread of knowledge should not be restricted only to the professionals that treat cancer patients, but also to the patients themselves and to the general public. The Foundation continues to provide support for »Radiology and Oncology«, a quarterly scientific magazine with a respectable impact factor that publishes research and review articles about all aspects of cancer. The magazine is edited and published in Ljubljana, Slovenia. »Radiology and Oncology« is an open access journal available to everyone free of charge. Its long tradition represents a guarantee for the continuity of international exchange of ideas and research results in the field of oncology for all in Slovenia that are interested and involved in helping people affected by many different aspects of cancer. The Foundation will continue with its activities in the future, especially since the problems associated with cancer affect more and more people in Slovenia and elsewhere. Ever more treatment that is suc­cessful reflects in results with longer survival in many patients with previously incurable cancer condi­tions. Thus adding many new dimensions in life of cancer survivors and their families. Borut Štabuc, M.D., Ph.D. Andrej Plesnicar, M.D., M.Sc. Viljem Kovac M.D., Ph.D. ONIVYDE pegylated liposomal je odobren za zdravljenje metastatskega adenokarcinoma trebušne slinavke v kombinaciji s 5-fluorouracilom (5-FU) in levkovorinom (LV) pri odraslih bolnikih, pri katerih je bolezen po zdravljenju na osnovi gemcitabina napredovala.1 ONIVYDE JE PEGILIRANI LIPOSOM Z IRINOTEKANOM, IZDELAN POSEBEJ ZA UCINKOVITO ZDRAVLJENJE TE AGRESIVNE BOLEZNI2–5 KLINICNI PODATKI ŠTUDIJE 3. FAZE POTRJUJEJO EDINSTVENO KLINICNO VREDNOST ZDRAVILA ONIVYDE V KOMBINACIJI S 5-FU/LV: 8 skladni podatki o ucinkovitosti pri vseh opazovanih dogodkih: pomembno podaljšanje preživetja in povecana stopnja odziva6–8 8 ohranjena kakovost življenja6,9 8 dobro poznan varnostni profil1,6,7 POMEMBNA UCINKOVITOST ONIVYDE + 5-FU/LV JE POTRJENA V KLINICNI PRAKSI10–12 ONIVYDE + 5-FU/LV PRIPOROCAJO VSE GLAVNE MEDNARODNE SMERNICE13–16 LITERATURA: 1. Povzetek glavnih znacilnosti zdravila ONIVYDE. 2. Lamb YN, Scott LJ. Drugs. 2017;77:785–792. 3. Drummond DC et al. Cancer Res. 2006;66:3271–3277. 4. Kalra AV et al. Cancer Res. 2014;74:7003–7013. 5. Carnevale J, Ko AH. Future Oncol 2016;12:453–464. 6. Wang-Gillam A et al. Lancet. 2016;387:545–557. 7. Wang-Gillam A et al. Eur J Cancer. 2019;108:78–87 Chen LT et al. Eur J Cancer. 2018;105:71–78. 9. Hubner RA et al. Eur J Cancer. 2019;106:24–33. 10. Kieler M et al. Ther Adv Med Oncol. 2019;11:1–13. 11. Yoo C et al. Ther Adv Med Oncol. 2019;11:1–9. 12. Pellino A et al. ESMO. 2019;P660. 13. Ducr al. Ann Oncol. 2015;25(suppl 5):v56–v68. 14. eUpdate Cancer of the Pancreas Treatment Recommendations. Published 2019. ESMO Guidelines Committee. Available at: https://www.esmo.org/Guidelines/Gastrointestinal-Cancers/Cancer Pancreas/eUpdate-Treatment-Recommendations. Last accessed June 2020. 15. Okusaka T et al. Pancreas. 