MASARYKOVA UNIVERZITA LÉKAŘSKÁ FAKULTA KLINIKA DĚTSKÉ ONKOLOGIE Diagnostické a léčebné pokroky v dětské onkologii Habilitační práce v oboru onkologie Autor: MUDr. Peter Múdry, Ph.D. Brno 2020 2 3 Diagnostické a léčebné pokroky v dětské onkologii Obsah Komentář ......................................................................................................................... 6 Commentary..................................................................................................................... 8 1 Inovativní léčebné postupy u vysoce rizikových malignit dětského věku .................... 10 1.1 Úvod..................................................................................................................................10 1.2 Personalizovaná onkologie .................................................................................................10 1.3 Klinická hodnocení nových léčiv v dětské onkologii .............................................................12 1.4 Nové metody získávání dat v personalizované onkologii......................................................13 1.5 „Drug repurposing“............................................................................................................14 1.6 Léčebné modality...............................................................................................................15 1.6.1 Tyrozinkinázové inhibitory .............................................................................................................. 15 1.6.2 Check-point inhibitory..................................................................................................................... 16 1.6.3 Jiné protilátkové imunoterapie ....................................................................................................... 17 1.6.4 Buněčné terapie .............................................................................................................................. 18 1.6.5 Neschválené terapie ve fázi klinického vývoje ................................................................................ 19 1.6.6 Antiangiogenní strategie ................................................................................................................. 20 1.7 Inovativní léčebné postupy na Klinice dětské onkologie LF MU a FN Brno.............................20 1.7.1 Imunoterapie dendritickou vakcínou .............................................................................................. 20 1.7.2 Personalizovaná léčba..................................................................................................................... 22 1.7.3 Antiangiogenní a metronomická léčba ........................................................................................... 24 1.7.4 Tyrozinkinázové inhibitory .............................................................................................................. 25 1.7.5 Rekurentní laryngeální papilomatosa ............................................................................................. 27 1.8 Souhrn...............................................................................................................................28 4 2 Antimykotická léčba u imunokompromitovaných pacientů ....................................... 29 2.1 Úvod..................................................................................................................................29 2.2 Komentář k publikovaným pracím ......................................................................................30 2.3 Souhrn...............................................................................................................................32 3 Léčba sarkomů měkkých tkání dětského věku........................................................... 33 3.1 Úvod..................................................................................................................................33 3.2 Epidemiologie a etiologie ...................................................................................................33 3.3 Lokální kontrola .................................................................................................................35 3.4 Systémová léčba ................................................................................................................36 3.5 Sarkomy měkkých tkání typické pro děti a adolescenty a jejich léčba...................................36 3.5.1 Rabdomyosarkom (RMS)................................................................................................................. 36 3.5.2 Infantilní fibrosarkom...................................................................................................................... 39 3.5.3 Inflamatorní myofibroblastický tumor (IMT) .................................................................................. 40 3.5.4 Synovialosarkom ............................................................................................................................. 42 3.5.5 Alveolární sarkom měkkých tkání ................................................................................................... 42 3.5.6 Světlobuněčný sarkom .................................................................................................................... 42 3.5.7 Desmoplastický kulatobuněčný nádor ............................................................................................ 43 3.5.8 Extrakraniální maligní rhabdoidní tumor ........................................................................................ 43 3.5.9 Epiteloidní sarkom........................................................................................................................... 43 3.6 Mezinárodní kooperativní skupina EpSSG ...........................................................................44 3.7 Komentář k publikovaným pracím ......................................................................................45 3.7.1 Udržovací terapie u rabdomyosarkomu.......................................................................................... 45 3.7.2 Prognostický vliv genové fúze u alveolárních rabdomyosarkomů s postižením regionálních lymfatických uzlin......................................................................................................................................... 46 3.7.3 Strategie léčby infantilních fibrosarkomů. ...................................................................................... 48 5 3.7.4 Léčba maligních rhabdoidních tumorů............................................................................................ 49 3.8 Souhrn...............................................................................................................................50 4 Seznam zkratek........................................................................................................ 51 5 Seznam příloh .......................................................................................................... 53 příloha č. 1 ....................................................................................................................................................56 příloha č. 2 ....................................................................................................................................................71 příloha č. 3 ....................................................................................................................................................83 příloha č. 4 ....................................................................................................................................................92 příloha č. 5 ..................................................................................................................................................106 příloha č. 6 ..................................................................................................................................................125 příloha č. 7 ..................................................................................................................................................131 příloha č. 8 ..................................................................................................................................................143 příloha č. 9 ..................................................................................................................................................150 příloha č. 10 ................................................................................................................................................166 příloha č. 11 ................................................................................................................................................167 příloha č. 12 ................................................................................................................................................170 příloha č. 13 ................................................................................................................................................183 příloha č. 14 ................................................................................................................................................192 příloha č. 15 ................................................................................................................................................199 příloha č. 16 ................................................................................................................................................202 příloha č. 17 ................................................................................................................................................212 příloha č. 18 ................................................................................................................................................221 příloha č. 19 ................................................................................................................................................230 6 Komentář ÚVOD: Po relativně stabilním období přichází v poslední dvou desetiletích rychlý vývoj diagnostických a molekulárních postupů v dětské onkologii. Zvláště je tento vývoj patrný jednak na implementaci molekulárně genetických metod do klinické praxe, a také na dostupnosti nových léčiv na jiných principech než dosud standardní chemoterapie. CÍLE: Cílem práce je podat přehled ve vývoji těchto metod za poslední dvě desetiletí a popsat na komentovaném souboru prací jejich přínos pro klinickou praxi. SOUBOR PACIENTŮ A METODY: Inovativní léčebné postupy, diagnostika a terapie invazivních mykóz a léčba sarkomů měkkých tkání jsou tři oblasti, které byly extenzivně zkoumány v publikovaných pracích. Na Klinice dětské onkologie Lékařské fakulty Masarykovy univerzity a Fakultní nemocnice Brno bylo v letech 2000–2020 více projektů, které se zabývaly uvedenými tématy. Pokrývají spektrum metronomické antiangiogenní terapie, použití tyrozinkinázových inhibitorů v nových indikacích, somatobuněčnou terapii autologními dendritickými buňkami, diagnostiky a terapie invazivních mykóz a diagnostické a terapeutické postupy u sarkomů měkkých tkání u dětí. VÝSLEDKY: Mnohá léčiva v kategorii inovativních postupů byla použita zcela poprvé, resp. s nejlepším publikovaným výsledkem, jako např. vakcinace proti HPV z indikace rekurentní laryngeální papilomatózy nebo léčba infantilní myofibromatózy u pacienta s germinální mutací PDGFRB genu. Zavedená antiangiogenní metronomická terapie má přínos pro pacienty s vysoce rizikovými nádory, personalizace léčby vedla i k neočekávaným kurativním výsledkům, které nebylo možné očekávat při použití konvenční léčby. Participace na projektech diagnostiky a léčby invazivních mykóz vedla ke kvalitní péči o tyto pacienty, jak je dokumentováno i v publikovaných pracích, i za použití inovativních léčebných postupů mimo klinická hodnocení. Rozvoj somatobuněčné terapie je spojen s novými technologickými postupy výroby dendritické vakcíny a se standardizací průběhu klinických hodnocení tohoto typu, ve formě protokolu KDO DC1311, dosud jediného provedeného u dětí do 18 let v České republice. Nové poznatky, které jsou tímto klinickým hodnocením získány, jsou inovativní a přispívají k dalšímu rozvoji této slibné léčebné metody. Participace na projektech mezinárodních akademických klinických studií diagnostiky a léčby sarkomů měkkých tkání vedly k velmi cenným výsledkům ve formě standardizace doporučených léčebných postupů u těchto diagnóz, k prokázání účinnosti udržovací léčby u rabdomyosarkomu jako první takový 7 důkaz u solidních tumorů, k nové stratifikaci léčby u alveolárních rabdomyosarkomů s postižením lymfatických uzlin a k dosud nejlepším léčebným výsledkům u extrakraniálních maligních rabdoidních tumorů. ZÁVĚR: V publikovaných pracích jsou shrnuty recentní výsledky rozvoje diagnostických metod a léčby u malignit dětského věku se zaměřením na vysoce rizikové pacienty bez možnosti konvenční léčby i standardizace léčebných postupů u invazivních mykóz a sarkomů měkkých tkání při participaci v mezinárodních multicentrických prospektivních akademických klinických hodnoceních. 8 Commentary INTRODUCTION: A quite standard treatments known two decades ago were followed by rapid progress in molecular diagnostics and treatment possibilities in paediatric oncology. New moleculal biology methods and new treatments based mainly on theranostics principles are used in daily practice. OBJECTIVES: The aim of this work is to review how far the paediatric oncology was updated during last two decades and to show this framework in commentary of published papers. PATIENTS AND METHODS: Innovative therapies, diagnostics and treatment of invasive fungal infections and treatment of soft tissue sarcomas are areas covered by published articles. There are a lot of projects on Paediatric Oncology Department of School of Medicine Masaryk University and University Hospital Brno during period 2000-2020, which cover topics as metronomic antiangiogenic therapy, tyrosinkinase inhibitors in new therapeutic indications, cell therapies with autologous dendritic cells, new therapeutic strategies of invasive fungal infections and diagnostics and therapeutic approaches in soft tissue sarcomas in children. RESULTS: Many of innovative therapies were used for first time ever or with the best results published. This is the case of human papillomavirus vaccination against recurrent laryngeal papillomatosis or successful management of infantile myofibromatosis in patients with germline mutation of PDGFRB gene with tyrosinkinase inhibitor. Introduced antiangiogenic metronomic therapy is successfully used in patients with high risk tumours. Personalised therapies were administered to patients with sometimes surprising results even curative which were not anticipated with conventional approach. Setting quality of diagnostic and therapeutic tools in patients with invasive fungal infections was substantial for good treatment results and was accompanied by novel therapies used off-label and off-clinical trial as published. New cell therapy with dendritic cells was introduced and this accelerated rapid biotechnology development and proper academic trial administration in era of new and tight law regulations. KDO DC 1311 trial is the only open for children in the Czech Republic, this trial is still active. It generated some new knowledges on monocyte response to anticancer therapies or host immune response against cancer which are used in clinical practice and support further development of such promising method. Multicentric prospective international clinical trials 9 on diagnostics and treatment of soft tissue sarcomas were essential for generating of standardized therapy across Europe, new treatments such as maintenance chemotherapy in rhabdomyosarcoma which is the first robust evidence about its efficacy in solid tumours, new treatment stratification in alveolar rhabdomyosarcomas with regional nodal involvement and improvement treatment of highly aggressive malignant rhabdoid tumours. CONCLUSIONS: Recent progress in diagnostics and therapies in childhood malignancies are described in published articles with focus to high risk patients without possibility of conventional treatment and to standardization of treatment of invasive fungal diseases and soft tissue sarcomas while participating in international multicentric prospective academic driven clinical trials. 10 1 Inovativní léčebné postupy u vysoce rizikových malignit dětského věku 1.1 Úvod Léčebné výsledky jsou u malignit dětského věku jako celku velmi dobré. Kurativní postupy vedou k trvalému vyléčení u více než 80 % dětských pacientů, byť za cenu jisté míry pozdních následků, které si s sebou pacienti do života odnáší. Existují ovšem velké rozdíly v přežití malignity mezi jednotlivými diagnostickými skupinami, které při celkovém hodnocení malignit dětského věku jako celku nejsou, z důvodu rozdílné incidence, na první pohled patrné: ve skupině akutních lymfoblastických leukémií, Wilmsových nádorů, germinálních nádorů, nízce rizikových sarkomů se úspěšnost léčby blíží 90 %. Naopak gliomy mozkového kmene, glioblastomy, metastatické sarkomy skeletu a měkkých tkání nebo relabované Burkittovy lymfomy jsou léčitelné v malém procentu případů s mediánem přežití do šesti měsíců. Právě tato skupina malignit je předmětem zájmu inovativních léčebných postupů. V posledních letech významným způsobem pokročilo poznání biologie nádorových onemocnění, které vedlo k identifikaci charakteristických znaků („hallmarks“) nádorů, poskytujících nádorové buňce a tkáni růstovou výhodu. Tyto aberace se v posledních letech snažíme léčebně ovlivňovat, a to tak, že pečlivě vyšetřeným pacientům je nabízena terapie na základě přítomnosti či nepřítomnosti určitých aberací či biomarkerů. 1.2 Personalizovaná onkologie Základní premisou personalizované onkologie je, že každý nádor je biologicky unikátní a v čase se může i velmi významně měnit. Množství poznatků o biologii nádorů, jejich genetickém pozadí, imunologických aspektech jejich vzniku a možnostech terapie rychle narůstá. Je pravděpodobné, že tradiční velké, tisícihlavé randomizované prospektivní studie budou často obsoletní dříve, než budou samy dokončeny. Toto je v případě léčby sarkomů známo u mnoha „nadějných“ kinázových inhibitorů, jako např. recentně u olaratumabu. Klasická paradigmata klinického výzkumu v onkologii začínají respektovat realitu, dokumentovanou obrovskou genetickou a epigenetickou heterogenitou morfologicky podobně vypadajících procesů. Tyto 11 informace dříve nebyly k dispozici. Dnes reflektujeme individuální variabilitu hostitele, tedy nositele nádorového onemocnění, a tím potřebu indikovat pacientovi v podstatě „léčbu na míru“, než abychom se drželi překonaného přístupu „one size fits all“ a monoterapií. Jednoznačným důvodem je snaha podávat správné léky ve správných kombinacích, ve správný čas a správnému pacientovi. Tato strategie se musí opírat o komplexní biologickou analýzu nádorové tkáně, musí být doplněna o detailní informace o hostiteli, např. o genotypizaci a fenotypizaci enzymů, ale také o mikrobiomu pacienta, který se zdá být velmi důležitý např. pro odpověď pacientů na protinádorovou imunoterapii. Výsledky řady studií naznačují, že cílené – „targeted“ – terapie lépe fungují na časné fáze nádorového onemocnění, jak ukazuje příklad obrovské efektivity inhibitorů bcr/abl u nově diagnostikované chronické myeloidní leukémie. A tím je vysoká pravděpodobnost selhání identické terapie, pokud je u stejné nemoci podávána v pozdních fázích. I přes tuto zkušenost je v oblasti solidních nádorů dětí i dospělých cílená léčba pacientům často nabízena až v pozdních, pokročilých fázích onemocnění, a většinou je přínos takové terapie velmi problematický, ne-li vůbec žádný. Imunoterapie, na rozdíl od cílené terapie, může být efektivní i u některých pacientů s pokročilým onemocněním, tedy často s vyšší mutační náloží. Další velmi problematickou oblastí je také dávkování a načasování podání cílených léků. V řadě případů se firmy stále drží konceptu maximálně tolerované dávky, i když např. dávka potřebná k zastavení fosforylace příslušné signální dráhy je mnohdy jen zlomkem dávky cytotoxické. Příliš vysoká dávka pak vede k významné toxicitě nových léků, zejména pokud jsou kombinovány se standardní chemoterapií. Nevhodné dávkování pak může vést k tomu, že potenciálně efektivní terapie se pak řadu let nepoužívá, a k renesanci dojde až po snížení podávaných dávek. Příkladem zde může být velmi komplikovaný příběh gemtuzumabozogamycinu, který se testoval již na přelomu tisíciletí, a po dlouhé přetržce se vrací na scénu jen ve významně redukovaných, a tedy lépe tolerovaných dávkách. Darwinistické vnímání procesů resistence je sice v medicíně teoreticky akceptováno, ale v reálné klinické onkologické praxi není reflektováno prakticky vůbec. Současná praxe podávání stejné terapie až do jasné klinické či radiologické progrese onemocnění bere onkologovi (a současně i pacientovi) výhodu zvažovat a realizovat léčbu alespoň jeden tah kupředu. Zajímavým konceptem by zde mohlo být například využití tzv. „liquid biopsies“, kdy je možno pomocí sekvenování nové generace detekovat cirkulující nádorovou DNA, a takto dříve detekovat objevení se nové, resistentní mutace či klonální evoluce – a tedy nástup resistence k podávané terapii. Identifikace klinicky relevantních informací pomocí sofistikovaných molekulárně genetických metod (např. NGS, 12 včetně „ultra deep“ sekvenování) z periferní krve či mozkomíšního moku může v některých případech nahradit rizikové, či třeba zcela nedostupné biopsie z nádorové tkáně a může pomoci ve sledování i klonální evoluce zpočátku minoritních klonů v průběhu času. 1.3 Klinická hodnocení nových léčiv v dětské onkologii Filozofie přístupu k inovativním léčebným postupům jsou prakticky dvě. Na jedné straně klasický model monoterapie novým léčivem s vyhodnocením preklinických experimentů, poté klinických fází I–III s vyhodnocením toxicity, popisem farmakologických vlastností a léčebných odpovědí. Takto nastavené testování je vhodné pro onemocnění s vyšší incidencí nebo z velkého počtu center, kdy je možné během relativně krátké doby vyhodnotit na reprezentativním vzorku pacientů účinnost léčby monoterapií. V dětské onkologii je toto testování v prvních fázích doménou velkých nadnárodních kooperativních skupin center dětské onkologie, největší z nich jsou severoamerická Children´s Oncology Group nebo evropské ITCC. Zatímco v počátcích klinické onkologie bylo toto testování základem kurativních postupů s použitím cytostatické léčby, která v dětské onkologii zůstává nadále dominantní a vedla k průkopnickým pracím, na jejichž základě byla chemoterapie v klinické onkologii vybudována i pro malignity dospělého věku, v poslední dekádě je patrný posun opačným směrem, kdy zkušenosti u malignit dospělého věku jsou rychle přenášeny do onkologie dětské. Příkladem jsou rychle rostoucí zkušenosti s imunoterapií „check-point“ inhibitory, u nichž se velmi rychle rozšiřují indikace u nádorů typických pro dospělý věk, ale velmi pomalu se získávají zkušenosti u nádorů dětského věku. Jednou z mála výjimek je použití principu CAR-T buněčné terapie, která vznikla jako výsledek akademického výzkumu v Dětské nemocnici ve Filadelfii ve Spojených státech amerických. Metodologie populačního testování je postavena na klasické statistické analýze. Druhým přístupem je kombinované léčba, kdy je předmětem zkoumání účinnost dosavadních postupů s přidáním nového léčiva. V dětské onkologii probíhá v posledních letech testování velkého počtu tyrozinkinázových inhibitorů, které jsou přidány ke stávající chemoterapii. Tento postup vede zřídka k úspěchu, neboť je zatížen značnou toxicitou. To je z větší části dáno nastavením chemoterapeutických léčebných schémat, tedy podáváním maximálně tolerovaných dávek, kdy přidání dalšího léčiva, byť s odlišným mechanismem účinku, již naráží na toleranci organismu a není možné konkomitantně takovou léčbu kombinovat. Legitimní přístup je u vzácných onemocnění a kombinované terapie použití tzv. „N-of-1 trial“ metodologie analýzy, která počítá s historií 13 léčby pacienta jako s hodnocenou proměnnou, a je možné díky ní zjistit, zda má použití léčebné intervence přínos pro jednotlivého pacienta, na rozdíl od populačních analýz, které určují benefit pro skupinu pacientů. 1.4 Nové metody získávání dat v personalizované onkologii Významným faktorem, který komplikuje další zlepšování léčebných výsledků v dětské onkologii, je orientace většiny klinických studií na samotné léčivo, především s cílem jeho registrace, což je pochopitelné u farmaceutických společností, ale méně to již reflektuje oprávněné zájmy konkrétního pacienta. Pacient je tak hledán pro potřeby studie, a daleko méně často míří studie či léčiva za pacientem. Právě zlepšení přežití, nejen léčebných odpovědí, bude vyžadovat mnohem větší dynamičnost a modulace léčby podle měnící se biologie nádoru, optimálně ještě před přirozeným nástupem resistence. To však bude samozřejmě vyžadovat také změny současného regulačního kontextu a charakteru klinických studií směrem k tzv. „Nof-1 trials“. Při této metodologii, na rozdíl od stávajícího populačního přístupu, je každý pacient sám sobě opakovaně v čase kontrolou, jako například při sledování krevního tlaku u individuálního pacienta („single patient trials“). Na stejném principu je založena i tvorba individuálního biologického profilu (pasu) u vrcholových sportovců. Populací je tedy jeden jediný pacient a vzorek pro analýzy se skládá z opakovaných měření efektu různé léčby u téhož pacienta. Jsou-li doba do progrese či přežití u pacienta na personalizované léčbě významně delší nebo srovnatelné a s lepší kvalitou života než při předchozím použití standardní (populační) léčby, pak to lze považovat za podporu takového přístupu. Je nutno zdůraznit, že zde není hlavním testovaným předmětem určitý lék, ale hlavním testovaným prvkem je právě přístup. Tedy to, zda komplexní molekulární charakterizace nádoru a jeho hostitele, a v případě potřeby i opakovaná, je tou správnou cestou. Tento přístup však zatím jen obtížně hledá uplatnění v rámci dosud velmi rigidně nastavených systémů regulací a úhrad léčivých přípravků, tak aby systém, který byl historicky nastaven s cílem ochraňovat pacienty, nebyl dnes spíše překážkou, bránící efektivní terapii respektující individualitu pacienta a jeho nemoci. Další možností, jak zlepšit šanci pacientů dostat se ke správné léčbě, je větší využívání klinických a laboratorních dat generovaných v reálném klinickém provozu. V rámci klinických studií je léčeno méně než 5 % onkologicky nemocných, tedy je zde obrovská populace pacientů, 14 kteří do klinických studií zařazování nejsou. Obrovskou, a dosud jen málo využívanou příležitostí je využít různé, ale dobře a řádně vedené registry, které mohou poskytovat nesmírně cenné informace. Tyto registry je pak třeba možno použít jako kontrolní ramena pro „single arm studies“, testující nové postupy na malých a biologicky dobře definovaných skupinách pacientů, kde klasické randomizované studie nejsou reálné. Zcela zásadní jsou v této souvislosti dostatečně spolehlivé biomarkery, které by měly zahrnovat optimálně multiomický přístup. Ani tehdy, kdy nenacházíme cílitelnou mutaci na úrovni DNA, to neznamená, že žádná genová aktivace přítomna není. Například gliomy jsou velmi náchylné k reaktivaci vývojových signálních drah i bez mutací na úrovni DNA. Stejně tak, imunitní únik nádoru („immune evasion“) a angiogenese nenastává na úrovni DNA, a tedy biomarkery opírající se výhradně o aberace na úrovni DNA, včetně stávajících komerčně dostupných panelů, zde mohou selhávat. Je proto zapotřebí zvažovat inkorporaci biomarkerů na úrovních RNA, proteinů, jejich fosforylace, včetně stanovení mutační nálože a mutačního podpisu nádoru („mutational signature“), a to vše v kontextu velmi pečlivě zhodnoceného nádorového mikroprostředí a cirkulujících biomarkerů, jako např. T-regulačních lymfocytů. Postupem času a se získáváním relevantních důkazů se z inovativních léčebných přístupů stávají standardní léčby. Níže je diskutován potenciál jednotlivých léčebných modalit, z nichž některé již můžeme v dospělé onkologii považovat za dnes standardní, nicméně data pro jejich použití v dětské onkologii chybí, jsou limitovaná nebo nekonkluzivní. Ve světle výše uvedeného se čeká na nové metodologické přístupy u „ultra orphan diseases“ a pomalu se ukazující důkazy i v malých souborech fází I a II klinických hodnocení. 1.5 „Drug repurposing“ Princip známý také jako „drug repositioning“. Jde o nalezení nové indikace k podání léčiva, které bylo původně schváleno k použití u jiného onemocnění. V klinické onkologii je jedním z nejznámějších valproová kyselina, valproát užívaný 50 let jako antiepileptikum. Byla zjištěna jeho schopnost inhibice histondeacetyláz, což ovlivňuje expresi genů ovlivňujících buněčný cyklus, diferenciaci a apoptózu i protinádorovou imunitu. Na jedné straně je z in- vitro studií 15 znám, že valproát na buněčné úrovni indukuje diferenciaci T-regulačních lymfocytů, které mají imunosupresivní účinek, využitelný např. při autoimunitních onemocněních, na druhou stranu existuje klinická evidence o jeho účinku na remodelaci chromatinu. Valproová kyselina přímo inhibuje histondeacetylázy (HDACs). Histony jsou v současnosti považovány z důležité aktéry epigenetické regulace prostřednictvím kovalentních modifickací na jejich N-terminálních koncích, které jsou na povrchu nukleosomu, což jim umožňuje interagovat s jadernými transkripčními faktory. Tento fenomén se jmenuje „histonový kód“ (angl.“histone code“) a jde o modifikace jednoho nebo více histonů tak, aby byl umožněn nebo naopak odmítnut přístup k transkripčním faktorům a regulačním proteinům, které modifikují proces aktivace nebo deaktivace genů, aniž by byl změněn genotyp. Valproát indukuje epigenetickou inhibici HDACs, čímž přispívá k vyšší acetylaci histonů H2, H3 a H4, které modifikují expresi genů asociovaných s apoptózou, buněčným cyklem, buněčnou diferenciací a protinádorovou obranou. Metabolomický efekt valproátu byl prokázán u AML, kdy jeho podání s nízce dávkovanou chemoterapií antimetabolity vedl ke změně metabolitů aminokyselin a mastných kyselin v séru. Recentně jsou publikovány práce, které prokazují, že podání valproátu je také spojeno se stimulací mechanismů buněčné imunity podmíněné protilátkami, tzv. ADCP a ADCC mechanismy. Toho lze využít při konkomitantím podávání s cílenými protilátkami jako je anti HER-2 trastuzumab, anti VEGF bevacizumab a další. 1.6 Léčebné modality 1.6.1 Tyrozinkinázové inhibitory Tyrozinkinázové inhibitory (TKI) blokují signální dráhu, která je aktivována pomocí příslušné fosforylované tyrozinkinázy. U některých maligních onemocnění je přítomen fúzní gen podmiňující patogenezi nemoci a jeho produkt ve formě konstitutivně aktivované tyrozininázy je na inhibitor citlivý. Skupina léčiv tyrozinkinázových inhibitorů je široce používána v dospělé onkologii. Jejich uvedení do praxe znamenalo značný přínos pro kvalitu života pacientů. Dříve používaná paliativní chemoterapeutická intravenózní léčba, která znamenala četné komplikace a nutnost 16 hospitalizací, doprovázená značnou morbiditou, byla vystřídána velmi dobře tolerovanou perorální léčbou. V dospělé hematoonkologii znamenala průlom, kdy se z dříve nezvladatelné choroby – např. CML – stala chronická nemoc pod kontrolou TKI s relativně dobrou kvalitou života. V dětské onkologii je rozšíření TKI menší. Pokud se s nimi setkáme, tak nejvíce u hematologických malignit s definovanými fúzními geny. Limitem TKI je v průběhu léčby vznikající rezistence a je nutné jednotlivé TKI měnit za preparáty dalších generací. U solidních nádorů dětského věku byl v roce 2001 prvním FDA schváleným preparátem imatinib mesylát jako účinný u gastrointestinálního stromálního nádoru (GIST), následovaly sunitinib a regorafenib v roce 2013. U refrakterních sarkomů existuje terapeutická indikace pro pazopanib, který prodloužil dobu do progrese o 3–4 měsíce, objektivní radiologickou odpověď je možné čekat pouze ve 4 % případů. Pokud pacient na léčbu odpoví, je medián odpovědi 9 měsíců. Velkou pozornost zasluhuje dávkování TKI. Zatímco standardně je k dávkování přistupováno se stejnou filozofií jako u chemoterapie, tj. v klinických hodnoceních byla testována maximálně tolerovaná dávka léčiva, v posledních letech přibývají důkazy, že snížená dávka léčiva až na 50 % i méně je stejně efektivní pro udržení v indukci navozené remise. V roce 2018 byl tento přístup matematicky modelován na základě dat ze dvou klinických studií fáze III u CML. V roce 2020 byl tento přístup validován u skupiny pacientů s CML, kteří byli léčeni redukovanými dávkami TKI (o padesát a více procent) a měli dobrou odpověď, a u nichž takové snížení dávky nevedlo ke zhoršení doby remise bez léčby ve srovnání s pacienty užívajícími standardní dávky TKI. Toto zjištění má velký potenciál v kombinované léčbě TKI a je nutné je validovat i u jiných onemocnění. V dětské onkologii otevírá tento přístup potenciál, který zatím není zkoumán, resp. přetrvává dogma podávání MTD. Na základě výsledků vysoké konkomitantní toxicity chemoterapie a standardních dávek TKI není toto dogma udržitelné a bude nutné ověřovat tyto kombinace i v nižších dávkách. 1.6.2 Check-point inhibitory Nádorová imunosuprese hraje jednu z klíčových rolí v multifaktoriální patogenezi malignity. Určující pro rezistenci nádoru k léčbě je nejen samotná nádorová buňka, ale komplex mechanizmů, kterými nádor uniká z kontroly imunitního systému hostitele. Maligní nádor 17 a nádorové mikroprostředí, jako heterogenní směs různých buněčných populací a nádorem produkovaných imunsupresivních cytokinů, jsou cílem inhibitorů drah CTLA-4 a PD-1. Checkpoint inhibitory jsou používány od března 2011, kdy byl k použití schválen preparát ipilimumab pro léčbu metastatického melanomu. O rozvoji této formy imunoterapie svědčí množství prací, které jsou dohledatelné v PubMed Central – https://www.ncbi.nlm.nih.gov/pmc/ – kdy heslo „ipilimumab“ je nalezeno v 17658 případech (19. prosince 2020). Při pátrání po kombinaci „ipilimumab“ a „child“ bylo nalezeno 669 článků. Teoretickým východiskem léčby check-point inhibitory je exprese CTLA-4 nebo PD-1 a PDL1 antigenů v maligním nádoru. PD-L1 exprese byla nalezena u různých typů dětských nádorů – u Hodgkinových lymfomů, difusního velkobuněčného B-buněčného lymfomu nebo gliomů. U maligního melanomu existuje korelace mezi účinností monoterapií blokádou PD-1 nebo PDL1 a vysokou mutační náloží (TMB-H). Kombinovaná terapie anti CTLA-4/anti PD-1/anti PDL1 protilátkami je účinná, aniž by nutně korelovala s výší TMB-H jako u monoterapie. Pembrolizumab Pembrolizumab je monoklonální protilátka proti PD-1. U dospělých je schválena k použití ve 20 indikacích a stále přibývají další. U dětí byla zkoumána v monoterapii u pokročilých sarkomů měkkých tkání a skeletu, prokázala objektivní odpověď u 18 % sarkomů měkkých tkání a 5 % sarkomů skeletu. V současnosti je u dětí schváleno použití pouze u refrakterního Hodgkinova lymfomu po třech nebo více liniích předchozí léčby. Ipilimumab Humanizovaná monoklonální IgG1 protilátka proti CTLA-4. Klinická účinnost byla potvrzena u pokročilého maligního melanomu s jednoletým celkovým přežitím 45,6 %. U dětí nad dvanáct let je její použití schváleno od roku 2017 ve stejné indikaci jako u dospělých. 1.6.3 Jiné protilátkové imunoterapie Blinatumomab – bispecifická protilátka proti znaku CD19, který je exprimován prakticky na všech B-buněčných akutních lymfoblastických leukemiích a lymfomech. FDA schválení pro léčbu relabovaných nebo refrakterníh ALL získal v r. 2016. 18 Dinutuximab je chimérická humanizovaná protilátka proti glykolipidu GD2, který je exprimován na většině neuroblastomů a na některých dalších embryonálních nádorech, jako rabdomyosarkoma a Ewingův sarkom, pak i u osteosarkomů nebo některých gliomů. U neuroblastomu prokázala kombinace dinutuximabu s GM-CSF a IL-2 přidaná ke standardní léčbě isotretinoinem lepší dvouleté přežití bez události i celkové přežití. Tato registrační studie COG vedla ke schválení použití dinutuximabu v roce 2016. 1.6.4 Buněčné terapie Tisagenlecleucel Jde o chimérický antigen receptor (CAR)-T buněčnou terapii, která využívá autologní pacientské T-lymfocyty, které jsou geneticky modifikovány ve vazebné extracelulární antigen rozpoznávající doméně k cílené vazbě na CD19 antigen. Zpětné podání pacientovi vede k invivo tisícinásobné expanzi této T-lymfocytární populace s následnou perzistencí po dobu několika měsíců. V pilotní studii fáze I/II byla podána dvěma pacientům, v následných studiích s několika desítkami pacientů vedla léčba k celkové odpovědi a půlročnímu přežití bez události u přibližně 70 % pacientů. Všichni pacienti, kteří na léčbu odpověděli, měli toxicity související s uvolněním cytokinů a B-buněčnou aplazií, při které je nutná IgG substituce. Regulační autority schválily tuto léčbu v roce 2017. Protinádorové vakcíny Rozvoj protinádorových vakcín je patrný na více úrovních. Technologie přípravy je široká, od buněčných vakcín, které obsahují nádorové lyzáty, přes vakcíny používající specifické nádorové peptidy jako cíl indukované simulace dendritických buněk, po ty, které pracují s induktorem imunitní odpovědi DNA nebo RNA nádoru nebo vakcíny využívající virového vektoru. Překvapivě dobré jsou výsledky kombinace nízko dávkovaného cyklofosfamidu a alogenní nádorové vakcíny v kombinaci s GM-CSF u relabovaných neuroblastomů. Jiný přístup zvolili výzkumníci na University of Florida u vysoce maligních gliomů, kdy kombinují dendritické buňky s alogenní gliomovou RNA spolu s aplikací GM-CSF, ve druhém kroku je pacientovi podána infuse tumor-specifických T-lymfocytů. Tato studie byla zahájena v roce 2017 a zatím nejsou známy výsledky. 19 U medulloblastomů a vysoce maligních gliomů je používána peptidová vakcína derivovaná z CMV, který je znám jako spouštěč onkogeneze u některých gliomů. U dospělých pacientů vedla k významnému prodloužení přežití bez události i celkového přežití. 1.6.5 Neschválené terapie ve fázi klinického vývoje Onkolytické viry Jde o buď nepatogenní „wild type“ viry nebo atenuované geneticky modifikované viry, které působí přímo cytotoxicky protinádorově nebo nepřímo stimulují protinádorovou imunitu. Ve fázi I klinického zkoušení byla prokázána bezpečnost použití modifikovaných virů herpes simplex a vakcinie u extrakraniálních nádorů dětského věku, i když při podané dávce nebyla pozorována objektivní odpověď. Jedinou FDA schválenou léčbou na bázi virové terapie je talimogene laherparepvec (T-VEC), atenuovaný herpesvirus typu 1 exprimující gen pro lidský GM-CSF v indikaci pokročilého melanomu. V kombinaci s pembrolizumabem prokázal vysoký potenciál pro dosažení léčebné odpovědi u pokročilého melanomu . NK buňky Na rozdíl od T-lymfocytů nepotřebují NK buňky (lymfocyty) předchozí stimulaci nádorem, aby byly aktivní. Jejich účinek je přímo cytotoxický a indukuje u nádorové buňky apoptózu. Aktivace NK buněk v nádorovém mikroprostředí se zdá být podmínkou pro migraci dendritických buněk a T-lymfocytů do nádoru. Mohou být separovány z periferní krve, pupečníkové krve, a v případě nutnosti početně expandovány ex-vivo. Jejich použití je bezpečné a dobře tolerované. Protinádorová účinnost je ovšem limitována pravděpodobně supresivním efektem nádorového mikroprostředí. Hlavními imunosupresivními faktory v nádorovém mikroprostředí jsou transforming growth factor beta (TGFβ), indoleamine 2,3-dioxygenase (IDO) a IL-10. Existují postupy genového inženýrství, které pomocí technologie CRISPR dokážou indukovat větší odolnost NK buněk vůči imunosupresivnímu působení cytokinů. CAR-NK manipulované anti CD19 buňky vykazují vysokou protinádorovou aktivitu proti B buněčným leukemiím. Velký potenciál NK buněčné terapie je v kombinaci jednotlivých typů imunoterapie. 20 1.6.6 Antiangiogenní strategie Klíčovým mediátorem angiogeneze je VEGF. Existují četné anti VEGF protilátky nebo tyrozinkinázové inhibitory, které jsou používány v klinické praxi. Jejich kombinace s imunoterapií je atraktivním přístupem, protože blokáda VEGF ipilimumabem vede k synergistickému efektu u pacientů s metastatickým melanomem. V preklinickém modelu xenograftu neuroblastomu byl prokázán synergistický efekt při kombinaci anti VEGF protilátky bevacizumabu a anti GD-2 CAR-T buněk. I nízké dávky bevacizumabu v této kombinaci vedly k efektu, který byl v monoterapii zanedbatelný. Antiangiogenní léčba vede k větší infiltraci a aktivaci imunokompetentních dendritických buněk do nádoru, spolu s redukcí imunosupresivních MDSC. 1.7 Inovativní léčebné postupy na Klinice dětské onkologie LF MU a FN Brno 1.7.1 Imunoterapie dendritickou vakcínou V roce 2013 byla na KDO FN Brno zahájena práce na realizaci klinického hodnocení KDO DC 1311s názvem „Kombinovaná protinádorová terapie s ex vivo manipulovanými dendritickými buňkami produkujícími interleukin-12 u dětských, adolescentních a mladých dospělých pacientů s progredujícími, relabujícími nebo primárně metastatickými malignitami vysokého rizika“, EudraCT No. 2014-003388-39. Vycházela z předchozí zkušenosti výzkumného týmu ACIU LF MU s výrobou nádorového lyzátu pro přípravu autologní vakcíny z dendritických buněk u dospělých pacientů s renálním karcinomem. Tento projekt, původně plánovaný v jiném složení řešitelů, nebyl dokončený pro legislativní a technologické změny – nutné pro legální a správnou klinickou praxi respektující postupy. Zpoždění v náboru pacientů vedlo k zastavení grantové podpory. Ovšem významné technologické pokroky a nastavení kvality výroby díky tomuto nedokončenému projektu umožnilo plánování a realizaci studie u dětských onkologických pacientů. Nábor pacientů byl v projektu KDO DC 1311 dostatečný. Vyhodnocení jednotlivých cílů doposud probíhá. Publikované práce reflektují výsledky primárních cílů studie. Vedle prokázání bezpečnosti výroby vakcíny je podstatné zjištění o 21 vyvolání měřitelné imunitní odpovědi, což může sloužit jako biomarker pro budoucí vývoj vakcíny. Analýza klinické účinnost je plánována na rok 2021, aby tak bylo možné vyhodnotit sekundární cíle výzkumu s adekvátním časovým odstupem. Proces výroby vakcíny začíná identifikací pacienta vysokého rizika, jehož šance na přežití jsou méně než 30 % ve 3 letech od diagnózy nebo relapsu. Po podepsání informovaného souhlasu je prvním krokem k výrobě vakcíny odebrání nádorové tkáně. Ve většině případů jde o diagnostickou nebo terapeutickou indikaci operačního výkonu, a nejedná se tedy o indikaci z důvodu inovativní terapie, ale naopak odběr na výrobu vakcíny je spojen s těmito indikacemi. Odběr tkáně na výrobu léčiva somatobuněčné terapie podléhá zákonným požadavkům na zacházení s lidskými tkáněmi a buňkami, proto je nutné splnit přísná kritéria kvality a příslušné odběrové místo musí mít povolení státních autorit. Toto povolení má v současné době (r. 2020) Fakultní nemocnice Brno. Odebraná nádorová tkáň je zpracována v čistých prostorách ACIU na formu nádorového lyzátu. Tento lyzát je uschován k dalšímu použití a zároveň probíhá kontrola mikrobiologické čistoty. Druhou fází výroby je odběr autologních monocytů pacienta. Tento odběr probíhá po zavedení centrálního venosního katetru na separátoru. Cílem separace je odběr minimálně 0,5 x 109 monocytů. Takto odebrané monocyty jsou dále ve výrobě zpracovávány s nádorovým lyzátem, cílem je indukce reaktivity monocytů derivovaných dendritických buněk proti nádoru, mechanisticky proti nádorovým neoantigenům. Dosavadní zjištění v publikované práci (příloha č. 1) se zabývá výrobním procesem dendritické vakcíny. Identifikovali jsme léčebné postupy před odběrem autologních monocytů, které vedou k horší výtěžnosti vakcíny a horší maturaci dendritických buněk a produkci IL-12. Nastavené parametry kontroly kvality pak nedovolí takovou vakcínu podat pacientovi. Chemoterapeutika cyklofosfamid a topotecan a tyrozinkinázový inhibitor pazopanib vedou k poruše diferenciace a poté inadekvátním imunostimulačním vlastnostem dendritických buněk. Kombinace temozolomidu a irinotecanu sice dovolí diferenciaci monocytů, ale výsledné dendritické buňky nemají adekvátní imunostimulační vlastnosti. Tato zjištění vedla ke změnám v logistice separace monocytů v návaznosti na druh podané léčby. Od listopadu 2017 byla zavedena pravidla odstupu separace monocytů od podané léčby. Tyrozinkinázové inhibitory musely být vysazeny s odstupem závisejícím na jejich biologickém poločasu: léčiva s krátkým poločasem, 3–14 hodin, nejméně 2 dny před leukaferézou (axitinib, dabrafenib, dasatinib, ibrutinib, 22 idelalisib, nintedanib, ruxolitinib, trametinib), léčiva se středním poločasem, 15–35 hodin, nejméně 7 dní před leukaferézou (alectinib, bosutinib, lapatinib, lenvatinib, nilotinib, osimertinib, pazopanib, ponatinib, regorafenib a mTOR inhibitor everolimus) a léčiva s dlouhým poločasem, 36–60 hodin, nejméně 12 dní před leukaferézou (afatinib, ceritinib, erlotinib, gefitinib, imatinib, cabozantinib, crizotinib, sorafenib, sunitinib, vemurafenib a mTOR inhibitor temsirolimus). Myelopoetické růstové faktory byly vysazeny nejméně 7 dní před leukaferézou. Následně se podařilo zrychlit proces mikrobiologické kontroly kvality lyzátu a v současnosti je možné po několika dnech od odběru nádorové tkáně provést i odběr monocytů. Těmito postupy se můžeme vyhnout negativním vlivům podávané protinádorové léčby na monocyty pacienta. Efektivita výroby vakcíny se tím významně zlepšila. Druhá publikovaná práce (příloha č. 2) se zabývá vlivem vakcinace dendritickými buňkami na imunologické parametry pacientů se sarkomy a popisuje jeden případ ilustrující validitu naměřených parametrů na klinickém průběhu nemoci. Kvantitativní parametry imunity byly hodnoceny při každém podání protinádorové vakcíny. Navíc bylo provedeno funkční testování odpovědi pacientových T-lymfocytů na autologní nádorový lyzát v tzv. auto-MLR reakci. V práci je uveden případ pacienta s diagnózou metastatický relabující Ewingův sarkom. Pacient prodělal dva relapsy onemocnění a bylo mu podáno 19 dávek vakcíny po prvním relapsu, spolu s konkomitantní onkologickou léčbou bylo dosaženo parciální remise. Opětovná revakcinace po druhém relapsu vedla ke stimulaci preexistující odpovědi na nádorové antigeny a T-buněčná reaktivita (měřeno auto-MLR reakcí) perzistovala i po předchozí vakcíně a byla zvýšena po revakcinaci. Tato zjištění představují zásadní vhled do imunologických mechanismů vyvolaných dendritickou vakcínou a jsou základem pro další vývoj této léčebné metody. 1.7.2 Personalizovaná léčba Využití personalizované léčby je publikováno ve dvou pracích. První z nich ukazuje využití molekulární diagnostiky u pacientů s Burkittovým lymfomem (příloha č. 3). Vyšetření pomocí celoexomového sekvenování nádorové tkáně, vyšetření transkriptomu a aktivity tyrozinkináz vede k identifikaci pacientů, jejichž nádor je cílitelný v současnosti dostupnými léčivy. V práci jsou popsány nálezy u tří pacientů, jejichž nádory s totožnou histologií mají zcela rozdílné biologické profily měřené uvedenými metodami. Ukazuje se, že klonální evoluce vedla u jednoho pacienta k TP53 mutaci a k chemorezistenci. Na základě molekulárního profilu 23 germinální mutace PI3K-delta, transkriptromikou zjištěné expresi HR23B, která je prediktorem účinnosti HDACs, byla pacientovi podána léčba PI3K inhibitorem idelalisibem spolu s ibrutinibem a valproátem, na základě exprese PD-1 v nádorové tkáni byl přidán nivolumab a personalizovaná dendritická vakcína. Tato kombinovaná léčba vedla u pacienta k navození a udržení kompletní remise, která byla v době publikace nejdelší z jeho tří intervalů přežití bez progrese. Jiný pacient dosáhl remise na základě indikace imunoterapie nivolumabem „offlabel“ při vysoké mutační náloži TMB-H 31 mutací/Mb. V druhé práci se autoři zabývají mutační náloží u 106 pacientů s 28 různými histologickými diagnózami (příloha č. 4). Jde o metodickou práci srovnávající vyšetření mutační nálože pomocí dvou různých metod – celoexomového sekvenování a standardizovaného panelu vyšetření mutační nálože, FoundationOne Heme. Byla nalezena významná variabilita výsledků, které závisí na použitých algoritmech analýz a laboratorních metodách. Práce přispívá svými výsledky k aktuální diskusi o zavedení harmonizované metodiky testování pro použití v klinických hodnoceních, což je důležité ke srovnatelné interpretaci výsledků léčby „checkpoint“ inhibitory. Jejich účinnost je v silné korelaci s mutační náloží nádoru. Přehledová práce na téma personalizované medicíny byla publikována ve spolupráci s řadou zahraničních spolupracovníků (příloha č. 5). Zabývá se tématem změny paradigmatu v onkologii s posunem k precizní a personalizované medicíně. Předpokladem k efektivnímu využití stávajících léčiv v jiných než schválených indikacích („drug repurposing“) nebo nových léčiv použitých bez ohledu na tkáňovou diagnózu (agnostický přístup) je přijetí principu „Nof-1 trials“, který se zásadně liší od typického klinického zkoušení ve fázích I–III. Tento nový přístup vychází z rychle se rozvíjejících poznatků nádorové biologie a postupuje dříve nevídanou rychlostí, se kterou se léčivo může k pacientovi dostat. Klasické paradigma zkoušení nového léčiva u tkáňově definované skupiny pacientů nezohledňuje individuální nádorovou biologii a biomarkery a zřídka vede k úspěchu. Nové paradigma konceptu klinických hodnocení naopak postupuje cestou definování biomarkerů, cílů terapie a zkoumání účinnosti léčiva na takto definované skupině pacientů, mnohdy bez ohledu na histologii. Metodologicky tento přístup využívá nové matematické modelování a je natolik komplexní, že není v silách „běžného“ klinika pochopit všechny jeho detaily. Zcela nezbytným se stává multidisciplinární přístup k diagnostice a návrhu terapie, kdy čím dál větší vliv v rozhodování o léčebném postupu mají molekulární biolog a klinický farmakolog. Nové jsou také kombinované léčby, které ve větší či menší míře zakomponují do léčby první linie genomické a biologické informace a vedou k individualizaci léčby. Mnohdy je kontraproduktivní podávat monoterapii cíleným léčivem 24 bez znalosti tkáňové biologie nádoru (nepersonalizované cílená terapie, „non-personalized targeted therapy“) a recentně se objevují důkazy, že takový přístup může vést k horším výsledkům než klasická cytotoxická terapie. Také kombinace maximálně tolerovaných dávek chemoterapie s přidaným cíleným léčivem je zpravidla kontraproduktivní, s vyšší toxicitou a bez lepší léčebné odpovědi. 