2020;49(3):326–335 16. NCCN Guidelines Version 1, 2020. Pancreatic Adenocarcinoma. Available at https://www2.tri-kobe.org/nccn/guideline/ pancreas/english/pancreatic.pdf. Published November 26, 2019. Last accessed June 2020. SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA ONIVYDE pegylated liposomal 4,3 mg/ml SESTAVA*: ONIVYDE pegylated liposomal 4,3 mg/ml koncentrat za disperzijo za infundiranje: ena viala z 10 ml koncentrata vsebuje 43 mg brezvodnega irinotekana (v obliki irinotekanijeve soli saharoznega oktasulfata v pegilirani liposomski formulaciji). TERAPEVTSKE INDIKACIJE*: Zdravljenje metastatskega adenokarcinoma trebušne slinavke v kombinaciji s 5-fluorouracilom (5-FU) in levkovorinom (LV) pri odraslih bolnikih, pri katerih je bolezen po zdravljenju na osnovi gemcitabina napredovala. ODMERJANJE IN NACIN UPORABE*: ONIVYDE pegylated liposomal (irinotekan) smejo bolnikom predpisati in dajati samo zdravstveni delavci, ki imajo izkušnje pri uporabi zdravil za zdravljenje raka. Priporoceni odmerek in režim odmerjanja zdravila ONIVYDE pegylated liposomal je 70 mg/m2 intravensko 90 minut, cemur sledi LV 400 mg/m2 intravensko 30 minut in nato 5FU 2400 mg/m2 intravensko 46 ur, vsaka 2 tedna. Zdravilo ONIVYDE pegylated liposomal se ne daje kot samostojno zdravilo. Pri bolnikih z znano homozigotnostjo za alel UGT1A1*28 je treba razmisliti o manjšem zacetnem odmerku zdravila ONIVYDE pegylated liposomal 50 mg/m2. Ce zdravilo bolniki dobro prenašajo, lahko v naslednjih ciklih razmislimo o odmerku zdravila ONIVYDE pegylated liposomal 70 mg/m2. Prilagajanje odmerkov se priporoca za obvladovanje toksicnosti 3. ali 4. stopnje, povezane z zdravilom ONIVYDE pegylated liposomal. KONTRAINDIKACIJE*: Anamneza hude preobcutljivosti na irinotekan ali katero koli pomožno snov. Dojenje. OPOZORILA*: Zdravilo ONIVYDE pegylated liposomal (irinotekan) ni enakovredno drugim neliposomskim formulacijam irinotekana, zato jih ne smemo zamenjevati. Mielosupresija/nevtropenija: Med zdravljenjem se priporoca nadziranje celotne krvne slike. Bolniki se morajo zavedati tveganja za nevtropenijo in pomena povišane telesne temperature. Febrilno nevtropenijo je treba nujno zdraviti v bolnišnici s širokospektralnimi intravenskimi antibiotiki. Pri bolnikih, ki doživijo hude hematološke neželene ucinke, se priporoca zmanjšanje odmerka ali prekinitev zdravljenja. Bolnikov s hudo odpovedjo kostnega mozga ne smemo zdraviti z zdravilom ONIVYDE pegylated liposomal. Imunosupresivni ucinki in cepiva: Dajanje živih ali atenuiranih cepiv bolnikom z oslabljenim imunskim sistemom lahko povzroci resne ali smrtne okužbe. Bolniki azijskega porekla imajo vecje tveganje za hudo in febrilno nevtropenijo. Posamezniki s homozigotnostjo 7/7 za alel UGT1A1*28 imajo povecano tveganje za nevtropenijo. Interakcije z mocnimi induktorji encima CYP3A4, mocnimi zaviralci encima CYP3A4 in mocnimi zaviralci encima UGT1A1: Zdravila ONIVYDE pegylated liposomal ne smemo dajati skupaj z mocnimi induktorji encima CYP3A4 (kot so antikonvulzivi, rifampicin, rifabutin in šentjanževka), mocnimi zaviralci encima CYP3A4 (npr. grenivkinim sokom, klaritromicinom, indinavirjem, itrakonazolom, lopinavirjem, nefazodonom, nelfinavirjem, ritonavirjem, sakvinavirjem, telaprevirjem, vorikonazolom) ali z mocnimi zaviralci encima UGT1A1, razen ce ni drugih terapevtskih