1.7.3 Antiangiogenní a metronomická léčba Léčba byla složena z kombinované antiangiogenní strategie COMBAT („combined oral maintenance biodifferentiating and antiangiogenic therapy“). V této strategii je používáno více antiangiogenních a imunomodulačních prvků podávaných metronomicky v nízké dávce trvale po dobu několika let – nízko dávkovaný cyklofosfamid, temozolomid, topotecan nebo vepesid, vinka alkaloid vinblastin nebo vinorelbin, COX-2 inhibitor celecoxib a variabilně hypolipidemikum fenofibrát, antiangiogenní bevacizumab a další. Komentované publikace na toto téma popisují dobrou toleranci léčby. Největšími benefity jsou ambulantní podávání umožňující běžné denní aktivity a absence toxicit, které by vyžadovaly hospitalizace. Pacienti na této léčbě běžně chodí do školy. Benefitem je také udržení celkového stavu pacienta, měřeno Lanského nebo Karnofského škálou. První generace protokolů COMBAT byla složena z nízko dávkovaného vepesidu a temozolomidu, celexocibu a biodiferenciační cis-retinové kyseliny podávané po dobu jednoho roku v jedenáctitýdenních cyklech léčby. Na souboru 22 pacientů z jednoho centra autoři publikovali benefit pro pacienta ve formě stabilizace onemocnění (příloha č. 6). Toxicita této kombinace se týkala především cis-retinové kyseliny ve formě cheilitid u 7 pacientů, hematologická toxicita stupně 3 se vyskytla u jednoho pacienta. Při zaznamenání takové toxicity byla v dalším cyklu redukována dávka chemoterapeutika. Ze 14 dětí s progredujícím onemocněním, u kterých bylo možné hodnotit léčebnou odpověď, byla zaznamenána u 6 dětí, u 3 pak stabilizace nemoci, odpovídající celkovému benefitu pro pacienty 64 %. V další práci byla publikována zkušenost s protokolem COMBAT jak na KDO FN Brno, tak v zahraničí na spolupracujících pracovištích v Košicích na Slovensku a v Marseille ve Francii (příloha č. 7). Změnou oproti původní verzi protokolu bylo přidání fenofibrátu a vitamínu D do metronomického schématu a prodloužení doby léčby na dva roky, vznikl protokol COMBAT II. Verze pro měkkotkáňové sarkomy se nazývala COMBAT IIS, ve kterém byl etoposid 25 s temodalem nahrazen nízce dávkovaným cyklofosfamidem, a byl přidán intravenózně vinorelbin. Pacienti vysokého rizika a bez možnosti kurativní léčby byly do této studie zařazeni od roku 2004 do roku 2010. Celkově se léčby zúčastnilo 74 pacientů. Klinický benefit pro pacienta byl zaznamenán ve 40 % případů. Toxicita léčby byla podobná, jak bylo publikováno v předchozí práci, zaznamenány byly především cheilitidy a zvýšené hodnoty jaterních testů. V další verzi protokolu COMBAT III byla cis-retinová kyselina nahrazena bevacizumabem, anti VEGF protilátkou podávanou ve dvoutýdenních intervalech. S nástupem tyrozinkinázových inhibitorů a rozvojem měření aktivity tyrozinkináz nebo MAP kináz byla do schématu COMBAT zakomponována i tato léčba-COMBAT III modif. Důležitá je titrace dávek léčiv tak, aby nedocházelo k neutropeniím vyžadujícím přerušení léčby nebo k dalším komplikacím, které vyžadují hospitalizaci. Data o účinnosti jsou aktuálně zpracovávána, do doby psaní tohoto textu je zpracována kohorta pacientů se sarkomy. Rámcově lze říct, že léčba COMBAT III modif. vede k prodloužení doby přežití u vysoce rizikových sarkomů. Konkomitance nízce dávkované chemoterapie se zakomponováním intravenózního vinblastinu s antiangiogenním účinkem a tyrozinkinázových inhibitorů je dobře snášena. Vyhodnocení efektivity u dalších diagnostických skupin je v plánu. 1.7.4 Tyrozinkinázové inhibitory Velká část klinického výzkumu efektivity tyrozinkinázových inhibitorů u solidních nádorů probíhá bez znalosti aktivace příslušných kináz v nádoru jednotlivého pacienta, tedy tzv. nepersonalizovaný cílený přístup. Vhodnější se zdá být přístup, kdy je jako biomarker měřena buď exprese tyrozinkinázy pomocí imunohistochemického histopatologického vyšetření, nebo některá z molekulárních metod pro určení aktivity příslušné tyrozinkinázy, např. aktivační mutace v kódujících exomech nebo na úrovni funkce proteinu podle míry jeho fosforylace (aktivovaný stav). Samostatné podání monoterapie tyrozinkinázovým inhibitorem je předmětem publikované práce (příloha č. 8). Pojednává o novorozenci, u kterého byly nalezeny mnohočetné měkkotkáňové, orgánové a kostní léze diagnostikované jako infantilní myofibromatóza. Konvenční léčba byla zatížena vysokou toxicitou s nutností modifikace dávek chemoterapie. Léčebná odpověď – parciální remise po této léčbě vydržela pouze tři měsíce. Vyšetřením 26 nádorové biologie byla zjištěna vysoká konstitutivní aktivita (fosforylace) tyrozinkinázy PDGFRβ. Dalším vyšetřením jsme zjistili nález aktivační mutace v kódujícím genu PDGFRB. Tato zjištění vedla k podání personalizované cílené léčby („personalized targeted therapy“) s excelentním výsledkem, kdy v řádu týdnů došlo k regresi myofibromatózy a i několik let od zahájení léčby pacient pokračuje v terapii; byť s nutností redukce dávek tyrozinkinázového inhibitoru a s několika infekčními komplikacemi a jednou symptomatickou hypoglykemií s nutností hospitalizace. Evoluční vývoj onemocnění vedl při redukci dávek k několika relapsům, které ovšem během času vyhasínaly, při zachování alespoň malé dávky tyrozinkinázového inhibitoru. Zajímavostí bylo, že pacientova 8letá sestra měla v anamnéze spontánně regredované nebioptované léze a v době léčby chlapce u ní došlo k novému vzplanutí choroby s relativně velkým ložiskem na bazi lební s velkými bolestmi. Biologie nádoru byla stejná jako u chlapce a stejně tak bylo velmi rychle dosaženo terapeutického úspěchu s tyrozinkinázovým inhibitorem sunitinibem. Tato práce ilustruje, jak velkým benefitem je pro pacienta personalizovaná cílená léčba. V případě nálezu aktivační mutace je možné podání inhibitoru v monoterapii s velmi dobrým výsledkem léčby, jak je v práci dokumentováno. Z tkáně nádoru pacienta popsaného v předchozím případě byla vytvořena buněčná linie, u které byla zkoumána fosforylace tyrozinkináz a citlivost na různé tyrozinkinázové inhibitory (příloha č. 9). V práci bylo experimentálně potvrzeno, že i buněčné linie derivované z nádoru si udržují vysokou míru fosforylace různých tyrozinkináz, nejvíce pak PDGFRβ podmíněnou mutací v genu PDGFRB. Inhibiční efekt sunitinibu byl zaznamenán při koncentracích, které jsou v klinické praxi dosažitelné. Fosforylace PDGFR nebyla podmíněna jenom přítomností aktivační mutace, ale v nepřítomnosti séra v kultivačním médiu došlo ke snížení exprese genu pro kinázu TGFA, nezměnila se exprese EGFR a PDGFRB, a překvapivě došlo ke zvýšení exprese genu PDGFRA. Jde pravděpodobně o adaptační autokrinní mechanismus pro přežití buňky při nedostatku živin. Potenciálně je tento mechanismus využitelný při kombinované terapii v klinické praxi. Případem úspěšného požití tyrozinkinázového inhibioru v monoterapii je případ batolete s vzácným onemocněním „fibrodysplasia progressiva ossificans“, FOP (příloha č. 10). Dvacetiměsíční dívenka přišla k lékaři s ložiskovými zarudnutími, otoky krku, axily a jugula, některé z nich po prvotním zarudnutí spontánně mizely, jiné se objevovaly, léze vedly k omezení mobility krku a pletence horní končetiny. Histologické vyšetření popsalo infantilní (lipo)fibromatózu měkkých tkání, při stagingu byly objeveny další asymptomatické léze na hrudníku a zádech. Byla zahájena léčba standardní nízkodávkovanou chemoterapií 27 methotrexate/vinblastin. Po čtyřech týdnech ovšem došlo k progresi lézí a objevování se nových, ve velmi rychlém sledu (v řádu hodin) doprovázených teplotou. Kortikoidy vedly k omezení těchto vzplanutí. Vyšetření biologické aktivity nádoru odhalilo vysokou míru fosforylace PDGFRβ. Dívce byla podána léčba sunitinibem, který cílí na tuto kinázu, v kombinaci s nízkodávkovaným vinblastinem a celecoxibem. Na této léčbě došlo ke zmenšení lézí, omezení frekvence a intenzity vzplanutí a ke zpomalení progrese omezení hybnosti. Při vyšetření celoexomovým sekvenováním byla odhalena patogenní mutce v AVCR1 genu, která je patognomonická pro onemocnění FOP, čímž změnila histopatologickou diagnózu. Dívka je na této léčbě již čtyři roky, během kterých je schopna s omezením mobility krku a horních končetin běžných denních činnost. V tomto případě nejde o cílenou léčbu, mechanismem účinku je inhibice prozánětlivých cytokinů a proliferativních kaskád včetně PDGFR α, PDGFRβ, c-kit, HIF1α a dalších. Tato kazuistika ilustruje, jak metody molekulární genetiky přispívají ke správné diagnostice ultravzácných onemocnění, kterým FOP je, a jak „off-label“ použití léčiva vede k léčebnému efektu. V době zahájení léčby byla dívka nejmladší pacientkou v dohledatelné anglickojazyčné literatuře, která tyrozinkinázový inhibitor v této indikaci dostala. 1.7.5 Rekurentní laryngeální papilomatosa Rekurentní papilomatosa hrtanu je onemocnění podmíněné lidským papilomavirem (HPV). Vyskytuje se ve všech věkových kategoriích. Cesta transmise viru je genitální. Vzácně se objevuje u malých dětí. V případě, že jde o onemocnění refrakterní na chirurgickou léčbu a opakovaně recidivuje, jde o devastující onemocnění, které pacientům nedovolí zapojení do běžného života. Pacienti trpí opakovanými ztrátami hlasu, nekonstantní barvou a zněním hlasu a vedou k psychosociální izolaci v kolektivu. Do léčby rekurentní laryngeální papilomatosy bylo zavedeno více lokálních nebo systémových přístupů, ale žádný z nich nedosáhl velké efektivity. Inovativní přístup léčby tohoto onemocnění je publikován v práci, kde je ukázána efektivita použití očkování pro HPV (příloha č. 11). Dvouletý chlapec, jehož matka neměla žádné známky HPV, postupně začal mít chraplavý hlas a při vyšetření ORL specialistou byla diagnostikována laryngeální papilomatosa. Během následujících dvou let bylo provedeno šest lokálně chirurgických zákroků. Tato lokální léčba vedla k dočasným úlevám. Pomocí PCR byla v papilomu detekována genotypická 28 přítomnost HPV-11. U chlapce nebyla detekována žádná porucha imunity. Bylo proto uvažováno o navození imunity proti HPV-11 po očkování, která by zajistila dlouhodobější efekt než lokální nebo toxické systémové léčby. Vzhledem k etiopatogenezi byla použita specifická vakcína cílená proti více genotypům HPV, mezi nimi HPV-11. Protilátková odpověď byla dostatečná a u chlapce došlo k vytvoření protilátkové imunity. Po očkování nedošlo k další recidivě papilomatosy po dobu 17 měsíců (v době psaní článku), což byl do té doby nejdelší zaznamenaný interval bez nemoci, hlas se chlapci udržel ve fyziologických mezích tónů. V době publikace šlo o „off label“ použití a fakticky o „drug repurposing“ použití léčiva schváleného pro jiné indikace. Tato práce má velký citační ohlas a vakcinace proti HPV je v současnosti akceptována jako léčebná metoda a strategie pro toto onemocnění. 1.8 Souhrn Inovativní léčebné postupy imunoterapie, personalizované léčby, antiangiogení metronomické terapie, tyrozinkinázových inhibitorů a vakcinací byly dokumentovány přiloženými pracemi. V klinické praxi vedly k rozšíření možností léčby maligních i jiných onemocnění, jako rekurentní laryngeální papilomotosa nebo „fibrodysplasia ossificans progresiva“. U malignit šlo v některých případech o kurativní přístup u do té doby beznadějných případů, jiným pacientům dokázala inovativní léčba alespoň udržet celkově uspokojivý stav po delší období, a to bez nutnosti hospitalizací nebo nežádoucích účinků, a významně tak přispěla ke kvalitě života pacientů. 29 2 Antimykotická léčba u imunokompromitovaných pacientů 2.1 Úvod Mykotické infekce jsou závažnou komplikací u pacientů léčených pro maligní onemocnění, především hematoonkologické. V dospělé onkologii se lze s mykotickou infekcí setkat vzácně. U hematoonkologických pacientů nebo dětských onkologických pacientů s vyšší intenzitu chemoterapie je četnost mykotických onemocnění vyšší. Nejrizikovější jsou pacienti s akutní myeloblastovou leukemií a pacienti po alogenní transplantaci kostní dřeně s kombinovanou imunosupresí a s nemocí štěpu proti hostiteli (GvHD). Historicky je prvním velmi účinným antimykotikem amphotericin B. Byl široce používaným přípravkem desítky let jako zlatý standard antimykotické léčby. Jde o makrocyklické polyenové protiplísňové antibiotikum produkované bakterií Streptomyces nodosum. V době jeho objevení v r. 1959 šlo o život zachraňující lék, k jeho používání nebyla nutná žádná randomizovaná studie. Naopak, dlouhá léta byl používán jako komparátor nově registrovaných a objevovaných antimykotik, jak echinokandinů, tak azolů, v pozdějším období pak byly stejně využívány jeho lipidové formy. Velkou nevýhodou konvenčního amphotericinu B je nefrotoxicita, která byla limitující pro dlouhodobé podávání. Proto byly na trh uvedeny lipidové formy, které mají menší afinitu k cholesterolu membrán na lidských buňkách a zároveň mají zachovánu afinitu k ergosterolu membrán hub. Na trh byly uvedeny tři formy – liposomální forma (Ambisom), lipidový komplex (Abelcet) a koloidní disperze (Amphocil). Všechny tři mají dobrou renální toleranci, která umožňuje dlouhodobé podávání. Spektrum účinnosti amphotericinu B sahá od candid, kryptokoků, blastomycet přes aspergillus spp. až po mukormycety. Z vlastní praxe autora je možné podávání těchto lipidových forem výjimečně i několik měsíců bez signifikantní toxicity, jako u pacientů s invazivní aspergilózou mozku. V posledních dvou desetiletích byla ke klinickému použití vyvinuta echinkandinová a nová azolová antimykotika. Jejich zavedení do praxe znamenalo rozšíření spektra účinných antimykotik. U aspergilózy byl nastaven nový standard terapie voriconazolem, v případě candidových infekcí je lékem volby echinokandin. Velkým přínosem je menší nebo žádná renální toxicita, malá hepatální toxicita a u azolů možnost perorální formy. V praxi se uplatňují jak v léčbě mykóz, tak v profylaxi. Profylaktické podávání azolů je standardem u vysoce rizikových hematoonkologických diagnóz jak u dospělých, tak u dětí. 30 V České a Slovenské republice je etablována síť spolupracujících hematoonkologických center. Jejich spolupráce je v oblasti antiinfekční léčby, koordinace léčebných postupů a společných databází mykotických onemocnění prováděna v rámci skupiny CELL – Czech Leukemia Study Group for Life, formálně jde o občanské sdružení. Klinika dětské onkologie FN Brno je jedním ze spolupracujících center. Cíli sdružení jsou tvorba společných diagnostických a léčebných protokolů, organizování klinických a experimentálních studií, zavádění nových poznatků do diagnostiky a léčby, navázání kooperace se zahraničními subjekty a rozšiřování poznatků i mezi laickou veřejnost (osvětová činnost). Zaměření tedy pokrývá celé spektrum hematoonkologické problematiky, nejen diagnostiku a léčbu mykóz u imunokompromitovaných pacientů. Autoři z KDO FN Brno se podíleli na definování doporučených postupů léčby invazivních mykóz, které byly publikovány v českých časopisech. Představují souhrn do té doby roztříštěných časopiseckých prací, klinických studií a výsledků kooperativních mezinárodních skupin a navazují na podobná úsilí, která byla vyvinuta v zahraničí. 2.2 Komentář k publikovaným pracím Množství dat o účinnosti a efektivitě voriconazolu u dětí je limitované. Pro rozšíření indikace voriconazolu na dětskou populaci byla provedeno klinické hodnocení (KH) voriconazolu u invazivní aspergilózy, invazivní kandidózy a esofageální kandidózy u dětí od 2 do 18 let (příloha č. 12). Na KDO probíhala dvě diagnóza specifická KH. První zkoumající voriconazol u invazivní aspergilózy (IA), NCT00836875, druhé v indikaci invazivní kandidózy a esofageální candidózy (IC/EC), NCT01092832. Obě KH byla multicentrická, sponzorována farmaceutickou firmou Pfizer, probíhala v 16 centrech v Evropě, Asii a Severní Americe v letech 2009–2013. Dávkování voriconazolu bylo nastaveno podle v té době nových znalostí o farmakokinetice, kdy u pacientů do 12 let nebo 12–14 let s hmotností pod 50 kg byla startovací dávka 9 mg/kg á 12 hodin první den, poté 8 mg/kg á 12 hodin, v případě esofageální kandidózy byla dávka 4 mg/kg. Bylo možno dávku modifikovat podle dosažených plazmatických hladin voriconazolu. Do studie IA bylo zařazeno 31 pacientů, do studie IC/EC bylo zařazeno 24 pacientů. Medián doby podávání antimykotika byl ve skupině IA 41 dní, u skupiny IC/EC 14 dní. Tolerance léčby byla dobrá. Celková léčebná odpověď byla zaznamenána u 78 % pacientů s IA ve věku 12–18 let, u mladších pacientů byla účinnost horší – 40 %, ale může jít o zkreslení vlivem malého vzorku, hodnocených pacientů bylo pouze pět. 31 Celková účinnost u IA byla 64,3 %. Profil toxicity byl podobný jako u dospělých, nicméně u mladších dětí byla zaznamenána častější hepatotoxicita, ale nikoliv zvýšení jejího stupně. Účinnost ve skupině pacientů s IC/EC byla lepší ve věkové skupině 2–12 let, 88,9 %, u starších byla 62,5 %, celkově 76,5 %. Interpretce těchto rozdílů mezi věkovými skupinami je limitována malou velikostí studované populace a faktem, že se jednalo o „open-label“ nekomparativní design studie. Celkově lze shrnout, že profil účinnosti a toxicity voriconazolu se v uvedených indikacích pro dětskou populaci neliší od dat získaných dříve u dospělých pacientů, a dovoluje indikaci tohoto azolu v dané věkové skupině. O retrospektivním výzkumu léčby invazivní aspergilózy u hematoonkologických pacientů v České a Slovenské Republice v letech 2005–2009 pojednává práce publikovaná v r. 2012 (příloha č. 13). Data byla získána v rámci projektu CELL. Autoři popisují diagnostiku a léčbu u 176 případů IA. U 15,3 % byla diagnostikována prokázaná IA, u 84,7% pravděpodobná IA, definice vycházely z EORST/MSG kritérií pro IA z roku 2002. Kompletní nebo parciální léčebné odpovědi bylo dosaženo u 53,2 % pacientů. Důležitým zjištěním je fakt, že pouze 53,7 % pacient mělo na diagnostických CT vyšetřeních léze považované za typické pro plicní IA dle EORTC/MSG 2008 kritérií. Nález na CT je významně ovlivněn přítomností nebo absencí neutrofilních segmentů. Vliv v tomto zjištění mohl mít i fakt, že v době studie se do praxe dostávaly HRCT přístroje, a diagnostické protokoly se tak lišily od předchozích CT technik. Významným zjištěním v této práci je role testování galaktomananu jak v séru, tak v tekutině z bronchoalveolární laváže, který byl pozitivní v 79,1 % resp. 78,8 % případů, tyto pozitivity přispěly k diagnostice IA. U čtvrtiny pacientů (26,3 %) byla IA léčena dvojkombinací antimykotik, nejčastěji voriconazolu a capfunginu, ovšem bez korelace s léčebným efektem. V současné době se za standardní postup považuje monoterapie voriconazolem nebo posaconazolem nebo isavconazolem, které mají schválení EMA v této indikaci. Velkým úspěchem byla léčba dvou invazivních mykóz publikovaných jako kazuistiky. V prvním případě byl pacientce s aspergilózou mozku během indukční léčby akutní lymfoblastické leukémie podáván lipidový komplex ampfotericinu B („amphotericin B lipid complex“; ABCD) spolu s lokální léčbou konvenčním amfotericinem B a chirurgickou resekcí reziduálního aspergilomu (příloha č. 14). Dívka toto onemocnění, fatální až v 80 % případů, zvládla a stejně tak úspěšně ukončila léčbu základního onemocnění. Kumulativní dávka ABCD byla 2,3 g/kg a nevedla k nefrotoxicitě, což je možné považovat za úspěch adekvátní hydratační léčby s pečlivou korekcí ionogramu, především hypokalemií. 32 Druhým případem je sterkorální kandidová peritonitida u chlapce s nehodgkinským lymfomem, opět během indukční léčby (příloha č. 15). S obtížemi léčená mykóza byla zvládnuta s konkomitantním podáním výzkumné terapie anti HSP-90 protilátkou efungumab v rámci firemního programu. Chlapec svou mykózu zvládnul a je vyléčen i ze své malignity. Publikované práce s efungumabem referují v den 10 terapie kandózy v 84 % případů zlepšení příznaků s přidáním efungumabu, versus 48 % u pacientů s monoterapií amfotericinem B bez efungumabu, stejně tak byla menší i mortalita den 3 s přidaným efungumabem 4 %, verus 18 % bez efungumabu. Látka efungmab nakonec nesplnila kritéria schválení tržní autorizace pro obavy z fluktuace krevního tlaku a syndromu uvolnění cytokinů a není k použití schválena regulátory. 2.3 Souhrn Participace v multicentrických a mezinárodních projektech diagnostiky a léčby invazivních mykóz je pro pacienty přínosem v podobě použití standardizovaného postupu diagnostiky a léčby. V případě selhání standardní léčby je použití inovativního postupu plně indikováno. Vytvoření jednotných postupů léčby u imunokompromitovaných pacientů ve formě doporučených postupů je přínosem i pro spolupracující obory. 33 3 Léčba sarkomů měkkých tkání dětského věku 3.1 Úvod V dospělé populaci představují sarkomy měkkých tkání (STS) přibližně 1 % všech nádorových onemocnění. U populace dětí a mladých dospělých do 20 let jsou relativně častější a představují 7 % všech nádorových onemocnění, z toho polovina jsou rabdomyosarkomy (RMS). Ostatní nádory této velmi heterogenní skupiny jsou nazývány non-rabdomyosarkomy měkkých tkání (NRSTS, z angl. „non-rhabdomyosarcoma soft tissue sarcomas“). 3.2 Epidemiologie a etiologie Epidemiologické rozložení STS ve věku do 20 let je velmi pestré. Ve věku do 5 let dominují rabdomyosarkomy s podílem 60 %, naopak mezi 15. a 19. rokem je jejich podíl 23 %. Naopak, NRSTS představují více než 75 % ze všech sarkomů v adolescentním věku. Nejčastějším STS u kojenců je infantilní fibrosarkom, u starších dětí a adolescentů jsou nejčastější synovialosarkom, dermatofibrosarcoma protuberans, maligní nádor pochev periferních nervů (MPNST) a maligní fibrózní histiocytom. Predominance je mírně vyšší u mužského pohlaví v poměru 1,2 : 1. Významným faktorem pro vznik STS je některá z genetických predispozicí, jako Li-Fraumeni syndrom – až 10 % ze všech STS, germinální mutace RB genu. Familiární adenomatosní polypóza je v 25 % případů spojená s agresivní fibromatózou. Neurofibromatóza typu 1 má riziko vzniku MPNST mezi 6 a 13 %. Mutace v SMARCB1 genu je často spojená s extrarenálním maligním rhabdoidním tumorem. Jako sekundární malignity jsou sarkomy měkkých tkání i kostí známy v dospělé populaci po radioterapii. Při dlouhodobém sledování pacientů léčených pro nádor v dětství bylo zjištěno, že riziko vzniku sarkomu jako sekundární malignity je 9x vyšší než v běžné populaci. Největším rizikem jsou předchozí léčba pro sarkom, anamnéza jiného sekundárního nádoru a radioterapie, nebo vysoké dávkování antracyklinů či alkylancií. Z dalších rizikových faktorů vnějšího prostředí pro vznik angiosarkomu jater je známa předchozí expozice vinylchloridu (u dospělých), chronický lymfedém predisponuje ke vzniku lymfangiosarkomu. 34 Vyšetření nádorové tkáně, grading, staging Samozřejmostí při biopsii při podezření na sarkom v dětském věku je uchování biologického materiálu pro další diagnostická vyšetření. Materiál se odesílá na cytogenetické vyšetření, více alikvotů je zamraženo v tekutém dusíku, uchovávají se otisky tkáně pro potřeby FISH analýzy, při dostatečném množství materiálu je odeslán jeho vzorek ke kultivaci buněčných linií. Takto odebraný materiál umožňuje ve složitých případech efektivní diagnostiku pomocí molekulárně genetických metod, které dokáží specifikovat mutace, disrupce a amplifikace genů nebo pomocí RT-PCR detekovat specifické translokace. Staging STS u dětí je tradičně definován chirurgicko-patologickou klasifikací IRS. Tabulka 1: Staging dětských sarkomů měkkých tkání podle chirurgicko-patologická klasifikace IRS IRS skupina Definice I Nádor resekován kompletně mikroskopicky (R0) IIa IIb IIc Nádor resekován makroskopicky – S mikroskopickým reziduem (R1) a bez postižení lymfatických uzlin Pozitivní lymfatické uzliny kompletně resekovány Pozitivní lymfatické uzliny kompletně resekovány a distální uzlina mikroskopicky pozitivní III IIIa IIIb Nádor s makroskopickým reziduem po biopsii (R2) nebo parciální resekci nad 50 % (R2) IV Metastázy přítomny, nebo postiženy nadregionální lymfatické uzliny Maligní výpotek nebo implantační metastázy 35 Diagnóza STS se opírá o probatorní excizi, u menší části pacientů lze provést adekvátní resekci menších a povrchově uložených tumorů. V zásadě nevhodná je tenkojehlová biopsie. Množství materiálu takto odebraného je často limitované, mnohdy nereprezentativní a pro potřeby precizní molekulárně biologické diagnostiky nedostačující. 3.3 Lokální kontrola Obecný přístup k terapii STS v dětství je podobný jako u dospělých, s některými věkovými specifiky. Tak, jak se liší histologické spektrum STS mezi dospělou a dětskou populací, liší se i přístupy k léčbě. Chování některých STS je v obou populacích podobné, u jiných, jako například u infantilního fibrosarkomu, je diametrálně odlišné. Zda proběhne lokální kontrola chirurgicky, nebo radioterapií, závisí na místě vzniku tumoru a věku pacienta – hůře se dosáhne radikálního záchovného zákroku při menším množství okolních zdravých tkání, a ten může ve výsledku vést k doživotní mutilaci. Pozdní následky radioterapie jsou u dětí mnohem závažnější než u dospělých. Dávky záření efektivní v léčbě STS zastavují další růst ozářených zdravých tkání, které jsou poté hypotrofické. První důležitým rozhodnutím po diagnóze STS je, jak dosáhnout lokální kontroly. Kdykoliv je to bez mutilace možné, má být provedena chirurgická resekce. Pokud nelze lokální kontroly dosáhnout při akceptovatelné morbiditě, je možné zvážit adjuvantní radioterapii ke snížení rizika lokální recidivy po marginální resekci. Pacientům s primárně neresekabilním tumorem nebo metastázami při diagnóze je nutné nabídnout kombinaci chemoterapie a radioterapie, případně nových léčebných postupů, ať již v rámci paliativní léčby, nebo v současné době rychle se rozvíjejícími možnostmi cílené léčby. Radikality resekce jako u dospělých není možné zpravidla v končetinových lokalizacích dosáhnout s požadovanou hranicí 2 cm zdravé tkáně. Minimálním požadavkem je odstranění nádorové pseudokapsuly, jinak lze čekat vysokou míru lokálních recidiv, zvláště u STS bez prokázané chemosenzitivity. Naopak u chemosenzitivních STS je marginální resekce přípustná, přežití je u radikálně a neradikálně resekovaných STS srovnatelné. Postižení lymfatických uzlin není obecně u NRSTS časté, na rozdíl od rabdomyosarkomů. U vybraných vysoce maligních nádorů jako synovialosarkom, angiosarkom, světlobuněčný sarkom nebo extraskeletální Ewingův sarkom může být postižení lymfatických uzlin až v 15 % 36 případů. Některá centra doporučují ve sporných případech biopsii sentinelové uzliny. Nález pozitivity vede k indikaci resekce a radioterapie. Přibližně 20 % pacientů se STS má metastatickou nemoc. Predominantním orgánem metastáz jsou plíce. Pokud je možná resekce všech plicních metastáz, měla by být provedena. Dlouhodobého přežití dosahuje pouze 10 % těchto pacientů, ovšem značně se liší mezi jednotlivými typy STS, rabdomyosarkomy mají lepší prognózu než jiné subtypy. 