možnosti. Zdravljenje z mocnimi zaviralci encima CYP3A4 moramo prekiniti vsaj 1 teden pred zacetkom zdravljenja z zdravilom ONIVYDE pegylated liposomal. Driska: Pri bolnikih, ki doživijo zgodnji pojav driske (v = 24 urah po zacetku zdravljenja z zdravilom ONIVYDE pegylated liposomal), je treba razmisliti o terapevtskem in profilakticnem zdravljenju z atropinom, razen ce je kontraindicirano. Bolnike je treba opozoriti na tveganje za zapoznelo drisko (> 24 ur), ki je izcrpavajoca in v redkih primerih tudi življenjsko nevarna. Ce driska traja tudi, ko bolnik prejema loperamid vec kot 24 ur, je treba razmisliti o dodatni peroralni antibioticni podpori. Zdravljenje z zdravilom ONIVYDE pegylated liposomal je treba odložiti, dokler se driska ne umiri do = 1. stopnje (2–3 odvajanja/dan vec kot pred zdravljenjem). Zdravila ONIVYDE pegylated liposomal ne smemo dajati bolnikom z zaporo crevesja ali kronicno vnetno crevesno boleznijo, dokler se ta ne pozdravi. Holinergicne reakcije: Zgodnjo drisko lahko spremljajo rinitis, povecano slinjenje, zardevanje, diaforeza, bradikardija, mioza in hiperperistaltika. Uporabiti je treba atropin. Akutne infuzijske in povezane reakcije: V primeru hudih preobcutljivostnih reakcij je treba zdravljenje z zdravilom ONIVYDE pegylated liposomal prekiniti. Predhodna Whipplova operacija: Vecje tveganje za resne okužbe. Bolnike je treba spremljati glede znakov okužbe. Žilne bolezni: Zdravilo ONIVYDE pegylated liposomal je bilo povezano s trombembolicnimi dogodki, kot so pljucna embolija, venska tromboza in arterijska trombembolija. Treba je pridobiti podrobno zdravstveno anamnezo, da bi prepoznali bolnike z vec dejavniki tveganja poleg osnovne neoplazme. Bolnike je treba obvestiti o znakih in simptomih trombembolije in jim svetovati, da se v primeru katerega od teh znakov ali simptomov takoj obrnejo na svojega zdravnika ali medicinsko sestro. Pljucna toksicnost: Pri bolnikih, ki so prejemali neliposomski irinotekan, so se pojavili dogodki, podobni intersticijski pljucni bolezni (IPB), ki so vodili do smrtnih primerov. Pri bolnikih z dejavniki tveganja (obstojeco pljucno boleznijo, uporabo pnevmotoksicnih zdravil, kolonije stimulirajocimi dejavniki ali predhodnim zdravljenjem z obsevanjem) je treba pred zdravljenjem z zdravilom ONIVYDE pegylated liposomal in po njem skrbno nadzirati respiratorne simptome. Dokler ni opravljena diagnosticna ocena, je treba ob pojavu nove ali napredovale dispneje, kašlja in povišane telesne temperature zdravljenje z zdravilom ONIVYDE pegylated liposomal zacasno prekiniti. Pri bolnikih s potrjeno diagnozo IPB moramo zdravljenje z zdravilom ONIVYDE pegylated liposomal dokoncno prekiniti. Jetrna okvara: Bolniki s hiperbilirubinemijo so imeli povišane koncentracije skupnega SN-38, zato je tveganje za nevtropenijo povecano. Pri bolnikih z vrednostjo skupnega bilirubina 1,0–2,0 mg/dl je treba redno nadzirati celotno krvno sliko. Previdnost je potrebna pri bolnikih z jetrno okvaro (bilirubin > 2-kratna zgornja meja normalnih vrednosti [ULN]; aminotransferaze > 5-kratna ULN). Ledvicna okvara: Uporaba zdravila pri bolnikih s pomembno ledvicno okvaro ni bila ocenjena. Bolniki s premajhno telesno maso (indeks telesne mase < 18,5 kg/m2): Potrebna je previdnost. Pomožne snovi: En mililiter zdravila ONIVYDE pegylated liposomal vsebuje 0,144 mmol (3,31 mg) natrija. INTERAKCIJE*: Previdnostni ukrepi: Socasno dajanje z induktorji encima CYP3A4 lahko zmanjša sistemsko izpostavljenost zdravilu ONIVYDE pegylated liposomal. Socasno dajanje z zaviralci encima CYP3A4 ali encima UGT1A1 (npr. atazanavirja, gemfibrozila, indinavirja, regorafeniba) lahko poveca sistemsko izpostavljenost zdravilu ONIVYDE pegylated liposomal. PLODNOST* NOSECNOST*: Uporaba ni priporocljiva. DOJENJE*: Zdravilo je kontraindicirano. KONTRACEPCIJA*: Ženske v rodni dobi morajo med zdravljenjem in še 1 mesec po zdravljenju uporabljati ucinkovito kontracepcijo. Moški morajo med zdravljenjem in 4 mesece po zdravljenju uporabljati kondome. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA STROJEV*: Bolniki morajo biti med zdravljenjem pri vožnji in upravljanju strojev previdni. NEŽELENI UCINKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, hipokaliemija, hipomagneziemija, dehidracija, zmanjšan apetit, omotica, driska, bruhanje, navzea, bolecine v trebuhu, stomatitis, alopecija, pireksija, periferni edem, vnetje sluznic, utrujenost, astenija, zmanjšana telesna masa. Pogosti: septicni šok, sepsa, pljucnica, febrilna nevtropenija, gastroenteritis, oralna kandidoza, limfopenija, hipoglikemija, hiponatriemija, hipofosfatemija, nespecnost, holinergicni sindrom, dizgevzija, hipotenzija, pljucna embolija, embolija, globoka venska tromboza, dispneja, disfonija, kolitis, hemoroidi, hipoalbuminemija, akutna ledvicna odpoved, z infuzijo povezana reakcija, edem, zvišana raven bilirubina, zvišana raven alanin-aminotransferaze, zvišana raven aspartat-aminotransferaze, zvišano mednarodno umerjeno razmerje. Obcasni: biliarna sepsa, preobcutljivost, tromboza, hipoksija, ezofagitis, proktitis, makulopapulozni izpušcaj, obarvanje nohtov. PREVELIKO ODMERJANJE*: Za preveliko odmerjanje zdravila ni znanega antidota. Treba je uvesti maksimalno podporno nego, s katero preprecimo dehidracijo zaradi driske in zdravimo zaplete zaradi okužb. FARMAKODINAMICNE LASTNOSTI*: Ucinkovina zdravila ONIVYDE pegylated liposomal je irinotekan (zaviralec topoizomeraze I), inkapsuliran v vezikel z lipidnim dvoslojem oziroma liposom. Irinotekan je derivat kamptotecina. Kamptotecini delujejo kot specificni zaviralci encima DNA-topoizomeraza I. Irinotekan in njegov aktivni presnovek SN-38 se reverzibilno vežeta na kompleks topoizomeraze I in DNA ter sprožita poškodbe v enoverižni DNA, kar zaustavi replikacijske vilice pri podvajanju DNA in povzroca citotoksicnost. Irinotekan se presnavlja s karboksilesterazo do SN-38. SN-38 je približno 1.000-krat mocnejši zaviralec topoizomeraze I, ocišcene iz tumorskih celicnih linij cloveka in glodavcev, kot irinotekan. PAKIRANJE*: Pakiranje vsebuje eno vialo z 10 ml koncentrata. NACIN PREDPISOVANJA IN IZDAJE ZDRAVILA: Rp/Spec. DATUM ZADNJE REVIZIJE BESEDILA: avgust 2020. Imetnik dovoljenja za promet: Les Laboratoires Servier, 50, rue Carnot, 92284 Suresnes cedex, Francija. Številka dovoljenja za promet z zdravilom: EU/1/16/1130/001. *Pred predpisovanjem preberite celoten povzetek glavnih znacilnosti zdravila. Celoten povzetek glavnih znacilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., Podmilšcakova ulica 24, 1000 Ljubljana, tel: 01 563 48 11, www.servier.si. Samo za strokovno javnost. Datum priprave informacije: avgust 2020. ONI AD1 C1 20-21 glave in vratu1 References: 1. Keytruda EU SmPC SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih znacilnosti zdravila! Ime zdravila: KEYTRUDA 25 mg/ml koncentrat za raztopino za infundiranje vsebuje pembrolizumab. Terapevtske indikacije: Zdravilo KEYTRUDA je kot samostojno zdravljenje indicirano za zdravlje nje: napredovalega (neoperabilnega ali metastatskega) melanoma pri odraslih; za adjuvantno zdravljenje odraslih z melanomom v stadiju III, ki se je razširil na bezgavke, po popolni kirurški odstranitvi; metastatskega nedrobnocelicnega pljucnega raka (NSCLC) v prvi liniji zdravljenja pri odraslih, ki imajo tumorje z = 50 % izraženostjo PD-L1 (TPS) in brez pozitivnih tumorskih mutacij EGFR ali ALK; lokalno napredovalega ali metastatskega NSCLC pri odraslih, ki imajo tumorje z = 1 % izraženostjo PD-L1 (TPS) in so bili predhodno zdravljeni z vsaj eno shemo kemoterapije, bolniki s pozitivnimi tumorskimi mutacijami EGFR ali ALK so pred prejemom zdravila KEYTRUDA morali prejeti tudi tarcno zdravljenje; odraslih bolnikov s ponovljenim ali neodzivnim klasicnim Hodgkino vim limfomom (cHL), pri katerih avtologna presaditev maticnih celic (ASCT) in zdravljenje z brentu ksimabom vedotinom (BV) nista bila uspešna, in odraslih bolnikov, ki za presaditev niso primerni, zdravljenje z BV pa pri njih ni bilo uspešno; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, predhodno zdravljenih s kemoterapijo, ki je vkljucevala platino; lokalno napredova lega ali metastatskega urotelijskega raka pri odraslih, ki niso primerni za zdravljenje s kemoterapijo, ki vsebuje cisplatin in imajo tumorje z izraženostjo PD-L1 = 10, ocenjeno s kombinirano pozitivno oceno (CPS); ponovljenega ali metastatskega plošcatocelicnega raka glave in vratu (HNSCC) pri odraslih, ki imajo tumorje z = 50 % izraženostjo PD-L1 (TPS), in pri katerih je bolezen napredovala med zdravljenjem ali po zdravljenju s kemoterapijo, ki je vkljucevala platino. Zdravilo KEYTRUDA je kot samostojno zdravljenje ali v kombinaciji s kemoterapijo s platino in 5-fluorouracilom (5-FU) indicirano za prvo linijo zdravljenja metastatskega ali neoperabilnega ponovljenega plošcatocelic nega raka glave in vratu pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS = 1. Zdravilo KEYTRUDA je v kombinaciji s pemetreksedom in kemoterapijo na osnovi platine indicirano za prvo linijo zdravljenja metastatskega neplošcatocelicnega NSCLC pri odraslih, pri katerih tumorji nimajo pozitivnih mutacij EGFR ali ALK; v kombinaciji s karboplatinom in bodisi paklitakselom bodisi nab -paklitakselom je indicirano za prvo linijo zdravljenja metastatskega plošcatocelicnega NSCLC pri odraslih; v kombinaciji z aksitinibom je indicirano za prvo linijo zdravljenja napredovalega raka ledvic nih celic (RCC) pri odraslih. Odmerjanje in nacin uporabe: Testiranje PD-L1 pri bolnikih z NSCLC, urotelijskim rakom ali HNSCC: Za samostojno zdravljenje