3.4 Systémová léčba Systémová léčba patří do léčebného postupu u chemosenzitivní choroby. Blíže je uvedena u jednotlivých histologických podtypů níže. 3.5 Sarkomy měkkých tkání typické pro děti a adolescenty a jejich léčba 3.5.1 Rabdomyosarkom (RMS) Jde o nejčastější STS u dětí a adolescentů s podílem přes 40 %, incidence je 4,3 případu v populaci 1 milionu do věku 20 let. Téměř dvě třetiny případů jsou diagnostikovány u dětí do 5 let. Vyrůstají prakticky v kterékoliv lokalizaci, i když lze pozorovat seskupení do několika skupin. Například lokalizace hlava a krk je typická pro pacienty do 8 let věku. RMS orbity je zpravidla embryonálního typu s velmi dobrou prognózou. Končetinové nádory jsou typické pro adolescenty, převažují u nich alveolární subtypy. Etiologie je podobná jako u všech STS. V rodinách dětí se sarkomem (dvě třetiny z nich byly RMS) a anamnézou spontánního abortu a úmrtím dítěte do jednoho roku věku byla zjištěna v jedné třetině případů některá z forem Li-Fraumeniho syndromu. V souladu s možnými interakcemi mezi genetickou predispozicí a vlivy vnějšího prostředí je zajímavé pozorování až trojnásobně vyššího rizika RMS pro dítě matky, která rok před jeho narozením užívala marihuanu, totéž platí pro otce dítěte. Užívání kokainu bylo spojeno s pětinásobným rizikem. Charakteristické pro RMS jsou genetické změny. U 70 % případů alveolárního subtypu (ARMS) nalézáme typickou translokaci t(2;13)(q35;q14) nebo t(1;13)(p36;q14) vedoucí ke 37 vzniku fúzního genu PAX3-FOXO1 nebo PAX7-FOXO1. Produkty těchto fúzních genů spolu se ztrátou funkce lokusu CDKN2 vedou k abnormální aktivaci transkripce vedoucí k finálnímu malignímu fenotypu. Podílí na tom i inhibice drah RB a p53 genů, amplifikace genu MYCN a zvýšená exprese receptorové tyrozinkinázy C-MET. Druhý subtyp RMS nazývaný embryonální (ERMS) má typicky ztrátu heterozygozity lokusu 11p15 se ztrátou maternální alely. Na tomto lokusu je gen IGF-2, který kóduje růstový faktor. Ten se pravděpodobně podílí na patogenezi ERMS. Patognomonická je exprese proteinů z rodiny MyoD, které se nevyskytují v jiných než mezenchymálních liniích určených k myogenní diferenciaci. Mutace v genu MyoD1 je spojená s extrémně špatnou prognózou i u lokalizované nemoci. Časté jsou mutace N-RAS a K-RAS onkogenů, které jsou u RMS věkové závislé. První se vyskytuje u novorozenců, druhá má maximum výskytu kolem 14. roku věku. Téměř 40 % RMS vyrůstá v oblasti hlavy a krku. Důležitá pro prognózu je tzv. parameningeální lokalizace – baze lební, nosní a paranasální dutiny, fossa pterygopalatina/infratemporalis, nosohlatan a střední ucho. V těchto lokalizacích je vysoké riziko lokální recidivy a je indikována radioterapie. Naopak v orbitě nebo v jiných lokalitách hlavy a krku, tzv. nonparameningeálních, je možné radioterapii vynechat z léčebného postupu bez větších rizik a s celkovým přežitím až 95 % pacientů. Další typickou lokalizací jsou genitálie a vývodné cesty močové – trigonum močového měchýře, děloha nebo pochva u dívek nebo nadvarle a prostata u hochů. Končetinové RMS jsou často spojené s postižením regionálních lymfatických uzlin a mají tendenci růst podél fascií. Postižení trupu je rizikové pro lokální recidivy. Obtížně a pozdě jsou diagnostikovány RMS v tělních dutinách nitrohrudí nebo v pánvi. Rychle rostoucí nádor v řádu týdnů vede k dušnosti a je zachycen na prostém RTG snímku. Pomaleji rostoucí nádor může vyplnit celý hemithorax bez dechové tísně, respiračně kompenzační mechanismy dokáží distress eliminovat, a pacient pak přichází s nádorem v celém hemithoraxu s metastázami na pleuře nebo s maligním výpotkem. Z orgánových lokalizací dosahují RMS žlučového traktu zpravidla intraparenchymatózně v játrech obrovských rozměrů, ale mají výbornou prognózu s dobrou senzitivitou na chemoterapii a bez nutnosti velkých chirurgických výkonů. Důvod této biologické vlastnosti znám není. Léčba rhabdomyosarkomu Rabdomyosarkom je chemosenzitivní malignita. Kombinovaná léčba chirurgická, radioterapií a chemoterapií je standardem. Intenzita chemoterapie a výběr cytostatik závisí na klinickém stádiu. Poměrně složitý systém stagingu počítá s parametry, které stratifikují pacienty s lokoregionálním onemocněním do šesti rizikových skupin, z nichž každá vyžaduje jinou 38 intenzitu terapie. Příznivými faktory jsou věk pod 10 let, velikost nádoru méně než 5 cm, lokalizace jiná než parameningeální, končetinová a trup, histologie jiná než alveolární. U choroby metastatické se také vyskytují faktory, které stratifikují pacienta do skupiny s relativně příznivou přibližně 40% prognózou pro vyléčení. Jsou to věk pacienta do 9 let, s nejvýše jednou metastatickou lokalizací (např. plíce) a bez postižení skeletu nebo kostní dřeně. Chemoterapeutická schémata jsou postavena na vinkristinu a actinomycinu D samotných u pacientů nízkého rizika. Pacienti středního rizika jsou léčení také vinkristinem s aktinomycinem D a s přidáním alkylancia, v Severní Americe cyklofosfamidem (VAC), v Evropě ifosfamidem (IVA). Role antracyklinu je historicky potvrzena jako efektivní, nicméně nevede ke zlepšení léčebných výsledků, jak bylo prokázáno v randomizované studii u vysoce rizikových rabdomyosarkomů. Vzhledem ke kardiální toxicitě se u lokoregionální choroby nepoužívá. Doxorubicin je vyhrazen pro léčbu metastatické nemoci v kombinaci s cyklofosfamidem a vinkristinem (VDC) nebo jako čtyřkombinace ifosfamid, vinkristin, aktinomycin D, doxorubicin (IVADo). Další efektivní kombinací léčiv používaných v terapii metastatické nemoci jsou ifosfamid a etoposid (IE) a vinkristin s irinotekanem (VI). Načasování chemoterapie záleží na typu chirurgického výkonu při diagnóze. Téměř polovina pacientů spadá do skupiny s neoadjuvantní léčbou. Chemosenzitivní nádor je jednak s výhodou objemově redukován, průkaz chemosenzitivity je biologicky ověřený benefit adjuvantní léčby v trvání až půl roku. Délka adjuvantní léčby je ve všech případech minimálně 6 cyklů opakovaných po třech týdnech. Recentní výsledky randomizované studie s udržovací terapií vinorelbinem a nízkodávkovaným cyklofosfamidem (denně 25 mg/m2 tělesného povrchu perorálně) podávanými půl roku prokázaly zlepšení přežití u vysoce rizikových pacientů, s dosud nejlepším pětiletým celkovým přežitím 86,5 %. Pacienti s postižením lymfatických uzlin a pozitivitou fúzního genu PAX3-FOXO1 nebo PAX7-FOXO1 mají prognózu srovnatelnou s metastatickou chorobou s pětiletým přežitím bez události 43 % oproti pacientům s N1 chorobou bez pozitivity fúzního genu. Tito pacienti budou v další studii léčeni pomocí delší udržovací terapie. Relapsy rabdomyosarkomu jsou léčitelné přibližně u třetiny pacientů. Opět záleží na prognostických faktorech a možnostech lokální léčby. Tam, kde byla při primární diagnóze provedena záchovná operace, je u relapsu indikována ztrátová nebo mutilující, pokud vede k remisi. Radikalita je oprávněná i u radioterapeutických schémat. Léčebné režimy chemoterapie reflektují předchozí linii. Vedle výše zmíněných režimů je efektivní také 39 kombinace vinorelbinu s blokovým cyklofosfamidem (1,2 g/m2 ). Kombinace VI s temodalem (VIT) zlepšuje celkové přežití u refrakterních a relabovaných RMS o přibližně čtyři měsíce (10,3 vs. 14,5). Pacienti s refrakterním nebo časně po léčbě první linie relabujícím RMS a pacienti s metastatickou progresí mají velmi malou šanci na dosažení dlouhodobého přežití a měli by být léčení individualizovaně na základě výsledků analýzy nádorového exomu, transkriptomu a profilu aktivity drah proteinkináz a MAP kináz, metylačního profilu a mutační nálože nádoru. Vybrané non-rhabdomyosarkomy měkkých tkání u dětí a adolescentů Všechny níže uvedené nádory mají incidenci splňující kritérium vzácného nebo ultra vzácného onemocnění s prevalencí menší než 1 případ v populaci 2 tisíce, resp. 50 tisíc obyvatel. Léčebný přístup tomu odpovídá, mnohdy neexistují žádné údaje o léčbě srovnatelné s populačními randomizovanými studiemi, jako u četnějších diagnóz. Léčba je postavena na historické empirii a publikovaných souborech pacientů s několika desítkami, vzácně stovkami případů, soubory jsou heterogenní. Společným jmenovatelem jsou genetické abnormity a využití cílených léčiv. I léčebná doporučení je nutno touto optikou vnímat jako dosud nejlepší možné standardy péče, včetně precizní diagnostiky a individualizované léčby. Alespoň částečně dávají pacientům s těmito nádory šanci na delší přežití případně kurativní léčbu multikinázové inhibitory cílené na geneticky podmíněné změny ve fúzních genech nebo receptorových tyrozinkinázách nebo MAP kinázách. Ty, pokud jsou v nádoru potvrzeny, bývají dobře zaměřitelné a vedou k dlouhodobé stabilizaci nebo i remisi onemocnění, jako například u NTRK fúzí v případě infantilního fibrosarkomu nebo ALK nebo PDGFRB mutovaných inflamatorních myofibroblastických tumorech nebo agresivních fibromatózách. 3.5.2 Infantilní fibrosarkom Vyskytuje se výhradně u pacientů mladších čtyř let, 60 % z nich je diagnostikováno do třetího měsíce života, až polovina vzniká in utero a je vrozená. Pro infantilní fibrosarkom (IFS) je charakteristická translokace t(12;15)(p13;25), kterou vznikne fúzní transkript ETV6-NTRK3 (stejná fúze je u vrozeného nádoru ledviny, mesoblastického nefromu). Tato fúze ovšem není stoprocentně určující, některé IFS ji nemají. 40 Naopak může v patogenezi hrát roli i jiný mechanizmus. Byly nalezeny zvýšené aktivace receptorových tyrozin kináz PI3-Akt, MAPK a SRC bez uvedené fúze. Histopatologicky jde o vřetenobuněčný vysoce buněčný nádor s častými mitózami, mohou být přítomny okrsky nekróz, zvýšená vaskularita je v okrscích podobných hemangiopericytomu. Původ je pravděpodobně v primitivní mezenchymální vřetenobuněčné buňce, jde o prekurzorovou buňku fibroblastické/myofibroblastické buněčné linie. Klinický nález je ve dvou třetinách případů měkkotkáňový tumor na končetině, méně často na trupu, krku nebo na kalvě, vzácné je postižení orgánové. Nádor obvykle roste rychle do relativně velkých rozměrů, dvě třetiny pacientů mají nádor větší než 5 cm. Metastatický potenciál IFS je nepatrný. Metodou léčby je v případě resekabilních IFS chirurgická resekce, pokud v plánovaném rozsahu nepovede k mutilujícímu výsledku. Akceptovatelná je i marginální resekce bez adjuvantní léčby. Vzhledem k relativní velikosti a lokalizaci nádoru to není vždy možné. Mnohem přijatelnější z hlediska funkčních následků léčby je neoadjuvantní chemoterapie. Největší publikované série pacientů s IFS shodně uvádí velmi dobrou léčebnou odpověď kolem 70 % na kombinaci vinkristin a actinomycin D. Pouze přibližně čtvrtina pacientů je tímto postupem indikována k primární resekci. Intenzifikace léčby o alkylans cyklofosfamid nebo ifosfamid je zpravidla vynucena při stabilním nebo progredujícím nálezu u neresekabilních nádorů. Radioterapie není vzhledem k věku pacientů indikována. Celkové tříleté přežití dosahuje 93 %. Recentní práce ukazují, že elektivní inhibitor TRK kináz larotrectinib prokázal aktivitu u pacientů s fúzí některého z genů NTRK 1–3. U pacientů s pokročilým nebo metastatickým a relabovaným nebo refrakterním sarkomem s touto genovou fúzí je objektivní radiologická odpověď dosažitelná monoterapií tímto inhibitorem v 93 % případů, což jako recentní poznatek povede k další eliminaci intenzity chemoterapie, zvláště alkylancií. 3.5.3 Inflamatorní myofibroblastický tumor (IMT) Popisován je také jako zánětlivý pseudotumor nebo zánětlivý (inflamatorní) fibrosarkom. Jde o intermediární nádor s lokálně agresivním růstem. Věkový vrchol incidence je kolem devíti let. Může se vyskytnout v měkkých tkáních i orgánech, v plicích i dutině břišní. Typický je 41 nespecifickými příznaky doprovázený indolentní růst buď v hrudníku, nebo v retroperitoneu. Diagnóza je uvedena dramatickým nálezem na zobrazovacích metodách (obr. 1). Až třetina pacientů má paraneoplastické projevy, jako zvýšenou sedimentaci erytrocytů, teploty, trombocytózu, anemii, polyklonální hypergamaglobulinemii. Přesná patogeneze není známa. Příležitostně se uvádí růst po operaci nebo zranění. Histologicky se skládá z myofibroblastů a zánětlivé infiltrace. Poměr těchto složek je proměnlivý a pohybuje se od obrazu fasciitidy k obrazu podobnému buněčnému fibrohistiocytomu nebo hypocelulárnímu desmoidnímu nádoru. Vzácně může být přítomna i kulatobuněčná komponenta. Imunohistochemicky je u 50 % případů pozitivní tyrozinkináza ALK. Podkladem pozitivity je přestavba genu ALK s fúzními partnery. Pacienti s takovou přestavbou odpovídají na léčbu ALK inhibitorem crizotinibem. Naopak druhá polovina pacientů, ALK negativních (podle imunohistochemie), má agresivnější průběh nemoci. Recentně bylo zjištěno, že tyto ALK negativní nádory mají ALK fúzi s řadou dalších genů. Výsledkem je konstitutivní aktivace fúzního transkriptu s potenciálem terapeutického efektu jinými inhibitory tyrozinkináz. Klinický průběh onemocnění je variabilní, od benigního po multifokální nebo infiltrativní nádory s tendencí k relapsům. Velikost nádoru s klinickým chováním nekoreluje. Horší průběh je popisován u nádorů vyrůstajících z mezenteria nebo u aneuploidních a s cytologickými atypiemi. Léčba IMT není vzhledem k variabilnímu klinickému chování jednotná. Resekce, pokud je možná, je metodou volby. Vzácně jsou popisovány i odpovědi na nesteroidní antiflogistika. U relapsů nebo inoperabilních nádorů je indikována radioterapie nebo chemoterapie širokého spektra, od nízce dávkované po alkylans a/nebo antracyklin obsahující režimy s variabilními léčebnými výsledky. Nově jsou indikovány tyrozinkinázové inhibitory korelující s fúzním stavem genu ALK a jeho partnery, pokud je přítomna konstitutivní aktivace výsledné tyrozinkinázy citlivé na podaný inhibitor. Pacienti, kteří takto definovaný cíl nemají, a i přes veškeré léčebné úsilí progredují, by měli být léčeni individualizovanými přístupy. 42 3.5.4 Synovialosarkom Synovialosarkom je u dětí a adolescentů nejčastějším NRSTS, představuje 7,7 % všech STS. Charakteristická je translokace t(X;18)(q11; Xp11) s výslednou fúzí genu SS18 a SSX1 nebo SSX2 vzácně i SSX4. Synovialosarkom nejčastěji vzniká na dolních končetinách (přibližně 60 %) a na horních končetinách (přibližně 20 %). Jde o chemosenzitivní nádor. Léčbou volby je chirurgie, případně radioterapie u marginálně resekovaných nádorů. Nádory do 5 cm mají výbornou prognózu i bez systémové léčby, pokud jsou kompletně resekovány. Metastazování je v těchto případech vzácné. Pacienti s nádory většími než 5 cm mají benefit ze systémové léčby kombinací doxorubicinu a ifosfamidu. Neoadjuvantní léčba je výhodná i jako in vivo debulking před záchovnou operací. 3.5.5 Alveolární sarkom měkkých tkání Jde o nádor s maximem výskytu ve třetí dekádě života. U dětí má maximum výskytu do pěti let života. Typický je nález nebalancované translokace der(17)t(X;17)(p11;q25) s výsledným vznikem fúzního genu APSL-TFE3, který působí jako aberantní transkripční faktor, aktivující dráhu MET kinázy. Potenciálně terapeuticky významná je role TFE3 v indukci imunosupresivního nádorového mikroprostředí, s možností regulace T-efektorovými a T-regulatorními lymfocyty. Průběh bývá zpravidla indolentní. Nádor je chemorezistentní a léčbou volby je chirurgie, případně s adjuvantní radioterapií. Pacienti s lokalizovanou a resekovanou nemocí přežívají v přibližně 70 % případů. Dlouhodobé přežití pacientů s metastázami je přibližně 10 %. Cílená léčiva jako sunitinib, pazopanib, crizotinib vedou ke stabilizaci choroby u většiny MET pozitivních nádorů. Jednoroční celkové přežití v sérii pacientů léčených pro pokročilou nebo metastatickou nemoc crizotinibem bylo 97,4 %. 3.5.6 Světlobuněčný sarkom Je popisován u dětí mezi 2. a 20. rokem věku, maximum výskytu je u mladých dospělých. Typická je v 90 % detekovaná translokace t(12;22)(q13;q12) s výsledným fúzním genem EWS-ATF1. Ta vede k aktivaci MET kinázy. Vyrůstá v okolí šlach a aponeuróz, histologický obraz 43 je podobný kožnímu melanomu. Tendence k šíření do lymfatických uzlin vede k nutnosti sentinelové biopsie. Principy terapie jsou chirurgické s možným podílem chemoterapie jako předoperační léčby. Přežití pacientů léčených MET inhibitorem crizitinibem bylo podobné jako u neselektované skupiny se STS léčených doxorubicinem. Vhodný je tento přístup u MET pozitivních nádorů u pacientů s premorbidní kardiomyopatií. 3.5.7 Desmoplastický kulatobuněčný nádor Vysoce agresivní nádor vyskytující se v tělních dutinách u adolescentů a mladých dospělých se specifickou translokací t(11;22)(q13;q12) s fúzním genem EWS-WT1. Většina pacientů má nádor v břišní dutině nebo v pánvi, často s infiltrací orgánů nebo implantačními metastázami. Volenou léčebnou metodou je multimodální chemoterapie. Resekce víceložiskových nádorů zpravidla není možná. Chemoterapie vede k prodloužení doby do progrese. I přes intenzivní léčbu je pětileté přežití přibližně 15 %, lepší šance mají děti s extraabdominální chorobou. Pokud je dosaženo remise, je šance na přežití v pěti letech přibližně 57 %. 3.5.8 Extrakraniální maligní rhabdoidní tumor Jde o vysoce agresivní nádor dětského věku, maximum výskytu je do dvou let. Typická je přestavba SMARCB1 genu. Léčba je multimodální. Tendence k časnému metastazování je vysoká. Systémová léčba chemoterapií je efektivní u resekovatelných lokoregionálních onemocnění, je kombinovaná bloky VDC a cyklofosfamid s karboplatinou a etoposidem (CyCE). Čtyřleté přežití je přibližně 40 % u nemetastatické choroby. Metastázy znamenají velmi špatnou prognózu s dvouletým přežitím 13 %. Rizikový je věk do 1,5 roku. 3.5.9 Epiteloidní sarkom Ze všech STS u dětí představuje epiteloidní sarkom 2 %. Vyskytuje se s maximem kolem 30. roku života, ale je popsán i u kojenců a starších dětí. Typická je genetická aberace – disrupce SMARCB1 genu (dříve popisovaný jako INI1). Histogeneze je nejasná. Vyskytuje se ve dvou 44 formách – tzv. distální typ, zpravidla jako kožní nebo podkožní léze, šířící se podél aponeuróz a fascií nebo perineurálně a perivaskulárně; může být zaměněn s granulomatózním procesem jiné etiologie, a tzv. konvenční (proximální) typ se znaky podobnými malignímu rhabdoidnímu tumoru včetně disrupce SMARCB1 genu. Má tendenci metastazovat do regionálních lymfatických uzlin, také do plic. Léčba je chirurgická, s adjuvantní radioterapií v případě marginální resekce. Chemoterapie neprokázala benefit, i když může vést ke stabilizaci nemoci. Kinázové inhibitory dosud neprokázaly reprodukovatelný benefit v léčbě této nemoci. Anekdoticky je uváděn benefit eribulinu s minimem nežádoucích účinků. 3.6 Mezinárodní kooperativní skupina EpSSG Mezinárodní spolupráce v oblasti léčby sarkomů měkkých tkání dětského věku probíhá od časné éry dětské onkologie. V německy mluvících zemích, Švédsku a Polsku je etablována skupina Coperative Weichteilsarkom Studiengruppe der GPOH (CWS). Úzká spolupráce probíhala od 70. let minulého století mezi partnery z Itálie, Velké Británie a Francie a položila základ kooperativní skupiny The International Society of Paediatric Oncology (SIOP), která dala základ studiím Malignant Mesenchymal Tumors (MMT). Postupnou konvergencí jednotlivých národních skupin z prakticky celé Evropy, Izraele, Brazílie, Argentiny a nově Austrálie, došlo v roce 2003 ke sjednocení diagnostických a léčebných postupů pod nově založenou kooperativní skupinou European Pediatric Soft Tissue Sarcoma Study Group (EpSSG, viz https://www.epssgassociation.it/en/). Německy mluvící země si ponechaly organizaci v rámci CWS. V rámci EpSSG probíhá spolupráce i v České republice. Od roku 2005 byla provedena dvě rozsáhlá akademická klinická hodnocení. Léčba rabdomyosakromů u dětí – studie EpSSG RMS 2005 a léčba non-rabdomyosarkomů u dětí – EpSSG NRSTS 2005. Z obou těchto studií jsou výstupem dosud nejlepší léčebné výsledky ve srovnání s historickými daty a na to navazující velká publikační aktivita. Studie EpSSG se z počátku potýkaly s rozsáhlými administrativními problémy, které vyplývaly z nově zavedené regulace klinických hodnocení: Clinical trials – Directive 2001/20/EC. Ta klade velké nároky na zodpovědnosti, pojištění, schvalování a povolování a realizaci klinických studií. Problémem byla do té doby v České republice neexistující infrastruktura, která by uvedenou regulaci uvedla do praxe. Konkrétně v zemích s organizační participací národních 45 skupin onkologie (v Německu – GPOH, Deutsche Krebshilfe, v Itálii – AIEOP, ve Francii – SIOP, ve skandinávských zemích – SSG nebo ve Spojeném království – Cancer Research UK) byla aplikace uvedené legislativy podpořena možnostmi již zavedených sekretariátů, kontaktů, finanční a právní podpory. V České republice podobná organizační struktura nebyla k dispozici. Studie EpSSG byly v České republice v začátcích podporovány v rámci Nadačního fondu dětské onkologie Krtek, který je personálně spojen přímo s Klinikou dětské onkologie FN Brno a díky kterému se podařilo tato klinická hodnocení úspěšně zorganizovat a provést. V roce 2020 je již podpora pro organizaci těchto typů akademických studií zahrnuta ve vládním programu Ministerstva zdravotnictví České republiky. Studie EpSSG RMS 2005 byla akademická, multicentrická, mezinárodní, „open label“, prospektivní randomizovaná klinická studie prováděná ve 102 centrech ve 14 zemích (Argentina, Belgie, Brazílie, Česká republika, Francie, Irsko, Itálie, Izrael, Nizozemí, Norsko, Slovinsko, Spojené království, Španělsko, Švýcarsko). Obsahovala dvě randomizační otázky. První byla role doxorubicinu u vysoce rizikových pacientů. Ve studii nebyl prokázán vliv na přežití bez události a na celkové přežití pro tuto skupinu pacientů. Tím se doxorubicin vyřadil z léčby lokoregionálních rabdoymyosarkomů a dále není v této indikaci používán. V Evropě zůstává součástí terapeutických schémat pro metastatické rabdomyosarkomy a některé nonrabdomyosarkomy. Druhou otázkou byla role udržovací chemoterapie. 3.7 Komentář k publikovaným pracím 3.7.1 Udržovací terapie u rabdomyosarkomu V randomizované části RMS 2005 byli do ramene s udržovací chemoterapií zařazeni pacienti, kteří dosáhli indukční léčbou kompletní remise, a jejich vstupní charakteristiky byly nemetastatický rabdomyosarkom buď alveolární histologie bez postižení lymfatických uzlin, nebo embryonální histologie po nekompletní resekci v nepříznivé lokalizaci a věku nad 10 let nebo velikosti tumoru 5 cm, nebo embryonální rabdomyosarkom s uzlinovým postižením (příloha č. 16). V období 20. 4. 2006 až 20. 12. 2016 bylo screenováno 670 pacientů, z nichž 299 nesplnilo vstupní kritéria zařazení do randomizované části studie. Celkově byl randomizováno 371 46 pacientů, z nichž 185 léčbu ukončilo v kompletní remisi po intenzivní chemoterapii sestávající z devíti MTD bloků, a u 182 léčba pokračovala ve formě nízkodávkované chemoterapie cyklofosfamid p.o. a vinorelbin i.v. Kritéria modifikace léčby obsahovala podmínky redukce léčiv tak, aby nedocházelo k neutropeniím s absolutním počtem neutrofilů pod 1 000/µl. I přes takto nastavená kritéria byla teplota v neutropenii zaznamenána ve 24 % případů, nonneutropenická infekce v 5 % případů. Po těchto epizodách byla chemoterapie redukována tak, aby nezpůsobovala další neutropenii, raději, než aby byla zcela vysazena. Medián sledování pacientů byl v době analýzy 60,3 měsíců. Výsledky analýzy přežití ukázaly, že pacient s udržovací chemoterapií měli přežití bez onemocnění 77,6 %, pacienti bez udržovací chemoterapie 69,8 % (HR 0,68, p = 0,061), celkové pětileté přežití bylo 86,5 % pro pacienty s udržovací chemoterapií a 73,7 % bez udržovací chemoterapie (HR 0,52, p = 0,0097). Toto zjištění je po třech dekádách kooperativních projektů v oblasti dětské onkologie u solidních nádorů poprvé, kdy byl prokázán přínos pro celkové přežití u nového chemoterapeutického schématu. Je možné, že udržovací chemoterapie má vliv na přežití i v případě, že pacient bude mít v budoucnu relaps rabdomyosarkomu. V analyzované populaci byl u pacientů s relapsem po udržovací terapii zjištěn relaps o 3 měsíce později než u pacientů bez udržovací chemoterapie. Mechanismem účinku této udržovací chemoterapie by mohl být antiangiogenní a imunomodulační efekt, což by mohlo vysvětlit, proč je relaps zaznamenán později u těch pacientů, kteří udržovací chemterapii měli. Pozorovaným efektem je v této studii menší riziko lokoregionálních relapsů než metastatických. Lokoregionální relapsy jsou hlavní příčinou selhání léčby a mortality. Udržovací chemoterapie je významným prvkem léčby u dětských akutních lymfoblastických leukémií. U solidních tumorů je tato studie první, která prokázala její efekt. Dávka cyklofosfamidu je relativně nízká, 25 mg/m2 tělesného povrchu. I přesto je nutné další sledování pacientů k vyloučení pozdní toxicity, především ve formě poškození gonád, známého u blokově podávaného cyklofosfamidu a ifosfamidu a sekundárních malignit. 3.7.2 Prognostický vliv genové fúze u alveolárních rabdomyosarkomů s postižením regionálních lymfatických uzlin Součástí vyšetření nádorové tkáně bylo v randomizované části studie EpSSG RMS2005 i vyšetření stavu fúzních genů typických pro alveolární rabdomyosarkom (ARMS) PAX3- 47 -FOXO1 nebo PAX7-FOXO1 a určení jejich prognostické hodnoty (příloha č. 17). U 70 % alveolárních rabdomyosarkomů je jedna z těchto fúzí přítomna. Předpokládá se její horší prognostický vliv na přežití. Cílem této části studie bylo zjistit, zda má stav fúzních genů vliv na parametry přežití při prospektivním sledování. Léčba sestávala z podání 9 bloků MTD chemoterapie ifosfamid/vinkrisitn/actinomycin D a v prvních čtyřech blocích byla intenzifikována o doxorubicin. Lokoregionální léčba byla podána po čtvrtém bloku chemoterapie a sestávala z odložené chirurgické resekce a radioterapie jak na místo primárního nádoru, tak na postižené lymfatické uzliny, bez ohledu na radikalitu resekce (s výjimkou ztrátových končetinových výkonů). Následovala udržovací chemoterapie vinorelbin a nízkodávkovaný cyklofosfamid. Molekulárně biologická analýza byla prováděna v každé z participujících zemí. Jako fúze pozitivní byly označeny nádory s FISH nebo RT-PCR prokázanou pozitivitou v PAX3-FOXO1 nebo PAX7-FOXO1. Do kohorty ARMS/N1 bylo zařazeno 103 pacientů, z nichž u 85 byla analýza provedena. FOXO1 disrupce byla detekována u 56 pacientů, u 28 byla negativní a u 1 pacienta byl vzorek neadekvátní pro analýzu. Medián sledování pacientů byl 64,9 měsíců. Pětileté přežití bez události bylo lepší ve skupině pacientů bez přítomnosti disrupce FOXO1 genu, 74,4 %, u pacientů s pozitivní disrupcí FOXO1 genu bylo 43 %. Pětileté celkové přežití bylo v těchto skupinách 74,7 % a 43,5 %. Při multivariantní analýze je přítomnost FOXO1 disrupce negativním prognostických faktorem. V univariantní analýze byly jako nepříznivé prognostické faktory identifikovány nepříznivá lokalizace primárního nádoru (tj. jiná než orbita, neparameningeální hlava a krk, vagína, uterus, paratestikulární), invazivita primárního nádoru (T2), přítomnost FOXO1 translokace a klinické stádium IRS III. Z analýzy přežití vyplývá, že pacienti, kteří progredovali po léčbě první linie, měli šanci na přežití pouze 5 %, a proto je legitimní těmto pacientům nabízet inovativní nebo experimentální léčbu prakticky ihned v době relapsu. Lokoregionální relapsy byly zaznamenány ve 42 % případů všech událostí. Lokoregionální staging by měl zahrnovat i „vmezeřené“ uzlinové oblasti, ideálně vyšetřené pomocí FDG-PET, tak aby v případě pozitivit mohl být proveden odběr k vyšetření i těchto uzlin (např. na předloktí, a nejen v kubitě v případě nádoru na ruce) a tím aplikována radioterapie na celou postiženou skupinu lymfatických uzlin. Pro další generaci diagnostických postupů je doporučeno vyšetření i na jiné fúzní partnery u genu PAX3 pomocí FISH ke zjištění jeho disrupce. Praktickým výstupem této studie je 48 budoucí zařazení pacientů s ARMS a N1 postižením do stejně rizikové skupiny jako metastatické rabdomyosarkomy, a tím intenzifikovat léčbu s šancí na zlepšení přežití. 