z zdravilom KEYTRUDA je priporocljivo opraviti testiranje izraženosti PD-L1 tumorja z validirano preiskavo, da izberemo bolnike z NSCLC ali predhodno nezdravljenim urotelijskim rakom. Bolnike s HNSCC je treba za samostojno zdravljenje z zdravilom KEYTRUDA ali v kombinaciji s kemoterapijo s platino in 5-fluorouracilom (5-FU) izbrati na podlagi izraženosti PD-L1, potrjene z validirano preiskavo. Odmerjanje: Priporoceni odmerek zdravila KEYTRUDA za samostojno zdravljenje je bodisi 200 mg na 3 tedne ali 400 mg na 6 tednov, apliciran z intravensko infuzijo v 30 minutah. Priporoceni odmerek za kombinirano zdravljenje je 200 mg na 3 tedne, apliciran z intravensko infuzijo v 30 minutah. Za uporabo v kombinaciji glejte povzetke glavnih znacilnosti socasno uporabljenih zdravil. Ce se uporablja kot del kombiniranega zdravljenja skupaj z intravensko kemoterapijo, je treba zdravilo KEYTRUDA aplicirati prvo. Bolnike je treba zdraviti do napredovanja bolezni ali nesprejemljivih toksicnih ucinkov. Pri adjuvantnem zdra vljenju melanoma je treba zdravilo uporabljati do ponovitve bolezni, pojava nesprejemljivih toksicnih ucinkov oziroma mora zdravljenje trajati do enega leta. Ce je aksitinib uporabljen v kombinaciji s pembrolizumabom, se lahko razmisli o povecanju odmerka aksitiniba nad zacetnih 5 mg v presledkih šest tednov ali vec. Pri bolnikih starih = 65 let, bolnikih z blago do zmerno okvaro ledvic, bolnikih z blago okvaro jeter prilagoditev odmerka ni potrebna. Odložitev odmerka ali ukinitev zdravljenja: Zmanjšanje odmerka zdravila KEYTRUDA ni priporocljivo. Za obvladovanje neželenih ucinkov je treba uporabo zdravila KEYTRUDA zadržati ali ukiniti, prosimo, glejte celoten Povzetek glavnih znacilnosti zdravila. Kontraindikacije: Preobcutljivost na ucinkovino ali katero koli pomožno snov. Povzetek posebnih opozoril, previdnostnih ukrepov, interakcij in neželenih ucinkov: Imunsko pogojeni neželeni ucinki (pnevmonitis, kolitis, hepatitis, nefritis, endokrinopatije, neželeni ucinki na kožo in drugi): Pri bolnikih, ki so prejemali pembrolizumab, so se pojavili imunsko pogojeni neželeni ucinki, vkljucno s hudimi in smrtnimi primeri. Vecina imunsko pogojenih neželenih ucinkov, ki so se pojavili med zdravljenjem s pembrolizumabom, je bila reverzibilnih in so jih obvladali s prekinitvami uporabe pembrolizumaba, uporabo kortikosteroidov in/ali podporno oskrbo. Pojavijo se lahko tudi po zadnjem odmerku pembrolizumaba in hkrati prizadanejo vec organskih sistemov. V primeru suma na imunsko pogojene neželene ucinke je treba poskrbeti za ustrezno oceno za potrditev etiologije oziro ma izkljucitev drugih vzrokov. Glede na izrazitost neželenega ucinka je treba zadržati uporabo pem brolizumaba in uporabiti kortikosteroide za natancna navodila, prosimo, glejte Povzetek glavnih znacilnosti zdravila Keytruda. Zdravljenje s pembrolizumabom lahko poveca tveganje za zavrnitev pri prejemnikih presadkov cvrstih organov. Pri bolnikih, ki so prejemali pembrolizumab, so porocali o hudih z infuzijo povezanih reakcijah, vkljucno s preobcutljivostjo in anafilaksijo. Pembrolizumab se iz obtoka odstrani s katabolizmom, zato presnovnih medsebojnih delovanj zdravil ni pricakovati. Uporabi sistemskih kortikosteroidov ali imunosupresivov pred uvedbo pembrolizumaba se je treba izogibati, ker lahko vplivajo na farmakodinamicno aktivnost in ucinkovitost pembrolizumaba. Vendar pa je kortikosteroide ali druge imunosupresive mogoce uporabiti za zdravljenje imunsko pogojenih neželenih ucinkov. Kortikosteroide je mogoce uporabiti tudi kot premedikacijo, ce je pembrolizumab uporabljen v kombinaciji s kemoterapijo, kot antiemeticno profilakso in/ali za ublažitev neželenih ucinkov, povezanih s kemoterapijo. Ženske v rodni dobi morajo med zdravljenjem s pembrolizuma bom in vsaj še 4 mesece po zadnjem odmerku pembrolizumaba uporabljati ucinkovito kontracepcijo, med nosecnostjo in dojenjem se ga ne sme uporabljati. Varnost pembrolizumaba pri samostojnem zdravljenju so v klinicnih študijah ocenili pri 5.884 bolnikih z napredovalim melanomom, kirurško odstranjenim melanomom v stadiju III (adjuvantno zdravlje nje), NSCLC, cHL, urotelijskim rakom ali HNSCC s štirimi odmerki (2 mg/kg na 3 tedne, 200 mg na 3 tedne in 10 mg/kg na 2 ali 3 tedne). V tej populaciji bolnikov je mediani cas opazovanja znašal 7,3 mesece (v razponu od 1 dneva do 31 mesecev), najpogostejši neželeni ucinki zdravljenja s pembro lizumabom so bili utrujenost (32 %), navzea (20 %) in diareja (20 %). Vecina porocanih neželenih ucinkov pri samostojnem zdravljenju je bila po izrazitosti 1. ali 2. stopnje. Najresnejši neželeni ucinki so bili imunsko pogojeni neželeni ucinki in hude z infuzijo povezane reakcije. Varnost pembrolizuma ba pri kombiniranem zdravljenju s kemoterapijo so ocenili pri 1.067 bolnikih NSCLC ali HNSCC, ki so v klinicnih študijah prejemali pembrolizumab v odmerkih 200 mg, 2 mg/kg ali 10 mg/kg na vsake 3 tedne. V tej populaciji bolnikov so bili najpogostejši neželeni ucinki naslednji: anemija (50 %), navzea (50 %), utrujenost (37 %), zaprtost (35%), diareja (30 %), nevtropenija (30 %), zmanjšanje apetita (28 %) in bruhanje (25 %). Pri kombiniranem zdravljenju s pembrolizumabom je pri bolnikih z NSCLC pojavnost neželenih ucinkov 3. do 5. stopnje znašala 67 %, pri zdravljenju samo s kemoterapijo pa 66 %, pri kombiniranem zdravljenju s pembrolizumabom pri bolnikih s HNSCC 85 % in pri zdravljenju s kemoterapijo v kombinaciji s cetuksimabom 84 %. Varnost pembrolizumaba v kombinaciji z aksiti nibom so ocenili v klinicni študiji pri 429 bolnikih z napredovalim rakom ledvicnih celic, ki so prejemali 200 mg pembrolizumaba na 3 tedne in 5 mg aksitiniba dvakrat na dan. V tej populaciji bolnikov so bili najpogostejši neželeni ucinki diareja (54 %), hipertenzija (45 %), utrujenost (38 %), hipotiroidizem (35 %), zmanjšan apetit (30 %), sindrom palmarno-plantarne eritrodisestezije (28 %), navzea (28 %), zvišanje vrednosti ALT (27 %), zvišanje vrednosti AST (26 %), disfonija (25 %), kašelj (21 %) in zaprtost (21 %). Pojavnost neželenih ucinkov 3. do 5. stopnje je bila med kombiniranim zdravljenjem s pembrolizumabom 76 % in pri zdravljenju s sunitinibom samim 71 %. Za celoten seznam neželenih ucinkov, prosimo, glejte celoten Povzetek glavnih znacilnosti zdravila Nacin in režim izdaje zdravila: H Predpisovanje in izdaja zdravila je le na recept, zdravilo se