3.7.3 Strategie léčby infantilních fibrosarkomů. Jedním z témat v léčebném protokolu EpSSG NRSTS 2005 byla strategie léčby infantilních fibrosarkomů (příloha č. 18). Tyto nádory se vyskytují v časném věku, jde o nejčastěji se vyskytující nádor měkkých tkání ve věku do jednoho roku, charakteristická je u nich translokace ETV6-NTRK3, vyskytující se u většiny případů. Cílem projektu bylo prospektivně vyhodnotit konzervativní strategii léčby. Od října 2005 do června 2012 byli do studie prospektivně zařazení pacienti z EpSSG center, staging byl proveden podle TNM a IRS klasifikace. Zařazení byli pacienti s nálezem ETV6-NTRK3 fúze nebo s negativní fúzí, ale konfirmovanou histopatologickou diagnózou s centrálním mezinárodním čtením. Léčba byla doporučena chirurgickou resekcí v případě, že bylo možné očekávat čisté resekční okraje a chirurgický zákrok nevedl k mutilaci nebo kosmeticky nepřijatelnému efektu. Pokud byl pooperační výsledek IRS I nebo II (R0 nebo R1 resekce), byli pacienti dále sledováni. Neoadjuvatní chemoterapie byla podána pacientům jako vinkristin a aktinomycin D, kromě pacient mladších 3 měsíců, u kterých byla zvolena strategie „wait and watch“, protože i spontánní regrese jsou možné. Do studie bylo zařazeno 50 pacientů, z toho 19 pacientů ve skupině IRS I-II a 31 pacientů ve skupině IRS III. Chemoterapie byla podána 27 pacientům s mediánem trvání léčby 4,14 měsíce, u 4 pacientů byla zvolena vyčkávací strategie. Cekově byla provedena resekce u 40 pacientů (80 %), z toho 19 jich mělo resekci samotnou, 21 s chemoterapií, tři výkony byly mutilující. V době analýzy bylo 35 pacientů v první kompletní remisi, 7 pacientů ve druhé kompletní remisi, 2 pacienti s reziduálním nálezem, 3 zemřeli a 3 byli ztraceni pro další sledování. Jedno z úmrtí bylo zapříčiněno 100násobným předávkováním aktinomycinu D. Celková odpověď na chemoterapii byla 62,9 %. Adherence k protokolárním doporučením byla velmi dobrá, protože 94,7 % pacientů ve skupině IRS I-II bylo iniciálně léčeno operačním zákrokem a 93,3 % dostalo chemoterapii vinkristin a aktinomycin D. Tím bylo prakticky dosaženo standardizace terapie infantilních fibrosarkomů v centrech napříč Evropou. Diagnosticky byla fúze ETV6-NTRK3 49 vyšetřena u 87,2 % pacientů. Dříve v některých případech v první linii používané chemoterapeutické režimy s alkylanciem nebo antracyklinem mohou být na základě této studie opuštěny, což vede k významně menší zátěži pacientů s ohledem na akutní i pozdní následky léčby. 3.7.4 Léčba maligních rhabdoidních tumorů Dalším projektem v rámci EpSSG NRSTS 2005 protokolu byla léčby maligních rhabdoidních tumorů (příloha č. 19). Extrakraniální maligní rhabdoidní tumory jsou velmi agresivní malignita, s nízkou incidencí 0,6 případů na milion dětí, letalita je vysoká s přežitím do 33 % a poslední dekády nedošlo k jakémukoliv zlepšení ve výsledcích přežití. Do studie byli zařazeni pacienti, kteří měli histopatologickou diagnózu podpořenou buď imunohistochemicky negativitou barvení INI1 nebo delecí SMARCB1 genu. Plán léčby byla 30týdenní intenzivní chemoterapie spolu s radioterapií na místo primárního tumoru nebo metastáz, bez ohledu na chirurgickou radikalitu. Od prosince 2005 do června 2014 bylo do studie zařazeno 110 pacientů, z nich 10 nebylo dále analyzováno pro non-adherenci doporučeného postupu, buď diagnostického, nebo terapeutického. Medián věku byl 1,4 roku, většina ze 77 pacientů měla lokoregionální nemoc, 13 pacientů mělo nádor vrozený (diagnostikován do 4 týdnů věku). Kompletní resekce v první době byla provedena u pouze 8 pacientů, 54 pacientů nedostalo radioterapii, z toho 39 progredovalo dříve, než dospěli k doporučenému termínu radioterapie, a 15 z důvodu rozhodnutí lékaře pro velmi nízký věk (do 1 roku). Medián sledování pacientů byl 44,6 měsíců. Pro celou kohortu bylo zjištěno tříleté přežití bez události 32,3 % a celkové přežití 38,4 %. Pro pacienty s lokoregionální nemocí bylo čtyřleté celkové přežití 40,1 %, pro metastatickou chorobu bylo dvouleté celkové přežití 13 %. Pacienti se stagingem IRS II měli identifikován jako významný prognostický faktor pro přežití dosažení kompletní remise s čtyřletým celkovým přežitím 66,3 %. Významně horší přežití měli pacienti diagnostikovaní do jednoho roku života, jejich čtyřleté přežití bylo 21,1 %. Iniciální chirurgická resekce nebyla spojena s výhodou pro přežití oproti pacientům s dosažením chirurgické remise později během léčby. 50 Ve srovnání s dříve publikovanými sériemi bylo v EpSSG skupině více pacientů s extrarenálními nádory, považovanými za prognosticky horší. I přesto bylo dosaženo lepších než dříve publikovaných výsledků přežití. V současné době je k dispozici cílené léčivo tazemetostat, který je efektivním inhibitorem EZH2 metyltransferázy. Ta je významnou katalytickou podjednotkou komplexu PRC2, mediátoru trimetylace H3K27, který je významný hráčem v onkogenní transformaci. V případě SMARCB1 a SMARCA4 mutovaných nádorů dochází v chromatin remodelujícím komplexu SWI/SWF, který je epigenetickým tumor supresorovým komplexem, k vychýlení rovnováhy diferenciace a proliferace buňky, což vede k tumorigenezi mediované komplexem PCR2. Inhibice podjednotky EZH2 tazemetostatem vede k navození původně vychýleného rovnovážného stavu mezi diferenciací a proliferací. Klinická aktivita tazemetostatu byla potvrzena u celého spektra nádorů, u nichž je EZH2 aktivita zvýšena – nehodgkinských lymfomů, synoviálního sarkomu, mesotheliomu a INI1 negativních nádorů. Klinické použití je od léta 2020 schváleno FDA pro pacienty s epiteloidním sarkomem a folikulárním lymfomem s mutací v EZH2 genu. Pro dětské pacienty je možné podání v rámci firemního „early access“ programu, do kterého jsou na KDO FN Brno zařazení pacienti s INI1 negativními nádory bez možnosti prioritní léčby. 3.8 Souhrn Léčba sarkomů měkkých tkání dětí a adolescentů je komplexí a vyžaduje multidisciplinární přístup. Nové poznatky z randomizovaných prospektivních studií, na kterých se pracoviště KDO LF MU a FN Brno podílí, jsou příkladem, že zařazení pacienta do multicentrické studie vede k lepším léčebným výsledkům při adekvátní adherenci k doporučeným postupům a generuje nové léčebné postupy rychle uváděné do klinické praxe. Spolu s inovativními léčebnými postupy a přístupem k firemním programům a klinickým hodnocením může pacientovi nabídnout maximum možného v případě refrakterních a relabujících nádorů, a to s reálnou vyhlídkou na léčebný úspěch. 51 4 Seznam zkratek ABCD amphotericin B lipid complex; lipidový komplex amfotericinu B ADCC antibody-dependent cell-mediated cytotoxicity ADCP antibody-dependent cellular phagocytosis AML akutní myeloidní leukemie ARMS alveolar rhabdomyosarcoma;alveolární rabdomyosarkom CAR chimeric antigen receptor; chimerický antigenní receptor CELL Czech Leukemia Study Group for Life; Česká leukemická skupina - pro život COMBAT combined oral maintenance biodifferentiating and antiangiogenic therapy; kombinovaná perorální udržovací biodiferenciační a antiangiogenní terapie COX-2 cyklooxygenáza -2 CML chronická myeloidní leukémie CMV cytomegalovirus CRISPR clustered regularly interspaced short palindromic repeats HDACs histone deacetylase inhibitors HPV human papiloma virus; lidský papilomavirus FDA food and drug administration FOP fibrodysplasia ossificans progressiva GIST gastrointestinal stromal tumor; gastrointestinální stromální nádor IA invazivní aspergilóza IC invazivní kandidóza IFS infantilní fibrosarkom IMT inflamatorní myofibroblastický tumor ITCC innovative therapies in children with cancer 52 IRS intergroup rhabdomyosarcoma studies KH klinické hodnocení MDSC myeloid-derived supressor cells MPNST malignant peripheral nerve sheet tumour MTD maximum tolerated dose; maximálně tolerovaná dávka NRSTS non-rhabdomyosarcoma of soft tissue; non-rabdomyosarkom měkkých tkání RMS rhabdomyosarcoma; rabdomyosarkom STS soft tissue sarcoma; sarkom měkkých tkání TKI tyrozinkinázový inhibitor TMB-H tumour mutation burden-high; vysoká mutační nálož 53 5 Seznam příloh [1] HLAVACKOVA, Eva, Katerina PILATOVA, Dasa CERNA, Iveta SELINGEROVA, Peter MUDRY, Pavel MAZANEK, Lenka FEDOROVA, Jana MERHAUTOVA, Lucie JURECKOVA, Lukas SEMERAD, Rita PACASOVA, Lucie FLAJSAROVA, Lenka SOUCKOVA, Regina DEMLOVA, Jaroslav STERBA, Dalibor VALIK a Lenka ZDRAZILOVA-DUBSKA. Dendritic Cell-Based Immunotherapy in Advanced Sarcoma and Neuroblastoma Pediatric Patients: Anticancer Treatment Preceding Monocyte Harvest Impairs the Immunostimulatory and AntigenPresenting Behavior of DCs and Manufacturing Process Outcome. Frontiers in Oncology [online]. 2019, 9, 1034. ISSN 2234-943X. Dostupné z: doi:10.3389/fonc.2019.01034. [2] FEDOROVA, Lenka, Peter MUDRY, Katerina PILATOVA, Iveta SELINGEROVA, Jana MERHAUTOVA, Zdenek REHAK, Dalibor VALIK, Eva HLAVACKOVA, Dasa CERNA, Lucie FABEROVA, Pavel MAZANEK, Zdenek PAVELKA, Regina DEMLOVA, Jaroslav STERBA a Lenka ZDRAZILOVA-DUBSKA. Assessment of Immune Response Following Dendritic Cell-Based Immunotherapy in Pediatric Patients With Relapsing Sarcoma. Frontiers in Oncology [online]. 2019, 9, 1169. ISSN 2234-943X. Dostupné z: doi:10.3389/fonc.2019.01169. 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Case report: rapid and durable response to PDGFR targeted therapy in a child with refractory multiple infantile myofibromatosis and a heterozygous germline mutation of the PDGFRB gene. Bmc Cancer [online]. 2017, 17, 119. ISSN 1471-2407. Dostupné z: doi:10.1186/s12885-017-3115-x. [9] SRAMEK, Martin, Jakub NERADIL, Petra MACIGOVA, Peter MUDRY, Kristyna POLASKOVA, Ondrej SLABY, Hana NOSKOVA, Jaroslav STERBA a Renata VESELSKA. Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis. International Journal of Molecular Sciences [online]. 2018, 19(9), 2599. ISSN 1422-0067. Dostupné z: doi:10.3390/ijms19092599. [10] ROHLEDER, O., P. MUDRY, J. NERADIL, H. NOSKOVA, O. SLABY a J. STERBA. Letter to Editor: FS Kaplan, et al., Early clinical observations on the use of imatinibmesylate in FOP: A report of seven cases, Bone (2017). Bone [online]. 2018, 116, 171–171. ISSN 8756-3282. Dostupné z: doi:10.1016/j.bone.2018.08.003. 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ORIGINAL RESEARCH published: 25 October 2019 doi: 10.3389/fonc.2019.01034 Frontiers in Oncology | www.frontiersin.org 1 October 2019 | Volume 9 | Article 1034 Edited by: Christina Annunziata, National Cancer Institute (NCI), United States Reviewed by: Daniel Green, Kite Pharma, United States Oladapo Yeku, Massachusetts General Hospital Cancer Center, United States *Correspondence: Lenka Zdrazilova-Dubska dubska@mou.cz †These authors have contributed equally to this work and are listed in alphabetical order Specialty section: This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology Received: 19 July 2019 Accepted: 24 September 2019 Published: 25 October 2019 Citation: Hlavackova E, Pilatova K, Cerna D, Selingerova I, Mudry P, Mazanek P, Fedorova L, Merhautova J, Jureckova L, Semerad L, Pacasova R, Flajsarova L, Souckova L, Demlova R, Sterba J, Valik D and Zdrazilova-Dubska L (2019) Dendritic Cell-Based Immunotherapy in Advanced Sarcoma and Neuroblastoma Pediatric Patients: Anti-cancer Treatment Preceding Monocyte Harvest Impairs the Immunostimulatory and Antigen-Presenting Behavior of DCs and Manufacturing Process Outcome. Front. Oncol. 9:1034. doi: 10.3389/fonc.2019.01034 Dendritic Cell-Based Immunotherapy in Advanced Sarcoma and Neuroblastoma Pediatric Patients: Anti-cancer Treatment Preceding Monocyte Harvest Impairs the Immunostimulatory and Antigen-Presenting Behavior of DCs and Manufacturing Process Outcome Eva Hlavackova1,2† , Katerina Pilatova1,3† , Dasa Cerna2 , Iveta Selingerova3 , Peter Mudry2 , Pavel Mazanek2 , Lenka Fedorova1,3 , Jana Merhautova1 , Lucie Jureckova1 , Lukas Semerad4 , Rita Pacasova5 , Lucie Flajsarova1 , Lenka Souckova1,2 , Regina Demlova1 , Jaroslav Sterba1,2 , Dalibor Valik1,3 and Lenka Zdrazilova-Dubska1,3 * 1 Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czechia, 2 Department of Pediatric Oncology, University Hospital and Faculty of Medicine, Masaryk University, Brno, Czechia, 3 Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czechia, 4 Department of Internal Medicine-Hematology and Oncology, University Hospital and Medical Faculty, Masaryk University, Brno, Czechia, 5 Transfusion and Tissue Department, University Hospital Brno, Brno, Czechia Despite efforts to develop novel treatment strategies, refractory and relapsing sarcoma, and high-risk neuroblastoma continue to have poor prognoses and limited overall survival. Monocyte-derived dendritic cell (DC)-based anti-cancer immunotherapy represents a promising treatment modality in these neoplasias. A DC-based anti-cancer vaccine was evaluated for safety in an academic phase-I/II clinical trial for children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors, mainly sarcomas and neuroblastomas. The DC vaccine was loaded with self-tumor antigens obtained from patient tumor tissue. DC vaccine quality was assessed in terms of DC yield, viability, immunophenotype, production of IL-12 and IL-10, and stimulation of allogenic donor T-cells and autologous T-cells in allo-MLR and auto-MLR, respectively. Here, we show that the outcome of the manufacture of DC-based vaccine is highly variable in terms of both DC yield and DC immunostimulatory properties. In 30% of cases, manufacturing resulted in a product that failed to meet medicinal product specifications and therefore was not released for administration to a patient. Focusing on the isolation of monocytes and the pharmacotherapy preceding monocyte harvest, we show that isolation of monocytes by elutriation is not superior to adherence on plastic in terms of DC yield, viability, or immunostimulatory capacity. Trial patients having undergone monocyte-interfering pharmacotherapy prior to monocyte harvest was associated with an impaired DC-based immunotherapy product outcome. Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome Certain combinations of anti-cancer treatment resulted in a similar pattern of inadequate DC parameters, namely, a combination of temozolomide with irinotecan was associated with DCs showing poor maturation and decreased immunostimulatory features, and a combination of pazopanib, topotecan, and MTD-based cyclophosphamide was associated with poor monocyte differentiation and decreased DC immunostimulatory parameters. Searching for a surrogate marker predicting an adverse outcome of DC manufacture in the peripheral blood complete blood count prior to monocyte harvest, we observed an association between an increased number of immature granulocytes in peripheral blood and decreased potency of the DC-based product as quantified by alloMLR. We conclude that the DC-manufacturing yield and the immunostimulatory quality of anti-cancer DC-based vaccines generated from the monocytes of patients were not influenced by the monocyte isolation modality but were detrimentally affected by the specific combination of anti-cancer agents used prior to monocyte harvest. Keywords: dendritic cells, anti-cancer medications, sarcoma, neuroblastoma, cell-based medicinal products, investigator-initiated clinical trial, manufacturing outcome variability INTRODUCTION Several progressive and relapsing malignancies in pediatric patients have dismal life prognosis. Refractory neuroblastoma and refractory or metastatic sarcoma have an especially poor prognosis, with no consistently curative treatments available. Oberlin et al. (1) published a meta-analysis of North American and European studies on primary metastatic sarcomas and welldefined risk factors that—where two or more are present at presentation—distribute patients into a subgroup with only a 14% event-free and overall survival probability at 3 years from diagnosis. Patients over 10 years of age with limb primary or “other site” primary tumors with the alveolar subtype of rhabdomyosarcoma, bone marrow or bone involvements, and more than three metastatic sites are defined as having markers for a worse prognosis (1). Similar results were published in a study of relapsed rhabdomyosarsomas, with the prognosis for survival being < 10% at 5 years (2). In high-risk neuroblastoma, survival after relapse is poor, and the usual life expectancy is < 6 months. Based on our experience, patients with neuroblastomas with a high MIBG score after induction therapy have very poor 2-year survival (3). High-risk rhabdomyosarcomas are treated according to several globally accepted protocols with a combination of chemotherapy, surgery, and radiotherapy. Chemotherapy regimens consist of the alkylating agent ifosfamide or cyclophosphamide and vinca alkaloids combined with either etoposide or doxorubicin and actinomycin D. The cytotoxic chemotherapy regimens for relapsed and refractory neuroblastoma typically use a combination of camptothecins, topotecan, and irinotecan with agents such as cyclophosphamide and temozolomide, and achieve objective tumor responses but poor long-term outcomes. For such poor-prognosis patients, treatments with innovative and metronomic therapies (e.g., COMBAT, METRO) (4, 5), cell-based immunotherapies (6, 7), and novel molecularly targeted agents (8) are justified and are also effective in many cases, although their long-term effect has yet to be demonstrated. DCs are essential antigen-presenting cells for the initiation, maintenance, and regulation of immune response (9). Active cancer immunotherapy directs the immune system to attack tumor cells by targeting tumor-associated antigens. We manufacture a fully personalized monocyte-derived dendritic cell-based vaccine that was evaluated in the investigatorinitiated clinical trial “Combined antitumor therapy with ex vivo manipulated dendritic cells producing interleukin-12 in children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors” (EudraCT number 2014-003388-39). The primary endpoint of the trial was an assessment of safety by analysis of the frequency of occurrence of AESI (adverse events of special interest). Vaccines that meet quality control (QC) requirements are registered for use and applied intradermally every 2–4 weeks for up to 35 doses. Dendritic cell-based medical products are mostly manufactured through derivation from monocytes. Autologous monocytes are readily accessible and can be obtained from peripheral blood in sufficient amounts to prepare 107-108 DCs. Monocytes arise from hematological precursors in bone marrow, with a maturation time of 50–60 h (10), and enter the bloodstream for several days until their recruitment into tissues, where they possess the property to mature into tissue macrophages (11). Specifically, the classical CD14++ CD16– subpopulation representing 80–95% of circulating monocytes has a 1-day lifespan in circulation, the intermediate CD14+ CD16+ subpopulation (2–8% of circulating monocytes) has a 4-day lifespan, and the non-classical CD14+ CD16++ subpopulation (2–11% of circulating monocytes) has a 7-day lifespan in circulation (12–14). Monocyte count and function are influenced by various anti-cancer agents. Nevertheless, the published data on the impact of particular anti-cancer agents on the development and function of monocytes are scarce in comparison with those on hematologic toxicity Frontiers in Oncology | www.frontiersin.org 2 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome toward neutrophils and lymphocytes. As most anti-cancer agents target DNA, they interfere with dividing cells including hematopoetic cells. Also, tyrosine kinase inhibitors (regorafenib, sunitinib, sorafenib) are associated with adverse events including hematological toxicities (15). Regorafenib hematological toxicity has been explained by the TK inhibition of FMS like tyrosine kinase 3 (FLT-3) and stem cell factor (c-KIT ligand), which represent hematopoietic growth receptors (15, 16). Reduction in the circulating monocyte count after sunitinib has been shown (17). Monocytes are also highly sensitive to the methylating agent temozolomide (TMZ) (18, 19). Cisplatin and carboplatin have been shown to alter monocyte differentiation to favor the generation of IL-10-producing M2 macrophages (20). Various chemotherapeutics affect cell differentiation and the antigen presentation of DCs when treated in vitro during the differentiation process (21). Data are lacking on the potential in vivo impact of hematotoxic agents on the properties of medicinal products from monocyte-derived DCs. During the manufacture of DC-based anti-cancer immunotherapy under stringent GMP-compliant conditions, we experienced highly variable final product parameters in terms of both DC yield and immunostimulatory properties, and we hypothesized that hematotoxic anti-cancer therapy preceding monocyte harvest may influence the quality of DC-based medicinal products. The issue of the effect of pharmacotherapy on the quality of human monocyte-derived DCs cannot be reliably assessed in mimicked conditions by in vitro pretreatment of monocytes by anti-cancer agents. Thus, data addressing this issue can only be gathered retrospectively from real-life clinical conditions, such as our clinical trial, though with a limited number of patients included. Here, the Phase-I/II clinical trial protocol designed for heavily pre-treated cancer patients with heterogenic anti-cancer therapeutic protocols allows us to observe and analyze the effect of pharmacotherapy on the quality and presumably also on the anti-cancer action of ex vivo-manufactured DCs. Therefore, our primary aims were to analyze the impact of (i) cytotoxic and targeted anti-cancer therapy preceding monocyte harvest and (ii) variability in the complete blood count on the quality of DC-based anti-cancer immunotherapy in high-risk sarcoma and neuroblastoma patients, representing the two main diagnoses in the DC clinical trial. A secondary aim was to reveal whether monocyte isolation by elutriation is superior to the isolation of monocytes through their adherence to plastic cultivation flasks. METHODS Patients and Clinical Trial Clinical Trial Eligibility and Allowed Medication Patient eligibility/inclusion criteria for the clinical trial included being 1–25 years old male/female with histologically confirmed refractory, relapsing, or primarily metastatic high-risk tumors and having a performance status (Karnofsky or Lansky score) ≥ 50 and a life expectancy of longer than 10 weeks. Patients had to be clinically eligible for the surgical procedure to harvest tumor tissue for histological verification and tumor antigen extraction. Female patients had to have had a negative pregnancy test. All patients had to have adequate bone marrow, kidney, liver, and heart function, defined as absolute neutrophil count (ANC) ≥ 0.75 × 109/L, thrombocytes ≥ 75 × 109/L, hemoglobin 80 g/L, estimated glomerular filtration rate (eGFR) ≥ 70 mL/min/1.73 m2, serum creatinine ≤ 1.5-fold the upper limit for the appropriate age, bilirubin ≤ 1.5-fold the upper limit for the appropriate age, AST and ALT ≤ 2.5-fold the upper limit for the appropriate age, ejection fraction ≥ 50%, and fractional shortening ≥ 27% as assessed by echocardiography. In the case of bone marrow infiltration, the allowable ANC was ≥ 0.5 × 109/L and blood platelets 40 × 109/L. In case of liver metastases, AST and ALT had to be ≤ 5fold the upper limit for the appropriate age. The exclusion criteria were as follows: seropositivity to HIV1,2, Treponema pallidum, hepatitis B or C, known hypersensitivity to the study medication, autoimmune disease that was not adequately treated, uncontrolled psychiatric disease, or uncontrolled hypertension defined as systolic and diastolic blood pressure over the 95th percentile for the appropriate age and height (patients ≤ 17 years old) or ≥ 160/90 mmHg or diastolic blood pressure ≥ 90 mmHg (patients ≥ 17 years old). Patients previously treated with dendritic cells or participating in another clinical trial during the 30 days before enrollment were not eligible to enter this clinical trial. The allowed medication prior to monocyte harvest (leukapheresis) was as follows: metronomic chemotherapy, immune checkpoint inhibitors, and anti-CD20 antibodies were allowed as concomitant medication for any time before leukapheresis. Monoclonal antibodies (except anti-CD20), high-dose chemotherapy, and high-dose corticoids had to have been withdrawn at least 3 weeks prior to leukapheresis with the exception of corticoid treatment of brain edema, which was allowed. Since November 2017, an amendment has been made to the procedure for monocyte harvest, and tyrosine kinase inhibitors have to be withdrawn according to their half-life: drugs with a short half-life of 3–14 h must be withdrawn at least 2 days before leukapheresis (axitinib, dabrafenib, dasatinib, ibrutinib, idelalisib, nintedanib, ruxolitinib, and trametinib), drugs with a medium half-life of 15–35 h at least 7 days before leukapheresis (alectinib, bosutinib, lapatinib, lenvatinib, nilotinib, osimertinib, pazopanib, ponatinib, regorafenib, and non-TKI everolimus), and drugs with a long half-life of 36–60 h at least 12 days before leukapheresis (afatinib, ceritinib, erlotinib, gefitinib, imatinib, cabozantinib, crizotinib, sorafenib, sunitinib, vemurafenib, and non-TKI temsirolimus). Myelopoietic growth factors have to be withdrawn at least 7 days before leukapheresis/monocyte harvest. Evaluation of Preceding and Concomitant Therapy A precise analysis was performed of preceding and/or concomitant therapy 60 days before monocyte harvest for clinical trial subjects with neuroblastoma and sarcoma diagnoses. Data were mined from the clinical trial electronic case report form and the subjects’ medical records. We particularly focused on therapeutic agents with a potential impact on the generation of DCs from monocytes and on DC immunostimulatory properties. These agents and the reports on their role in monocyte biology are summarized in Supplementary Table 1. Frontiers in Oncology | www.frontiersin.org 3 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome DC Manufacture and Quality Control Dendritic cell vaccine manufacture encompassed two phases— (i) preparation of tumor lysate as a source of the patient’s tumor antigens and (ii) preparation of monocyte-derived DCs and their loading with tumor lysate. Quality control tests evaluated safety (negativity for pathogens), identity (cell immunophenotype), viability, and functions (cytokine production, stimulation of Tcells). The flow and decision tree of the manufacturing process is shown in Supplementary Figure 1. Self-Tumor Antigen Extraction Tumor lysate was prepared from the tumor tissue obtained from the patient during curative surgery or extended biopsy. In Clean Rooms, necrotic areas and connective tissue were removed from the tumor tissue with a surgical scalpel, keeping the specimen immersed in buffered solution. The remaining tissue was sliced into fragments of about 0.5 mm with a scalpel and forceps and then further crushed with the back of a syringe. Each suspension of tumor fragments and cells in HBSS was lysed through repeated (5 times) freezing in liquid nitrogen and thawing at 37◦C. The crude tumor lysate was centrifuged at 450 g/7 min/4◦C to remove particulate components. The tumor lysate was released for DC manufacture if the following criteria were met: (i) presence of viable tumor cells reported by a histopathologist, (ii) protein concentration, and (iii) microbiological sterility. Peripheral Mononuclear Cell Collection Monocytes were harvested as part of the mononuclear white blood cell (WBC) fraction. Mononuclear cells were collected from the peripheral blood of the patient using the Terumo BCT Spectra Optia Apheresis System. For collection, we used either an intermittent or continuous leukapheresis system. Due to its superior collection efficacy and easier procedure settings, we have preferred the continuous leukapheresis system since April 2018. A citrate dextrose solution, solution A (ACD-A), was used as an anticoagulant. In patients with a body weight of < 20 kg, anticoagulation with heparin was used to prevent citrate toxicity. The requirement for the minimal WBC count was 3 × 109/L before the initiation of leukapheresis. To prevent risk of bleeding or ischemic complications during and after the procedure, hemoglobin of at least 80 g/L and platelets of at least 30 × 109/L were required. In case of a patient with a body weight of < 20 kg, the leukapheresis set was pre-filled with donor erythrocytes. The aim of the leukapheresis was to obtain 60– 80 mL of concentrate of mononuclear cells with a content of at least 0.5 × 109 monocytes. Subsequent addition of 5% human albumin to the minimum required volume of 80 mL for further processing was allowed. DC Manufacture in Clean Rooms The numbers of WBCs, B-cells and T-cells, monocytes, and granulocytes in the leukapheretic product were evaluated using a hematology analyzer (XT-4000i, Sysmex) and flow cytometer (FC-500, Beckman Coulter) with staining for CD3 (clone UCHT1, Beckman Coulter) and CD19 (clone J3-119, Beckman Coulter). Monocytes for DC manufacture were separated from the leukapheresis product by either elutriation or adherence to a plastic surface. During elutriation (using an Elutra cell separator, Gambro BCT), blood cells were separated on the basis of sedimentation velocity into six fractions, where the last fraction rich in monocytes was used for DC manufacture. Contaminating cells after elutriation were mainly granulocytes with similar sedimentation velocity to monocytes. Five hundred million monocytes adhered for 2–4 h in three 175-cm2 tissue culture flasks with 35 mL of CellGenix R GMP DC Medium at 37◦C/5% CO2 and were then washed with HBSS and processed further. Monocytes seeded from the elutriation product or attached by plastic adherence were then cultivated in three 175cm2 tissue culture flasks with 70 mL of CellGenix R GMP DC medium supplemented with GM-CSF (1000 U/mL, CellGenix R ) and IL-4 (320 U/mL, CellGenix R ) at 37◦C/5% CO2/6 days. On day 3, a fresh 70 mL of medium supplemented with the same concentration of GM-CSF and IL-4 was added to the culture. On day 6, immature DCs were exposed to autologous tumor lysate antigens (10 µg/mL) with added keyhole limpet haemocyanin (KLH, 1 µg/mL), IL-4 (320 U/mL), and GMCSF (1000 U/mL) at 37◦C/5% CO2/for 1.5–2 h. Maturation was induced by lipopolysaccharide (200 U/mL) and interferon-γ (50 ng/mL) for an additional 6 h at 37◦C/5% CO2. Finally, cells were collected using accutase (Accutase R , Corning), counted in a Bürker cell chamber and frozen in aliquots of 2 × 106 DCs in 100 µL of freezing medium CryoStor R CS2 at -80◦C. All doses of the DC-based investigational medical product (IMP) named “MyDendrix R ” were stored at -150◦C until administration to the patient. Quality Control of DC-Based Investigational Medicinal Product DC characteristics were evaluated as a part of the quality control process of IMP from an aliquot of manufactured DC from each batch. The cryotube with DC was removed from a deep freezing box (-150◦C) into a laminar flow box, quickly and gently thawed in hand while avoiding shaking, 1 mL of cold (2– 8◦C) DC medium (CellGenix R GMP-grade) was slowly added to the thawed DCs, and the DC suspension was transferred into 2 mL of cold DC medium. The DC suspension was handled at room temperature and processed immediately. DCs (8 × 105 cells) were seeded into 1 well of a 6-well culture plate for sensitive adherent cells (Sarstedt, TC Plate 6-well, Cell+, growth area 8.87 cm2) and cultured in 3 mL of DC medium for 2 days (37◦C/5% CO2) to obtain (i) medium containing cytokines produced by DCs during cultivation and (ii) mature DCs for phenotypic evaluation after 2 days of postthaw cultivation. A 0.5 mL volume of medium containing DCproduced cytokines was collected after 23–25 h upon DC seeding and was centrifuged (10 min/410 g/4◦C), and the supernatant was stored at -25◦C for no longer than 30 days prior to analysis. For immunophenotypic evaluation of mature DCs, both detached and adherent DCs were harvested 47–49 h after DC seeding. The culture medium was collected and pooled with DCs harvested by accutase (0.5 mL/well 8.87 cm2/37◦C) and centrifuged (5 min/410 g/20◦C). The pellet was resuspended in 800 µL HBSS with 0.25% human albumin (Grifols) and processed immediately for immunophenotypic evaluation. Viability quantification was Frontiers in Oncology | www.frontiersin.org 4 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome performed by propidium iodide (PI) exclusion assay. Briefly, 105 DCs were stained with 10 µL of 1% PI in HBSS followed by immediate flow cytometric (Cytomics FC500) analysis of PIpositive events (= non-viable cells). The immunophenotype of DCs was evaluated in post-thaw DCs and in post-cultivation mature DCs. For the detection of each surface molecule, 0.5 × 105 DCs were incubated for 20 min in the dark with the following antibodies: CD80-PC7 (clone MAB104, 10 µL), CD83FITC (clone HB15e, 10 µL), CD86-PE (clone HA5.2B7, 10 µL), CD197-PE (clone G043H7, 10 µL), HLA-DR-PC5 (clone Immu357, 10 µL), CD14-PE (clone RMO52, 10 µL), or isotype controls IgG-PC5 (clone 679.1Mc7, 10 µL), IgG-PC7 (clone 679.1Mc7, 10 µL), IgG2a-FITC (clone 7T4-1F5, 10 µL), or IgG2a-PE (7T4-1F5, 10 µL), all from Beckman Coulter. Flow cytometric analysis was performed using a Cytomics FC500 with CXP software by manual gating on individual parameters, and the discrimination by appropriate isotype control was used to gate and quantify positive events. The concentrations of IL-12 and IL-10 in the DC culture medium were measured by flow cytometric bead assay (BD Biosciences) using internal quality controls (Quantikine R Immunoassay Control Group 1, R&D Systems). Absolute production of IL-12 or IL-10 per 106 DC and the IL-12/IL-10 ratio were calculated. The allogenic (allo) and autologous (auto) stimulatory properties of DCs were examined by mixed lymphocyte reaction (MLR). In allo-MLR, the target cells were the peripheral blood mononuclear cells (PBMCs) obtained from pooled buffy coats from healthy donors. In autoMLR, the target cells were the patient’s lymphocytes separated by centrifugation in a density gradient using Histopaque-1077 (SigmaAldrich, density 1,077 g/mL) from the leukapheresis product obtained for DC manufacture. These pre-vaccination lymphocytes were cryopreserved using CryoStor CS5 medium (BioLife solutions) at -150◦C and thawed prior to auto-MLR seeding. A sample of 107 target lymphocytes were stained with 250 µL 10 µM carboxyfluorescein succidimidyl ester (CFSE, SigmaAldrich) and seeded into a sterile 96-well culture plate (Sarstedt, TC Plate 96-well, Suspension, F) at 105 cells/well in 200 µL of complete X-vivo 10 medium (Lonza) containing 5% inactivated human male AB serum (SigmaAldrich) for the following: (i) 104 DC/well in 10:1 target:effector MLR, (ii) positive control (PC) with phytohemagglutinin (PHA, SigmaAldrich) at a final concentration of 10 µg/mL, or (iii) negative control (NC) with complete X-vivo medium only. MLR experiments were seeded in triplicate and cultured for 6 days at 37◦C/5% CO2. 2 × 104 cells from each well were stained with CD3-PC7 (clone UCHT1, 10 µL/test, Beckmann Coulter) for flow cytometric detection of CFSE fluorescence on CD3+ T cells. Discrimination for dividing cells was set up using NC. T-cell proliferation was calculated as follows: [(average % of dividing T-cells in 10:1 MLR) – (average % of dividing T-cells in NC)] × 100/[(average % of dividing T-cells in PC) – (average % of dividing T-cells in NC)]. Statistical Analysis The Spearman correlation coefficient with a significance test was used to measure the strength of the relationship between patient CBC prior to leukapheresis, the parameters of the leukapheresis product, the DC yield, and the quality control parameters. Differences in parameter values between groups were assessed by the non-parametric Mann-Whitney or KruskalWallis test. Hierarchical clustering analyses were performed using the complete linkage method with the distance based on the Spearman correlation coefficient. The Spearman correlation distance was used for clustering of batches, and the absolute Spearman correlation distance was used for clustering DC parameters. For clustering analyses, DC parameters were centered and scaled (Z-score of parameters). P < 0.05 were considered statistically significant. All statistical analyses were performed with R 3.5.3 software (22). RESULTS Clinical Trial Accrual and Course As of May 2019, 47 subjects were enrolled in the clinical trial, and the manufacturing process of DC-based vaccine was performed in 31 cases. Of these 31, the most common diagnoses were sarcoma, with 19 cases (61%), and high-risk neuroblastoma, with 4 cases (Table 1). In this group of 23 patients, we performed analysis of the manufacturing issues presented here. Sarcomas were specifically: seven Ewing sarcomas (36% of sarcoma pts), five (26%) osteosarcoma, two (11%) alveolar rhabdomyosarcoma, two (11%) embryonal rhabdomyosarcoma, and three (16%) synovial sarcoma (Table 1). The median enrollment age of the clinical trial was 14 years; 15 years for sarcoma patients and 5 years for neuroblastoma patients (Table 1). All 23 study subjects, i.e., 19 with sarcoma and four with neuroblastoma, underwent initial surgery to obtain tumor tissue for the tumor lysatemanufacturing process, and tumor lysates were manufactured without any tumor antigen extraction failure. Monocyte harvest and the subsequent manufacturing of DC-based IMP were performed for all 23 subjects. Out of the 23, 16 DC-based IMPs successfully passed through the manufacturing process and met the quality control criteria for administration to the patients. DC-based IMPs from seven subjects (six sarcoma, one neuroblastoma) were not manufactured or failed to pass quality control due to inadequate immunostimulatory properties (Table 1). The basic patient characteristics are described in Table 1, and the detailed clinical course is summarized in Supplementary Table 2. Dendritic Cell Manufacturing, Its Yield, and DC Quality Including Immunostimulatory Properties We achieved DC yields ranging from 0 to 43.6%, with a mean of 17.2% and an s.d. of 12.7% in this specific cohort. A DC yield equal to 0 represented a manufacturing process that was unsuccessful, with all DCs detached from the flasks. The quality control parameters involved microbial sterility and Mycoplasma spp. negativity, the viability and phenotype of thawed DCs, the phenotype of thawed DCs after 2-day cultivation, the production of IL-12 and IL-10 during 24-h cultivation of thawed DCs, and 6-day allo-MLR and auto-MLR. All batches of DCs fulfilled the microbiological criteria of QC and the criteria Frontiers in Oncology | www.frontiersin.org 5 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome TABLE 1 | DC-based vaccine-manufacturing outcome, basic patient characteristics, therapy preceding monocyte harvest. Primary diagnosis Date of study enrollment/Age in years at study enrollment/Pt No Treatment line prior to monocyte harvest/Treatment and its duration/Date of monocyte harvest DC-based vaccine- manufacturing outcome EWING SARCOMA Ewing sarcoma of the mandible 09/2015; 14; KDO-0101 2nd; VCR/Irino + pazopanib, 09/2015–04/2016; 01/2016 Passed QC Localized Ewing sarcoma of the left femur 02/2016; 12; KDO-0109 3rd; ARST08P1 + sunitinib, 03/2016–06/2016; 03/2016 Did not pass QC Localized Ewing sarcoma of the left distal humerus 02/2016; 12; KDO-0111 2nd; AEWS1031 + pazopanib, 02/2016–08/2016; 05/2016 Did not pass QC Localized Ewing sarcoma of the spine C5-Th2, extradural, and intraspinal involvement 08/2016; 24; KDO-0118 2nd; AEWS1031, 08/2016–02/2017, 2 cycles VTC, 2 cycles VCR/Irino; 01/2017 Passed QC Ewing sarcoma of the pelvis 12/2016; 14; KDO-0121 1st; Euro Ewing 2008, 11/2016–05/2017; 06/2017 Did not pass QC Ewing sarcoma of the left proximal tibia 12/2016; 15; KDO-0122 2nd; VTC cycles, 01/2017–05/2017; 03/2017 Did not pass QC Localized Ewing sarcoma of the left tibia 08/2018; 22; KDO-0144 2nd; 2x TMZ/Irino, 08/2018–10/2018; 10/2018 Did not pass QC OSTEOSARCOMA Localized high-grade osteosarcoma of the right distal femur 09/2015; 10; KDO-0102 4th; VCR/Irino + pazopanib; 12/2015 Passed QC High grade osteoblastic osteosarcoma of the left distal femur 10/2016; 8; KDO-0120 1st; AOST 0331, 10/2016–07/2017; 03/2017 Not manufactured Localized osteoblastic osteosarcoma of the right proximal tibia 01/2017; 18; KDO-0124 3rd; AOST 1321 + VBL + CPM, 02/2017–10/2017; 3/2017 Passed QC Localized osteosarcoma of the right proximal femur 02/2018; 25; KDO-0133 2nd; COMBAT III, 04/2018–12/2018; 04/2018 Passed QC High-grade osteoblastic osteosarcoma of the left distal femur 05/2018; 22; KDO-0139 2nd; AOST0331 – cycle IE 07/2018; 09/2018 Passed QC ALVEOLAR RHABDOMYOSARCOMA Alveolar rhabdomyosarcoma of the right calf 10/2015; 14; KDO-0103 2nd; ARST 0921 + TEM, 11/2015–01/2016; 12/2015 Passed QC Alveolar rhabomyosarcoma, primum ignotum 10/2016; 12; KDO-0119 1st; ARST08P1 + TEM, 10/2016–05/2018; 04/2017 Passed QC EMBRYONAL RHABDOMYOSARCOMA Embryonal rhabomyosarcoma of the pelvis 09/2017; 18; KDO-0131 1st; EpSSG RMS 2005, 09/2017–06/2018; 01/2017 Passed QC Localized embryonal rhabomyosarcoma of the pelvis 07/2018; 15; KDO-0143 3rd; - rEECur - Topo/CYC, 08/2018–12/2018; 09/2018 Passed QC (Continued) Frontiers in Oncology | www.frontiersin.org 6 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome TABLE 1 | Continued Primary diagnosis Date of study enrollment/Age in years at study enrollment/Pt No Treatment line prior to monocyte harvest/Treatment and its duration/Date of monocyte harvest DC-based vaccine- manufacturing outcome SYNOVIALOSARCOMA Synovial sarcoma of the left thigh 04/2016; 14; KDO-0114 1st followed by COMBAT III 05/2015–12/2016; 12/2016 Passed QC Localized synovial sarcoma of the neck 04/2018; 17; KDO-0137 2nd; Modified COMBAT III from 04/2018 + pazopanib from 08/2018; 06/2018 Passed QC Localized synovial sarcoma of the left calf 06/2018; 21; KDO-0141 2nd; COMBAT III modified, 08/2018–02/2019; 10/2018 Passed QC NEUROBLASTOMA Neuroblastoma in the retroperitoneum 04/2016; 12; KDO-0115 2nd; METRO-NB2012, 05/2016–10/2016; 07/2016 Passed QC High-risk neuroblastoma in the left glandula suprarenalis 02/2018; 4; KDO-0135 1st followed by dinutuximab + retinoic acid, 11/2018–02/2019; 02/2019 Passed QC Neuroblastoma in the right retroperitoneum 07/2018; 3; KDO-0142 2nd; ANBL 1221 - 3 cycles TMZ/Irino + dinutuximab, 08/2018–11/2018; 08/2018 Did not pass QC Neuroblastoma in the right glandula suprarenalis 10/2018; 6; KDO-0147 4th; METRO-NB2012, 05/2017–12/2018; 11/2018 Passed QC CPM, cyclophosphamide; Irino, irinotecan; TEM, temsirolimus; TMZ, temozolomide; Topo, topotecan; VBL, vinblastine; VCR, vincristine; IE, ifosfamide etoposid; VTC, vincristine, topotecan, cyclophosphamide; Pt. No., patient number; QC, quality control. Chemotherapy protocols: AEWS1031 (Ewing sarcoma)—vincristine, doxorubcin, cyclophosphamide, ifosfamide, etoposide; AOST0331 (osteosarcoma)—cisplatin, doxorubicine, methotrexate; AOST1321 (osteosarcoma)—denosumab; ARST0921 (refractory or relapsed rhabdomyosarcoma)—bevacizumab, vinorelbine, cyclophosphamide and temsirolimus; ARST1321 (non-rhabdomyosarcoma soft tissue sarcomas)—ifosfamide, doxorubicin, pazopanib; COMBAT III (metronomic)—celecoxib, etoposide, temozolomide, fenofibrate, ergocalciferol, bevacizumab, vinorelbine, cis-retinoic acid; EpSSG RMS 2005 (rhabdomyosarcoma)—ifosfamide, vincristine, actinomycin, doxorubicin; Euro Ewing (Ewing sarcoma)—vincristine, ifosfamide, doxorubicin, etoposide, actinomycin, cyclophosphamide; METRO-NBL2012 (metronomic treatment for neuroblastoma)—etoposide, celecoxib, propranolol, cyclophosphamide, vinblastine; rEECur protocol (relapsed soft tissue sarcoma)— topotecan, cyclophosphamide, irinotecan, temozolomide. Details on anti-cancer therapy dosing are summarized in Supplementary Table 2. of viability, ranging from 85 to 100% with a mean of 95%. Their variability in phenotype and immunostimulatory property is shown in Supplementary Table 3. The mean phenotype of the manufactured DCs immediately after thawing for selected parameters was as follows: CD8019 (range: 2–86%), CD86 91% (76–100%), CD83 21% (0–86%), CD14 20% (1–69%), and CD197 90% (73–99%). The mean phenotype of thawed DCs after 2day cultivation for selected parameters was as follows: CD80 77% (range: 25–97%), CD86 99% (95–100%), CD83 61% (12– 89%), and MHC II 93% (63–100%). Mean cytokine production was as follows: IL-12 8,327 pg/106 DC (range: 9–80,824 pg/106 DC), IL-10 280 pg/106 DC (6–1,731 pg/106 DC), and IL-12/IL- 10 ratio 35 (1–246). The mean in vitro proliferation of T-cells stimulated by manufactured DCs was 67% (29–98%) in allo-MLR and 9% (−3–37%) in auto-MLR. Due to inappropriate results for the immunostimulatory parameters of QC (phenotype, cytokine production, MLR), six out of 22 (27%) of the manufactured batches of DCs were not released for use in the clinical trial. The parameter values of the manufactured batches of DCs are shown in Supplementary Table 3. Isolation of Monocytes by Adherence vs. Elutriation and Its Impact on Manufacturing Process Yield and the Immunostimulatory Parameters of DCs Isolation of monocytes for DC manufacture was performed by elutriation in 14 cases and by plastic adherence in nine (39%) cases based on the real-world situation. Until March 2017, we performed elutriation of the leukapheresis product in all cases (11 cases: KDO-0101, -0102, -0103, -0109, -0111, -0114, -0115, -0118, -0120, -0122, -0124). Between April and September 2018, we performed elutriation in cases KDO-0121, -0137, and -0139, and adherence to plastic in cases KDO-0133, -0142, and -0144 due to there being > 10% neutrophils in the leukapheresis product or technical issues with the Elutra device for KDO-0119 and -0131. After October 2018, we isolated monocytes exclusively by adherence to the plastic surface in all cases: KDO-0135, -0141, -0144, and -0147. Addressing the issue of whether the elutriation process is superior to adherence to plastic retrospectively, we compared the proportions of batches passing QC and their DC yield and phenotypic and immunostimulatory properties under the Frontiers in Oncology | www.frontiersin.org 7 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome two methods. Adherence to plastic resulted in two (22%) batches not being released, and elutriation resulted in five (36%) batches not being released (four did not pass QC and one was not manufactured). The OR (odds ratio) for passing QC in the plastic-adherence modality was 1.94 (95% CI: 0.29–13.19). The DC yield, viability, phenotype, and immunostimulatory properties (IL-12, IL-10, the IL-12/IL-10 ratio, allo-MLR, autoMLR) in adherence to plastic vs. elutriation are summarized in Figure 1. A statistically significant difference was observed between QC results and monocyte isolation modality for the following post-thaw parameters (i) DC expression of CD86 on day 0 that was higher in the manufacturing process with plastic adherence, and (ii) borderline significant expression of CD14 on day 0 that was higher with elutriation. The values of both parameters were in favor of adherence to plastic. It is of note here that the subgroup with isolation of monocytes by the adherence to plastic was not biased by including a higher proportion of cases without potentially monocyte-interfering pharmacotherapy (“m” vs. “0” as described later; p = 0.643). Thus, we conclude that the isolation of monocytes by adherence to plastic is comparable to a manufacturing process with monocyte elutriation. Parameters of CBC Prior to Monocyte Harvest, and Parameters of the Leukapheresis Product and Their Impact on Manufacturing Process Yield and the Immunostimulatory Properties of DCs With the aim of identifying the CBC parameters (shown for each batch in Supplementary Table 3) associated with adequate DC characteristics and thus predicting whether the DCmanufacturing process would pass QC, we analyzed CBC prior to monocyte harvest in the context of batches that fail to pass QC and DC yield, phenotype, and immunostimulatory properties. The presence of immature granulocytes in CBC was associated with unsuccessful manufacturing (p = 0.046). DC yield was not associated with any single parameter of CBC. Expression of CD14 on manufactured cells was negatively correlated with relative lymphocyte count in CBC (p = 0.001) (Figure 2). The level of allogenic MLR was negatively associated with both the presence of immature granulocytes (p = 0.010) and NRBC (p = 0.018) FIGURE 1 | Comparison of two monocyte isolation modalities with respect to dendritic cell (DC) production. Elutriation (white box plots) and adherence to plastic (gray box plots) were compared based on QC parameters: (A) DC yield, and post-thaw: (B) viability, (C) DC phenotype on day 0: CD14, CD197, CD80, CD86, and CD83 and on day 2: MHC II, CD80, CD86, and CD83, and immunostimulatory properties presented by (D) IL-12 production, IL-10 production, and IL-12/IL-10 production ratio, (E) allo-MLR and auto-MLR. Median values are shown for each parameter for each monocyte isolation modality. Black dots show QC results of manufactured DCs that passed quality control, and red dots show results of manufactured DCs that did not pass quality control. Frontiers in Oncology | www.frontiersin.org 8 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome FIGURE 2 | Association of patient CBC prior to monocyte harvest and parameters of leukapheresis product with DC yield and quality control. Red color represents a positive correlation and blue color a negative correlation; strength of relationship is represented by size of square and intensity of color—larger squares with intense color have a stronger association; *p < 0.05, **p < 0.01, ***p < 0.001. in pre-leukapheresis CBC (Figure 2). The level of autologous MLR was positively associated with absolute leukocyte count (p = 0.016) (Figure 2). Similarly, a high proportion of monocytes (p < 0.001) and low proportion of T-cells (p = 0.001) in the leukapheresis product were associated with increased expression of CD14 on manufactured cells (Figure 2). A high proportion of monocytes in the leukapheresis product was associated with increased production of IL-10 by manufactured cells (p = 0.027) (Figure 2). Therapy Preceding and/or Concomitant With Monocyte Harvest and Its Association With Manufacturing Process Yield and the Immunostimulatory Properties of DCs The patient history of anti-cancer treatment and the outcome of DC manufacture were evaluated for an association between DC parameters and lines of therapy classified as 1st, 2nd, and 3rd or subsequent lines that were followed by monocyte harvest for DCs. The history of anti-cancer treatment had no observed impact on the quality of manufactured DCs (Supplementary Figure 2). Pharmacotherapeutics 60 days prior to and/or concomitant to monocyte harvest were classified into two groups and designated as follows (i) “m” (n = 17) for administration of therapy potentially interfering with monocyte viability and/or differentiation, namely TKI, mTOR inhibitors, chemotherapy in cell biology-interfering doses, i.e., MTD-based dose, anti-RANKL mAb, retinoic acid, and/or GCSF (Supplementary Table 1) < 60 days prior to monocyte harvest, (ii) “0” (n = 6) for metronomic therapy/chemotherapy or no potentially monocyte-interfering therapy concomitantly or < 60 days prior to monocyte harvest. All batches from the “0” category passed QC, whereas seven out of 17 (41%) monocytederived DCs from the “m” category failed to be released for patient administration. The OR for passing QC in category “0” was 9.3 (95% CI: 0.5–191). DC