uporablja samo v bolnišnicah. Imetnik dovoljenja za promet z zdravilom: Merck Sharp & Dohme B.V. , Waarderweg 39, 2031 BN Haarlem, Nizozemska. Merck Sharp & Dohme inovativna zdravila d.o.o., Šmartinska cesta 140, 1000 Ljubljana, tel: +386 1/ 520 42 01, fax: +386 1/ 520 43 50 Pripravljeno v Sloveniji, September 2020; SI-KEY-00145 EXP: 09/2022 Samo za strokovno javnost. H - Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih znacilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. BOLNIKU PRILAGOJENEGA ZDRAVLJENJA2-7 Odkrijte možnosti visoko kakovostnih storitev obširnega genomskega profiliranja FoundationOne®, ki olajšajo odlocitev o najustreznejšem zdravljenju za posameznega bolnika z rakom, v razlicnih klinicnih stanjih. 5-7 M-SI-00000141 (v1.0) Viri: 1. FDA Approves Liquid Biopsy Next-Generation Sequencing Companion Diagnostic Test; dostopano oktober 2020 na: https://www.fda.gov/drugs/drug-approvals-and­databases/fda-approves-liquid-biopsy-next-generation-sequencing-companion-diagnostic-test. 2. Frampton GM s sod. Nat Biotechnol 2013; 31:1023-1031. 3. Clark TA s sod. JMol Diagn 2018; 20:686-702. 4. He J s sod. Blood 2016; 127:3004-3014. 5. FoundationOne® CDx Technical Specifications; dostopano oktober 2020 na: https://assets.ctfassets.net/w98cd481qyp0/YqqKHaqQmFeqc5ueQk48w/0a34fcdaa3a71dbe460cdcb01cebe8ad/F1CDx_Technical_Specifications_072020.pdf. 6. FoundationOne® Liquid Technical Specifications; dostopano oktober 2020 na: https://assets.ctfassets.net/w98cd481qyp0/wVEm7VtICYR0sT5C1VbU7/cc6ac2109785d70fe6d91903b241006f/FoundationOne_Liquid CDx Technical Specifications.pdf. 7. FoundationOne® Heme Technical Specifications; dostopano oktober 2020 na: https://assets.ctfassets.net/w98cd481qyp0/42r1cTE8VR4137CaHrsaen/baf91080cb3d78a52ada10c6358fa130/FoundationOne_Heme_Technical_Specifications.pdfff8b70e90c292182/F1H_TechnicalInformation.pdf CDx - spremljevalna diagnostika, FDA - Uprava ZDA za hrano in zdravila (Food and Drug Administration) Informacija pripravljena: oktober 2020. Samo za strokovno javnost. DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o., Stegne 13g, 1000 Ljubljana rocheprotiraku.si instructions Instructions for authors The editorial policy Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers and review articles, pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, medical physics and radiation protection. 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The following are some examples of references from articles, books and book chapters: Dent RAG, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. doi: 10.1038/bjc.1981.71 Chapman S, Nakielny R. A guide to radiological procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology of macrophage. New York: Academic Press; 1976. p. 45-74. Authorization for the use of human subjects or experimental animals When reporting experiments on human subjects, authors should state whether the procedures followed the Helsinki Declaration. Patients have the right to privacy; therefore the identifying information (patient’s names, hospital unit numbers) should not be published unless it is essential. 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