yield, DC immunophenotype on day 0 and day 2, and production of IL-10 did not differ between Frontiers in Oncology | www.frontiersin.org 9 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome FIGURE 3 | Treatment prior to monocyte harvest and immunostimulatory properties of manufactured DCs. Manufacturing subgroup from monocytes harvested after MTD-based therapy potentially interfering with monocyte biology (listed in Supplementary Table 1; “m” treatment, gray box plots) and manufacturing subgroup from monocytes from untreated patients or after non-interfering treatment (“0” treatment, white box plots) were compared based on QC parameters: (A) IL-12 production, (B) IL-12/IL-10 production ratio, (C) allo-MLR and (D) auto-MLR. Median values are shown for each parameter for each treatment subgroup. Black dots show QC results of manufactured DCs that passed quality control, and red dots show results of manufactured DCs that did not pass quality control. the “0” and “m” categories (Supplementary Figure 3). Median IL-12 production was 2,424 pg/106 DCs in the “0” category and 743 pg/106 DCs in category “m” (p = 0.083). The median IL- 12/IL-10 ratio was 71 in the “0” category and 9 in the “m” category (p = 0.002). The median T-cell proliferation in alloMLR was 86% in the “0” category and 63% in the “m” category (p = 0.027), and the in auto-MLR was 12% in the category “0” and 5% in category “m” (p = 0.036) (Figure 3). In the analyzed study cohort, therapeutic regimens were heterogenic, with patients often treated with a combination of various compounds prior to monocyte harvest, and thus further categorization into single agent-defined subgroups and their analysis were impossible. Therefore, we performed cluster analysis of DC parameters in the context of therapy prior to monocyte harvest (Figure 4). Here we observed a cluster defined mainly by a superior IL-12/IL-10 ratio but low DC yield comprising batches KDO-0133 without any anti-cancer treatment, KDO-0137 treated with metronomic modified COMBAT with celecoxib, fenofibrate, low-dose cyclophosphamide, and low-dose vinblastine, and KDO-0115 treated with metronomic therapy with low-dose vinblastine, celecoxib, low-dose cyclophosphamide, and propranolol (see Supplementary Table 2 for details on the treatment schedule and dosing). Furthermore, we observed a very similar pattern in DC properties in two batches, KDO-0142 and KDO-0144, that were manufactured from monocytes obtained from patients treated with temozolomide and irinotecan. These batches exhibited robust monocyte differentiation, as represented by their low CD14 expression, but failed to produce IL-12 or an immunostimulatory phenotype when matured, as represented by CD80 on post-cultivation DCs on day 2, and therefore did not meet the QC criteria. A pattern of relatively low DC yield, high production of IL-12, and notable monocyte differentiation and DC immunostimulatory phenotype and function was observed for batches KDO-0147, generated from monocytes from patients treated with celecoxib, and KDO-0141, from patients pretreated with combined metronomic therapy with low-dose vinblastine, low-dose etoposide, celecoxib, cholecalciferol, and fenofibrate. Batches KDO-0103 and KDO-0122 similarly exhibited poor yield, poor monocyte differentiation, a rather low IL-12/IL-10 ratio, and very low immunostimulatory functions toward donor T-cells. Monocytes from both batches were pretreated with an MTD-based combination of topoisomerase inhibitor and alkylating agent, with last administration from day 21 to 17, namely etoposide and ifosfamide in KDO-0103 and topotecan and cyclophosphamide in KDO-0122. This was followed in both cases by 9 days of administration of G-CSF filgrastim up to 7 days prior to monocyte harvest. High DC yield and viability but low markers of differentiation, immunostimulatory phenotype and IL-12/IL-10 ratio were similarly observed for batches KDO-0111 and KDO-0109 treated with topotecan, cyclophosphamide, and pazopanib. Based on features such as good DC yield and viability but low monocyte differentiation and a below-average IL-12/IL-10 ratio, these two batches clustered with KDO-0139 (treated with etoposide, ifosfamide, and filgrastim), KDO-0121 (etoposide, ifosfamide, and filgrastim), KDO-0118 (irinotecan and sunitinib), and KDO-0119 (cyclophosphamide, temsirolimus, and filgrastim). Notably, monocytes affected by retinoic acid (KDO-0135) or anti-RANKL denosumab (KDO-0124) produced DCs of average quality. In summary, monocyte-interfering MTD-based treatment of the clinical trial patients prior to monocyte harvest was associated with an impaired DC-based immunotherapy manufacturing process outcome. Certain combinations of anti-cancer treatments elicited a similar pattern of inadequate DC parameters. Namely, a combination of temozolomide and irinotecan was associated with poor DC maturation and immunostimulatory features, and a combination of pazopanib, topotecan, and MTD-based cyclophosphamide was associated with poor DC differentiation maturation and immunostimulatory parameters. DISCUSSION Here we show that despite strict adherence to the validated manufacturing protocol, the outcome of the manufacture of Frontiers in Oncology | www.frontiersin.org 10 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome FIGURE 4 | Cluster analysis of DC parameters in the context of therapy prior to monocyte harvest. The heatmap on the right shows the immunostimulatory properties of manufactured DCs centered and scaled in the column direction (Z-score of parameters). Clusters are based on correlations. For clustering of DC parameters, but not batches, an equal meaning to positive and negative correlations was considered, and therefore strongly correlated parameters in the positive or negative manner clustered together. The left panel shows the treatment administered within 60 days of monocyte harvest. The day of the mononuclear harvest was set as day 0. An interactive version of the left panel with a detailed description of treatment including dosing is provided in Supplementary Material 1. Metronomic doses of chemotherapeutic drugs and supportive therapy such as vitamins and probiotics are not shown here but are summarized in Supplementary Table 2. Batches that did not pass quality control are indicated in red. the medicinal product with monocyte-derived DCs is highly variable in terms of both DC yield and immunostimulatory properties. Moreover, in 30% of cases, manufacture of DCbased immunotherapy for advanced sarcoma and high-risk neuroblastoma patients resulted in a product that did not meet the specifications for the medicinal product and therefore was not released for application. This product failure rate was higher than in published studies (23, 24). Thus, in an attempt to improve the manufacturing process, to predict DC-manufacturing outcome, and, subsequently, to avoid laborious and costly DC manufacture that would not meet QC specifications, we addressed key variables in the manufacturing process. Namely, we focused on the issues of (i) monocyte isolation from the mononuclear leukapheresis product, (ii) parameters of the patient’s CBC prior to monocyte harvest and parameters of the leukapheresis product, and (iii) anti-cancer therapy preceding monocyte harvest that may interfere with the ability of monocytes to differentiate into immunostimulatory DCs. Regarding the method of monocyte isolation, we assessed whether monocyte extraction by a simple method of adherence to a plastic surface is comparable to the elaborate method of elutriation. During elutriation, monocytes can be contaminated with granulocytes with a similar sedimentation velocity to monocytes. Based on this observation, we validated the DC-manufacturing process with isolation of monocytes by adherence to plastic (25) to avoid contaminants that may interfere with DC differentiation by altering the levels of pro-differentiation cytokines and/or the formation of a suppressing microenvironment through generating decay products during cultivation. By comparative analysis of DC yield and immunostimulatory properties from the manufacturing processes of isolation of monocytes by elutriation vs. adherence to plastic, we conclude that the adherence method is comparable to the elutriation method. The method of adherence to plastic is simple in terms of the equipment, material, and manufacturing steps required and therefore is less costly, less prone to errors, Frontiers in Oncology | www.frontiersin.org 11 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome and more GMP-friendly than the elutriation process. In healthy adult volunteers, monocyte-derived DC yield with monocyte elutriation has been shown to be superior to adherence to plastic (26); this was not observed under our manufacturing conditions of heavily pretreated pediatric sarcoma and neuroblastoma patients. With regards to the pharmacotherapy preceding monocyte harvest, we observed that therapy with agents interfering with the biology of monocytes 60 days prior to monocyte harvest was associated with reduced production of IL-12 and deficient functional immunostimulatory properties of the manufactured DC-based vaccine and subsequently often resulted in QC failure. It is of note here that failures in DC production occurred more often prior to the implementation of stricter criteria for non-allowed pharmacotherapy preceding monocyte harvest. Specifically, we observed impaired monocyte differentiation and, subsequently, inadequate immunostimulatory features in monocytes pretreated with a combination of an MTDbased dose of the alkylating agent cyclophosphamide, topoisomerase I inhibitor topotecan, and TKI pazopanib. We have previously shown that TKI pazopanib in vitro impairs the immunostimulatory properties of monocytes, including up-regulation of the immunoinhibitory surface molecule ILT-3 and decreased capability to up-regulate MHC II in response to LPS (27). Interestingly, however, pretreatment of monocytes in vivo with pazopanib without any other immediate treatment (KDO-0101) did not result in attenuated DC vaccine quality. Topotecan has been shown to partially activate monocytederived DCs but to prevent the full maturation of DCs stimulated with a cocktail of proinflammatory mediators (28). A different pattern was observed for DCs from cases treated with a combination of the alkylating agent temozolomide (TMZ) and the topoisomerase I inhibitor irinotecan (iri), and we observed monocyte differentiation but not DC immunostimulatory properties, resulting in a medicinal product that did not pass QC and was not administered. It is of note that one case was a sarcoma and one a neuroblastoma patient. Moreover, we also observed a similar pattern of poor DC parameters in a case of synovialosarcoma with TMZ/iri therapy in a cohort of patients outside this clinical trial. It has been shown that monocytes are particularly sensitive to the methylating agent temozolomide, undergoing apoptosis, while monocyte-derived DCs and macrophages are resistant to TMZ (19). Briegert and Kaina and Bauer et al. showed that monocytes accumulated single-strand DNA breaks due to failure of the re-ligation step in base excision repair and showed a lack of DNA repair protein expression (18, 19). Following TMZ treatment, monocytes demonstrated an unbalanced expression of DNA repair proteins, impairing base excision repair and the accumulation of doublestranded breaks (18, 19). In vitro studies of TMZ/iri cytotoxicity to neuroblastoma cells have revealed single- or double-stranded DNA damage to be mostly due to SN-38 (the active metabolite of irinotecan) and to be further enhanced through the addition of TMZ (29). Thus, we hypothesize that DNA damage caused by the combination of irinotecan and TMZ in the context of particular hypersensitivity of monocytes to temozolomide may underlie the unfavorable effect of anti-cancer therapy with TMZ/iri on the monocyte-derived immunostimulatory DC-manufacturing process. Monocytes from a patient treated with methotrexate, doxorubicin, and cisplatin failed to produce viable dendritic cells, but monocytes from another patient treated with methotrexate did not fail to produce DC vaccine. Methotrexate has reportedly inducedl apoptosis, reduced viability, induced differentiation, and reduced inflammatory properties of monocytes (30–33), and we may speculate, although based on anecdotal observation, that if combined with cisplatin, thereby shifting monocyte differentiation into an immunosuppressive phenotype (20), methotrexate may result in failure of monocyte-derived DC generation. Regarding the composition of pre-leukapheresis CBC and the derived leukapheresis product and the outcome of DC manufacture, we observed that three interconnected features, i.e., (i) a low relative lymphocyte count, (ii) a high relative neutrophil count in CBC, and (iii) a high proportion of monocytes in the leukapheresis product, were associated with unfavorably high expression of CD14 on the manufactured cell product. Moreover, the presence of an increased number of immature granulocytes was associated with decreased potency of the DC-based product as quantified by allo-MLR. These observations may be underlain by emergency myelopoesis stimulated by G-CSF, which leads to a quantitative and qualitative change in all circulating myeloid cell types including neutrophils, monocytes, and myeloid-derived suppressor cells (34, 35). While fostering granulocyte effector functions, G-CSF also seems to promote immunosuppressive and tolerogenic properties in monocytes and monocyte-derived cells including increased production of IL-10 (36–39). In this context, it is of note that six out of seven cases treated with GCSF within 60 days prior to monocyte harvest exhibited donor T-cell stimulation below the average and that the level of Tcell stimulation decreased with the intensity of G-CSF prior to monocyte harvest. Although the effect of G-CSF treatment on the DC-manufacturing process in our study cannot be dissected from the effect of preceding chemotherapy and targeted therapy, the tentative interpretation is that stimulation of myelopoesis with growth factors of granulocytes may have a rather negative impact on the outcome of the DC-based vaccine-manufacturing process. Here, we show that treatment of patients with certain anticancer agents in MTD-based doses prior to monocyte harvest often leads to failure of manufacture of the immunostimulatory DC-based vaccine. We propose that the optimal time for monocyte harvest for generating DCs is prior to a cellinterfering treatment. With respect to the DC-manufacturing workflow, this would mean, in a majority of cancer patients, the implementation of DC manufacture from cryopreserved monocytes. Several studies have investigated the effect of cryopreservation on monocyte differentiation into DCs, but results have been conflicting. Some studies observed cryopreservation to have no effect on monocyte-derived DC production (40, 41). On the other hand, Silveira et al. showed that, when compared to fresh monocytes, cryopreserved monocytes exhibited impaired differentiation into dendritic cells, with lower rates of maturation and cytokine production in response to LPS and lower lymphocyte proliferation in allo-MLR (42). Thus, the cryopreservation of monocytes for Frontiers in Oncology | www.frontiersin.org 12 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome DC generation may decrease the quality of manufactured DCs, and the level of this decrease needs to be specified for a particular manufacturing protocol. In case of a minor drop in DC maturation and immunostimulatory parameters and function due to the cryopreservation of monocytes, this manufacturing modality should be considered, as it would allow harvesting of therapy-naïve monocytes and avoid a potentially detrimental effect of certain anti-cancer and supportive treatment on the quality of DC-based anti-cancer immunotherapy. Another issue in the context of the concurrence of anti-cancer treatment and monocyte-derived DC manufacture is the length of the pharmacotherapy-free period prior to monocyte harvest. From our real-life experience gained on this study group, we conclude that a 30-day interval without treatment is not sufficient for the combination of temozolomide and irinotecan to sufficiently wash out the monocyte biology-interfering effect of this combination. However, the issue of a safe therapy-free window is not likely to be addressable through the establishment of a wash-out period for a particular drug. The fitness of monocytes and their capacity to differentiate and mature into DCs with high antigen-presenting effect is a matter of their biological function in the context of iatrogenic affection, which is complexly shaped by the need for immediate treatments, their combinations, their cumulative doses, and the long-term history of treatment. Therefore, identifying a marker revealed from a patient’s peripheral blood that predicts the outcome of DC-generation would help to avoid an unproductive anti-cancer DC-manufacturing process. Here we show that a high monocyte count in CBC is not predictive of an efficacious outcome for DC generation. Nevertheless, we find that the presence of immature granulocytes in CBC may predict decreased immunostimulation elicited by DCs and, subsequently, unsuccessful preparation of DC-based IMP. However, closer evaluation of monocyte function prior to their collection for DC generation may be considered. A surrogate marker for the immunostimulatory capacity of monocytes may be evaluated in (i) their phenotype, e.g., the level of HLA-DR or ILT-3 expression on monocytes or the proportion of particular monocyte subsets according to CD14 and CD16 expression, or (ii) their ability to produce pro-inflammatory cytokines upon TLR stimulation (27). In summary, monocytes represent a key starting material for anti-cancer DC-based vaccine manufacture. Therefore, monocyte conditions have an impact on the manufacturing yield, the differentiation into DCs, and the level of maturation and subsequent immunostimulatory functions. For DC manufacture from heavily pretreated pediatric patients with high-risk sarcomas and neuroblastoma, we conclude that the manufacturing yield and immunostimulatory quality of anticancer DC-based vaccine generated from patient’s monocytes were not influenced by the monocyte isolation modality but were detrimentally affected by certain combinations of anti-cancer agents. Thus, the combination of chemotherapy or targeted therapy with DC-based immunotherapy needs to be scheduled not only with respect to the likely beneficial role of anti-cancer agents on the immunogenicity of tumor antigens for both in vitro DC generation via induction of immunogenic cell death and in vivo for effector response of DC-activated T-cells but also with respect to optimal monocyte immunostimulatory functions. Finally, these findings may also have implications for the general pharmacology of anticancer treatment. As our model of ex vivo-activated DC preparation generally parallels the in vivo differentiation pathways of monocytes to the antigenpresenting cells, we may imply that drug combinations at doses used clinically may result in an impairment of patient DCs and possibly immune competence in general. In conclusion, these findings may stimulate further research on dose and mechanismof-action-based drug combination in patient-centered trials to optimize the treatment modalities currently available in clinical oncology. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the manuscript/Supplementary Files. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee, University Hospital Brno. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. AUTHOR CONTRIBUTIONS EH contributed to the trial design, contributed to the study design, participated in clinical data acquisition and analysis, contributed to Supplementary Material preparation, and drafted the manuscript. KP supervised IMP manufacture, contributed to laboratory data acquisition and analysis, contributed to data interpretation, and drafted the manuscript. DC participated in clinical data acquisition, contributed to Supplementary Material preparation, and revised the manuscript. IS performed statistical analysis, contributed to figure preparation and data interpretation, and drafted the manuscript. PMu contributed to the trial design, performed patient enrollment and treatment, contributed to data interpretation, and drafted the manuscript. PMa contributed to the trial design, participated in patient treatment, and drafted the manuscript. LFe contributed to laboratory data acquisition and analysis, contributed to Supplementary Material preparation, and drafted the manuscript. JM contributed to the trial design and drafted the manuscript. LJ participated in IMP manufacturing and revised the manuscript. LSe contributed to IMP manufacturing— monocyte harvest and drafted the manuscript. RP contributed to IMP manufacturing—starting material harvest and revised the manuscript. LFl contributed to IMP manufacturing— certification and revised the manuscript. LSo contributed to the trial design and revised the manuscript. RD, JS, and DV contributed to the trial design, contributed to data interpretation, and revised the manuscript. LZ-D conceived the study design, designed and supervised laboratory data acquisition and analysis, contributed to data analysis and interpretation, and drafted and finalized the manuscript. Frontiers in Oncology | www.frontiersin.org 13 October 2019 | Volume 9 | Article 1034 Hlavackova et al. Chemotherapy Impairs DC Manufacturing Outcome FUNDING This work was supported by the Czech Ministry of Education, Youth and Sport via Large infrastructure CZECRIN (LM2015090) and RECAMO (LO1413), by the European Regional Development Fund—project CZECRIN_4PATIENTY (Reg. No. CZ.02.1.01/0.0/0.0/16_013/0001826), and by the Czech Ministry of Health via DRO 00209805. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.01034/full#supplementary-material Supplementary Figure 1 | Decision tree for DC-IMP manufacturing workflow including in-process and quality controls. Supplementary Figure 2 | Number of anti-cancer therapy lines preceding monocyte harvest were compared based on QC parameters: (A) DC yield, and post-thaw: (B) viability, (C) DC phenotype on day 0: CD14, CD197, CD80, CD86, CD83 and on day 2: MHC II, CD80, CD86, CD83 and immunostimulatory properties presented by (D) IL-12 production, IL-10 production and IL-12/IL-10 production ratio, (E) allo-MLR and auto-MLR. Supplementary Figure 3 | Manufacturing subgroup from monocytes harvested after MTD-based therapy potentially interfering with monocyte biology and manufacturing subgroup from monocytes from untreated patients or after non-interfering treatment compared based on QC parameters: (A) DC yield, and post-thaw: (B) viability, (C) DC phenotype on day 0: CD14, CD197, CD80, CD86, CD83 and on day 2: MHC II, CD80, CD86, CD83 and immunostimulatory properties presented by (D) IL-12 production, IL-10 production and IL-12/IL-10 production ratio, (E) allo-MLR and auto-MLR. Supplementary Table 1 | Monocyte biology-interfering medications. Supplementary Table 2 | Study patient characteristics, disease course, and therapy. Supplementary Table 3 | Source data: CBC parameters, manufacturing details, and QC parameters. Supplementary Material 1 | html. Interactive—medications 60 days prior to monocyte harvest. REFERENCES 1. Oberlin O, Rey A, Lyden E, Bisogno G, Stevens MC, Meyer WH, et al. 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Ghanekar SA, Bhatia S, Ruitenberg JJ, Dela Rosa C, Disis ML, Maino VC, et al. Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors. J Immune Based Ther Vaccines. (2007) 5:7. doi: 10.1186/1476-8518-5-7 42. Silveira GF, Wowk PF, Machado AM, Duarte Dos Santos CN, Bordignon J. Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation. PLoS ONE. (2013) 8:e71291. doi: 10.1371/journal.pone.0071291 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2019 Hlavackova, Pilatova, Cerna, Selingerova, Mudry, Mazanek, Fedorova, Merhautova, Jureckova, Semerad, Pacasova, Flajsarova, Souckova, Demlova, Sterba, Valik and Zdrazilova-Dubska. 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Frontiers in Oncology | www.frontiersin.org 15 October 2019 | Volume 9 | Article 1034 CLINICAL TRIAL published: 14 November 2019 doi: 10.3389/fonc.2019.01169 Frontiers in Oncology | www.frontiersin.org 1 November 2019 | Volume 9 | Article 1169 Edited by: Andrew Zloza, Rush University Medical Center, United States Reviewed by: Pierpaolo Correale, Azienda Ospedaliera ‘Bianchi-Melacrino-Morelli’, Italy Simone Anfossi, University of Texas MD Anderson Cancer Center, United States Praveen Bommareddy, Rutgers, The State University of New Jersey, United States *Correspondence: Lenka Zdrazilova-Dubska dubska@mou.cz Specialty section: This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology Received: 18 June 2019 Accepted: 18 October 2019 Published: 14 November 2019 Citation: Fedorova L, Mudry P, Pilatova K, Selingerova I, Merhautova J, Rehak Z, Valik D, Hlavackova E, Cerna D, Faberova L, Mazanek P, Pavelka Z, Demlova R, Sterba J and Zdrazilova-Dubska L (2019) Assessment of Immune Response Following Dendritic Cell-Based Immunotherapy in Pediatric Patients With Relapsing Sarcoma. Front. Oncol. 9:1169. doi: 10.3389/fonc.2019.01169 Assessment of Immune Response Following Dendritic Cell-Based Immunotherapy in Pediatric Patients With Relapsing Sarcoma Lenka Fedorova1,2,3 , Peter Mudry4 , Katerina Pilatova1,2,3 , Iveta Selingerova3 , Jana Merhautova1 , Zdenek Rehak2,5 , Dalibor Valik1,2,3 , Eva Hlavackova4 , Dasa Cerna4 , Lucie Faberova4 , Pavel Mazanek4 , Zdenek Pavelka4 , Regina Demlova1,3 , Jaroslav Sterba1,4,6 and Lenka Zdrazilova-Dubska1,2,3 * 1 Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czechia, 2 Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Brno, Czechia, 3 Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czechia, 4 Department of Pediatric Oncology, University Hospital and Faculty of Medicine, Masaryk University, Brno, Czechia, 5 Department of Nuclear Medicine, Masaryk Memorial Cancer Institute, Brno, Czechia, 6 International Clinical Research Center, St. Anne’s University Hospital, Brno, Czechia Monocyte-derived dendritic cell (DC)-based vaccines loaded with tumor self-antigens represent a novel approach in anticancer therapy. We evaluated DC-based anticancer immunotherapy (ITx) in an academic Phase I/II clinical trial for children, adolescent, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors. The primary endpoint was safety of intradermal administration of manufactured DCs. Here, we focused on relapsing high-risk sarcoma subgroup representing a major diagnosis in DC clinical trial. As a part of peripheral blood immunomonitoring, we evaluated quantitative association between basic cell-based immune parameters. Furthermore, we describe the pattern of these parameters and their time-dependent variations during the DC vaccination in the peripheral blood immunograms. The peripheral blood immunograms revealed distinct patterns in particular patients in the study group. As a functional testing, we evaluated immune response of patient T-cells to the tumor antigens presented by DCs in the autoMLR proliferation assay. This analysis was performed with T-cells obtained prior to DC ITx initiation and with T-cells collected after the fifth dose of DCs, demonstrating that the anticancer DC-based vaccine stimulates a preexisting immune response against self-tumor antigens. Finally, we present clinical and immunological findings in a Ewing’s sarcoma patient with an interesting clinical course. Prior to DC therapy, we observed prevailing CD8+ T-cell stimulation and low immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSC) and regulatory T-cells (Tregs). This patient was subsequently treated with 19 doses of DCs and experienced substantial regression of metastatic lesions after second disease relapse and was further rechallenged with DCs. In this patient, functional ex vivo testing of autologous T-cell activation by manufactured Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients DC medicinal product during the course of DC ITx revealed that personalized anticancer DC-based vaccine stimulates a preexisting immune response against self-tumor antigens and that the T-cell reactivity persisted for the period without DC treatment and was further boosted by DC rechallenge. Trial Registration Number: EudraCT 2014-003388-39. Keywords: dendritic cells, anticancer immunotherapy, dendritic-cell (DC)-based vaccine, pediatric sarcoma, academic clinical trials, immunomonitoring, personalized medicine INTRODUCTION Patients with relapsed or refractory Ewing’s sarcoma have a very poor prognosis. No substantial improvement has been achieved in the therapy of sarcoma patients in the last two decades despite research, and long-term survival is still <25%. Immunotherapeutic approaches including antigen-presenting cell-based vaccines have been employed as single agent or as part of combination strategies having been substantiated by a report on immunogenicity of Ewing’s sarcoma with specific translocation resulting in EWS/FLI1 fusion. Following dendritic cell (DC) vaccine with untreated autologous lymphocytes, 39% of patients had measurable immune response against a neopeptide derived from the fusion gene (1). Promising results were reported after CD25+ regulatory T-cell depletion of an autologous lymphocyte infusion product augmented with interleukin (IL)- 7, where immune reconstitution correlated with an improved survival of 63% in Ewing’s sarcoma and rhabdomyosarcoma (2). Immunocompetent CD8+ T lymphocytes were observed within the tumor microenvironment of metastases after DC immunotherapy (ITx) but without direct cytotoxic efficacy probably due to expression of PD-1 on lymphocytes and PDL1 on tumor cells (3). Such immune suppression could be bypassed using recently developed anti-PD-1 and anti-PD-L1 agents, demonstrating improved survival in several malignancies, including anecdotal cases of sarcomas (4, 5). Proper antigen presentation has a key role in directing the immune system to attack tumor cells by targeting tumor-associated antigens. We manufacture fully personalized monocyte-derived DC-based vaccines that are evaluated in an academic investigator-initiated clinical trial for children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors (EudraCT 2014- 003388-39). As a part of clinical and research evaluation of patients, we performed DC characterization, peripheral blood immunomonitoring during DC treatment, and ex vivo assessment of T-cell cytotoxic function pre- and post-DC treatment. During peripheral blood immunomonitoring, we quantified circulating immune cells to evaluate both positive and negative players in cancer surveillance and eradication. We focused on absolute lymphocyte count (ALC) and neutrophilto-lymphocyte ratio (NLR). Both parameters are associated with the number of lymphocytes as key players in the immune response to tumors. Additionally, NLR reflects the number of neutrophils that is a negative prognostic factor often related to paraneoplastic immune response. The peripheral blood lymphocyte compartment contains conventional αβ TCR+ T-cells, B-cells, natural killer (NK) cells, and also minor specific effector and regulatory cell types, including regulatory T-cells (Tregs), CD56+ CD3+ NKT-like cells (6), γδ Tcells (7), and monocytic myeloid-derived suppressor cells (MMDSCs). These immune cell subsets constitute the actual clinical immunomonitoring, and their characteristics are reviewed in Supplementary Material 1. This study focuses on high-risk sarcoma patients representing a major diagnosis in this clinical trial. First, we evaluated quantitative association between basic cell-based immune parameters. Next, we described patterns of these parameters and their time changes during the DC vaccination course in the peripheral blood immunograms. As a functional testing, we evaluated immune response of patient T-cells to the tumor antigens presented by DCs in autoMLR proliferation assay. This analysis was performed with T-cells obtained prior to DC ITx initiation and with T-cells collected after administration of the fifth dose of DCs. Finally, we presented clinical and immunological findings from DC-based ITx after relapse in the case of the Ewing’s sarcoma patient. METHODS Clinical Trial Design and Methodology This nonrandomized, open-label, academic, investigatorinitiated, phase I/II clinical trial (EudraCT No. 2014-003388-39) was performed at a single center in Czechia in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. The protocol was approved by the local ethics committee at the site and by the designated authority of Czechia (the State Institute for Drug Control). Patients eligible for the clinical trial were children, adolescents, and young adults (1–25 years old) with histologically confirmed refractory, relapsing, or primarily metastatic high-risk tumors; Karnofsky or Lansky score ≥50; life expectancy longer than 10 weeks; and adequate function of bone marrow, kidney, liver, and heart defined as absolute neutrophil count (ANC) ≥0.75 × 103/µl, thrombocytes ≥75 × 103/µl, hemoglobin 80 g/l, estimated glomerular filtration rate (eGFR) ≥70 ml/min/1.73 m2, serum creatinine ≤1.5-fold upper limit for the appropriate age, bilirubin ≤1.5-fold upper limit for the appropriate age, Frontiers in Oncology | www.frontiersin.org 2 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients AST and ALT ≤2.5-fold upper limit for the appropriate age, ejection fraction ≥50%, and fractional shortening ≥27% assessed by echocardiography. In the case of bone marrow infiltration, ANC had to be ≥0.5 × 103/µl and thrombocytes ≥40 × 103/µl. In the case of liver metastases, AST and ALT must have been ≤5-fold upper limit for the appropriate age. Patients must not have had severe ongoing toxicity resulting from any previous treatment. Radiotherapy (RTx), myelosuppressive, and immunosuppressive treatment must have been withdrawn at least 3 weeks before tumor tissue harvesting; the only exception is corticoid treatment of brain edema that was allowed. Myelopoietic growth factors must have been withdrawn at least 7 days before tumor tissue harvesting. Targeted therapy must have been withdrawn at least 7 days for tyrosine kinase inhibitors (TKI) or at least 3-fold half-life of the drug (upper limit 6 weeks) before tumor tissue harvesting. The time interval between autologous transplantation and tumor tissue harvest must have been ≥12 weeks and in the case of allogeneic transplantation ≥26 weeks. Patients with seropositivity to HIV1, HIV2, Treponema pallidum, hepatitis B or C, known hypersensitivity to the study medication, an autoimmune disease that was not adequately treated, uncontrolled psychiatric disease, or uncontrolled hypertension were not eligible. Allowed medication prior to monocyte harvest (leukapheresis) was as follows: metronomic chemotherapy (CTx), immune checkpoint inhibitors, and anti-CD20 antibodies are allowed as concomitant medication for any time before leukapheresis. Monoclonal antibodies (except anti-CD20), high-dose CTx, and high-dose corticoids must have been withdrawn at least 3 weeks prior to leukapheresis with the exception of corticoid treatment of brain edema, which was allowed. Since November 2017, amendment of the procedure for monocyte harvest was made, and TKI must have been withdrawn according to their halflife: drugs with short half-life of 3–14 h at least 2 days before leukapheresis (axitinib, dabrafenib, dasatinib, ibrutinib, idelalisib, nintedanib, ruxolitinib, trametinib), drugs with medium halflife of 15–35 h at least 7 days before leukapheresis [alectinib, bosutinib, lapatinib, lenvatinib, nilotinib, osimertinib, pazopanib, ponatinib, regorafenib, and non-tyrosine kinase inhibitor (nonTKI) everolimus], and drugs with long half-life of 36–60 h at least 12 days before leukapheresis (afatinib, ceritinib, erlotinib, gefitinib, imatinib, cabozantinib, crizotinib, sorafenib, sunitinib, vemurafenib, and non-TKI temsirolimus). Myelopoietic growth factors must have been withdrawn at least 7 days before leukapheresis/monocyte harvest. Patients previously treated with DCs were not allowed to enter the trial. The primary endpoint of the trial was assessment of safety by analysis of incidence of adverse events of special interest (AESI; i.e., allergic reactions grade ≥3, acute or subacute autoimmune organ toxicity symptoms manifesting up to 30 days after administration of the vaccine, injection site reactions grade ≥4, infectious complications grade ≥3). The secondary safety endpoint was incidence of all adverse events assessed in relation to type, seriousness, and causality. Secondary efficacy endpoints were time to progression, overall survival, objective response to treatment at 12 and 24 months, and clinical benefit rate assessment at 6 and 12 months. Investigational medicinal product (IMP) was administered as an add-on therapy to standard treatment. The dose of IMP contains 2 × 106 DCs in 100 µl of cryopreservation medium. DC-based IMP was administered intradermally every 3 ± 1 weeks, up to 35 doses, to a predefined site on the left or right arm near the axillary lymph node. The evening before administration and two evenings after application, topical imiquimod, toll-like receptor (TLR)-7 agonist, was applied on the injection site as an adjuvant. On the day of administration, the patient had to have adequate bone marrow function (defined in the same way as in the entry criteria described above) and was not allowed the following therapy: more than a week systemically administered corticosteroids except treatment for cerebral or spinal edema (single administration of corticoids due to premedication, treatment of allergic reaction, and substitution treatment in secondary hypocortisolism are allowed), anticoagulants in therapeutical dose (prophylactic doses of low-molecular-weight heparins were allowed), erythropoietin, pegylated granulocytestimulating growth factors or other growth factors except for filgrastim, RTx to sites and regional lymph nodes, except radiation for pain control, the interval between vaccine application, and administration of conventional CTx must have been more than 72 h. Complete blood count, biochemical analysis, and immunomonitoring were performed on every patient visit associated with administration of IMP. DC Manufacturing and Quality Control The DC-based vaccine, called MyDendrix, was manufactured under GMP in Clean rooms of the Department of Pharmacology, Faculty of Medicine, Masaryk University. Briefly, mononuclear cells were collected by leukapheresis, and then monocytes were separated by elutriation or adherence to a plastic surface. Harvested monocytes were cultivated with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and differentiated into DC. Immature DCs were subsequently exposed to autologous tumor lysate antigens. The preparation of tumor lysate from the patient’s tumor obtained during curative surgery or extended biopsy preceded monocyte harvest. Maturation was induced by lipopolysaccharide and interferonγ. Manufactured DCs were aliquoted into IMP doses, each containing 2 × 106 DCs based on reports (8, 9), cryopreserved in DMSO-containing medium, and stored at −150◦C to −196◦C. Quality control (QC) of DC-based IMP included viability, cell phenotype, production of IL-12 and IL-10, and stimulation of allogeneic and autologous T-cells to reflect the level of stimulatory properties of DCs. Details on DC-based IMP manufacturing were described in Supplementary Material 2 (8, 10). DCs were stored frozen until the day of administration when a DC dose was shipped on dry ice for administration to a study patient, shortly thawed, and immediately injected intradermally to the patient. Ex vivo Assessment of Prevaccination and Postvaccination T-Cells Stimulatory properties of DCs were examined pre- and post-DC treatment by autologous mixed lymphocyte reaction (MLR). PreDC ITx lymphocytes were obtained during the manufacturing of Frontiers in Oncology | www.frontiersin.org 3 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients DCs of from the elutriation process or adherence of leukapheresis product obtained for separation of monocytes. The number of T-cells in the lymphocyte-rich fraction was quantified by flow cytometry: approximately 105 PBMCs were mixed with 10 µl of anti-CD45-PC7 (clone J33) and anti-CD3-FITC (clone UCHT1, both from Beckman Coulter), incubated 20 min in the dark, and analyzed on an FC500 flow cytometer (Beckman Coulter). PBMCs were aliquoted, cryopreserved in 1,000 µl of Cryostor CS5 (BioLife Solutions), frozen, stored at −150◦C to −196◦C, and thawed prior to auto-MLR seeding. For post-DC treatment assay, PBMCs were obtained from peripheral blood collected into K3EDTA tube (7 ml, Sarstedt) after application of at least five doses of DCs. Blood was layered onto Histopaque-1077 R (Sigma-Aldrich, density 1,077 g/ml) and centrifuged (450 g, 30 min, 20◦C, acceleration 3, brake 3). Fractions of mononuclear cells were collected and washed with Hank’s Balanced Salt Solution (HBSS, Lonza). 107 PBMCs were cryopreserved in 1,000 µl Cryostor CS5 (BioLife solutions) and stored at −150◦C. For pre- and post-DC treatment autoMLR, 107 target lymphocytes were stained with 250 µl 10 µM carboxyfluorescein succinimidyl ester (CFSE, Sigma-Aldrich) and seeded into sterile 96-well culture plate (Sarstedt, TC Plate 96-well, Suspension, F) at 105 cells/well in X-vivo 10 medium (Lonza) containing 5% inactivated human male AB serum (Sigma-Aldrich) at a 1:10 effector:target ratio (104 DC/well), positive control (PC) with phytohemagglutinin (PHA, Sigma-Aldrich) 1 mg/ml HBSS (final concentration 10 µg/ml in MLR), or negative control (NC) with complete X-vivo medium, final volume 200 µl/well. MLR experiments were seeded in triplicates and cultured for 6 days at 37◦C/5% CO2. Then 2 × 104 cells from each well were stained with CD3-PC7 (clone UCHT1, 10 µl/test, Beckman Coulter) for flow cytometric detection of CFSE fluorescence dilution on CD3+ T-cells. Discrimination for dividing cells was set up using the NC. T-cell proliferation was calculated as follows: [(average % of dividing T-cells in 10:1 MLR)−(average % of dividing T-cells in NC)] × 100/[(average % of dividing T-cells in PC)−(average % of dividing T-cells in NC)]. The medium from autoMLR was centrifuged, and pooled supernatant from triplicates was stored at −20◦C until analysis. The concentration of interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-17A was measured using a flow cytometric bead assay (BD Biosciences). Peripheral Blood Immunomonitoring Detailed peripheral blood immunomonitoring was performed at baseline (= before DC therapy initiation) and at each DC dose administration. The samples were collected on the day of vaccination just before the application of the vaccine. Blood was collected in a 7.5-ml S-Monovette R tube with K3EDTA anticoagulant. Lymphocytes (ALC) and neutrophils (ANC) were measured using a Sysmex XN hematology analyzer. NLR was calculated as ANC/ALC. Immunophenotype was analyzed by multiparameter multicolor flow cytometer and software (Navios, Beckman Coulter). Diagnostic antibodies were purchased from Beckman Coulter, premixed in equal amounts in five cocktails, and stored in the dark at 2–8◦C not longer than 7 days: 1/ CD14PE (RMO52), CD15-KrO (80H5), CD11b-APC (Bear1), CD33FITC (D3HL60.251), CD45-PB (J33), HLA-DR-PC5 (Immu357); 2/ CD3-FITC (UCHT1), CD4-PB (13B8.2), CD16-PC7 (3G8), CD56-PE (NKH-1); 3/ CD3-FITC (UCHT1), CD4-PB (13B8.2), CD27-AF750, CD45-KrO (J33), CD45RO-ECD (UCHL1), HLADR-PC5 (Immu357); 4/ TCR PAN γ/δ-FITC (IMMU510), TCR Vγ9-PC5 (IMMU360), TCR Vδ2-PB (IMMU 389), CD314-APC (ON72); 5/ CD3-FITC (UCHT1), CD4-PC7 (SFCI12T4D11), CD25-PC5 (B1.49.9), CD127-PE (R34.34). Blood (25 µl) was incubated with 10 µl of premixed antibody cocktail for 15 min in the dark at room temperature, hemolyzed by Versalyse R (Beckman Coulter) for 15 min and measured in five flow cytometric assays to detect: (1) M-MDSCs detected as CD45+ CD14+ CD11b+ CD33+ HLA-DR−, and their absolute count was calculated using the number of white blood cells (WBC) measured by the Sysmex XN hematology analyzer; (2) NK cells detected as CD3− CD56+ CD16+, NKT-like cells detected as CD56+CD3+; (3) circulating effector CD8+ T-cells were defined as CD3+ CD8+ CD27–, activated CD8+ T-cells were defined as CD8+ HLA-DR+; (4) γδ T-cell subsets classified as δ2+γ9−, δ2+γ9+, δ2−γ 9+, δ2−γ9− and evaluated for CD314; (5) Tregs defined as CD3+ CD4+ CD25+ CD127−/low+. 18F-FDG PET/CT Scan 18F-FDG PET/CT examination was performed using the hybrid scanner Biograph 64 HR+ (Siemens Erlangen, Germany). CT scan was provided in low-dose CT (25 mAs eff/120 kV). The patient had standard preparation prior to examination, including FIGURE 1 | Flow diagram for DC-based immunotherapy trial and study group definition. CONSORT flow diagram showing participant flow through each stage of the trial [enrollment, DC-based investigational medicinal product (IMP) manufacturing, treatment] and the analysis of sarcoma patients study group. Frontiers in Oncology | www.frontiersin.org 4 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients restriction of physical activity for 12 h, fasting for at least 6 h, capillary glycemia lower than 10 mmol/l (180 mg/dl) prior to 18F-FDG administration and peroral hydration with 500– 1,000 ml of plain water. 18F-FDG was administered at a dose of 262 MBq in study 7/2017 and at a dose of 260 MBq in 1/2018. After an in vivo accumulation time of 60 min, whole-body scanning from the proximal third of thighs to the vertex of the skull was performed in both studies. All images were iteratively reconstructed and corrected for attenuation. 18F-FDG uptake was assessed visually and also semi-quantitatively in the defined region of interest with calculation of target-to-liver ratios. A target-to-liver ratio higher than 1.0 was considered positive in all evaluated regions. Statistical Analysis Spearman correlation coefficient with significance test was used to measure the strength of the relationship between baseline circulating immune parameters. Graphic visualization of immunograms was performed using radar plot. Non-parametric Wilcoxon test for paired samples was used for analysis of pre- and post-treatment T-cell stimulation. P-values <0.05 were considered statistically significant. All statistical analyses were performed with R 3.5.3 software (11). RESULTS Clinical Trial Progress With Focus on Sarcoma Patients The first subject was enrolled in September 2015. As of May 2019, the clinical trial was still ongoing, but with the accrual suspended. From the overall 47 enrolled patients, 25 (53%) were sarcoma patients. Screening failure occurred in one subject, and tumor harvest was not performed in two subjects. Tumor was harvested in 44 subjects; among them, the harvested tissue contained no cancer cells in one subject, tumor antigen extraction failure presenting as low concentration of protein in tumor lysate in six subjects, participation in the trial ended in five subjects due to disease progression and/or death, monocyte harvest has been pending in two subjects, monocyte harvest and subsequent manufacturing of DC-based IMP was performed in 30 subjects. Of the 30, manufacturing failed in two subjects, IMP did not pass quality control specifications in five subjects (four of them are sarcoma patients) (10), and 22 DC-based IMPs were released for administration to the patients. Of the 22, one subject died before IMP administration, administration has been pending in two sarcoma patients until the completion of high-dose CTx, and DC vaccine was administered to 19 subjects, including 11 sarcoma patients. Of these 11, nine patients received at least six doses of DC-based IMP as of March 2019 and were analyzed in presented immunomonitoring study (Figure 1). The age of sarcoma patients in the study group ranged from 10 to 24 years at the DC ITx initiation (Table 1). Stage of the disease in the study group at the DC ITx initiation was as follows: one (11%) in complete remission, three (33%) subjects in partial remission, one (11%) with stable disease, four (44%) with progressive disease (Table 1). Detail clinical course TABLE1|Baselinepatientcharacteristicsandperipheralbloodimmunecelllevelsatdendriticcell(DC)therapyinitiation. Subjectno./Primarydiagnosis(primarylocalization)StageofthediseaseandPSatAgeatDCITxinit.Baselinecell-basedimmuneparametersatDCITxinitiation sexDCITxinit. ALC*Eff.CD8+Act.CD8+NK*NKT-like*GD%*Tregs*M-MDSCcount*NLR* 106 /ml%%%%%%106 /mlRatio KDO-0101/FEwingsarcoma(mandible)2ndCRKarnofsky10015years1.973.834.81.6↓4.92.95.90.070.8↓ KDO-0102/FOsteosarcoma(rightdistalfemur)PDLansky8010years1.3↓58.921.54.61.94.62.9↓0.071.2 KDO-0114/MSynovialsarcoma(leftthigh)PDKarnofsky8015years0.2↓34.372.00.5↓2.91.1↓12.00.63↑19.9↑ KDO-0118/FEwingsarcoma(spineC5-Th2)PRKarnofsky10024years0.6↓31.311.68.12.23.24.50.25↑5.2↑ KDO-0119/FAlveolarrhabdomyo-sarcoma(primumignotum)PRKarnofsky8013years0.6↓25.110.85.84.62.613.40.242.7 KDO-0124/FOsteosarcoma(rightproximaltibia)2ndmtsrelapseKarnofsky10019years0.8↓7321.96.41.562.9↓0.041.6 KDO-0131/MEmbryonalrhabdomyosarcoma(pelvis)PRKarnofsky7019years0.6↓94.960.53.5↓14.83.13.0↓0.001.7 KDO-0133/MOsteosarcoma(rightproximalfemur)PDKarnofsky10024years0.9↓49.12.76.51.52.83.0↓0.26↑2.9 KDO-0139/FOsteosarcoma(leftdistalfemur)SDKarnofsky9022years0.5↓14.7337.94.9↓1.20.6↓7.90.42↑10.7↑ Cell-basedimmuneparametersandtheirage-specificreferencerange(*ifavailable):ALC,absolutelymphocytecount(referencerange110–16years1.4–4.2×106/ml,>16years1.2–4.1×106/ml);NLR,neutrophil-to-lymphocyteratio (referencerange21–3);EfCD8+,circulatingeffectorcytotoxicT-cells(CD27-/CD8+,%ofCD8+T-cells);ActCD8+,activatedcytotoxicT-cells(HLA-DR+/CD8+;%ofCD8+T-cells);NKcells,naturalkillers(referencerange110–16years 4–51%oflymphocytes,>16years5–49%oflymphocytes);NKT-like,circulatingCD3+CD56+cells(referencerange110–16years0.64–15%oflymphocytes,>16years1–18%oflymphocytes);GD-T,gamma-deltaT-cells(reference range110–16years2–17%oflymphocytes,>16years0.8–11%oflymphocytes);Treg,regulatoryT-cells(referencerange110–16years4–20%ofCD4+T-cells,>16years4–17%ofCD4+T-cells);M-MDSC,monocyticmyeloid-derived suppressorcells(referencerange30–0.24×106/ml).Numbersinboldrefertothevalueswithinthereferencerange,↑–abovetheupperlimitofthereferencerange,↓–bellowthelowerlimitofthereferencerange.1Referencerange originatedfromSchatorjeetal.(12).2Estimatedfromreferencerangesforrelativedifferentialcellbloodcount(13).3Ownreferencevalue,sourcegroupdescribedinPilatovaetal.(13).init.,initiation;F,female;M,male. Frontiers in Oncology | www.frontiersin.org 5 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients of disease in nine sarcoma study patients is summarized in Supplementary Material 3. No immune or infection-related AESIs were reported for all 15 evaluated subjects receiving DC ITx by the date of analysis. Peripheral Blood Immunomonitoring of DC-Treated Sarcoma Patients First, we evaluated the possible association of cell-based immune parameters in sarcoma patients before DC ITx and during DC treatment, up to six doses of DCs (Figure 2). Based on positive and negative correlations, immune parameters clustered de facto into two groups with inverse relation; a group consisting of ALC, proportion of effector cytotoxic Tcells among all T-cells, proportion of CD56+ CD3+ NKTlike cells among lymphocytes, proportion of γδ T-cells among lymphocytes, and an inversely correlated group with neutrophilto-lymphocyte ratio (NLR), proportion of regulatory T-cells among CD4+ cells, number of M-MDSC, proportion of activated HLA-DR+ CD8+ cells among CD8+ cells, and proportion of CD56+ CD16+ CD3− NK cells among lymphocytes (Figure 2). Baseline circulating immune parameters in nine sarcoma patients are shown in Table 1. At baseline, eight of nine patients had lymphopenia with mean ALC of 0.81 × 106/ml (Table 1). An exception was patient KDO-0101 (ALC 1.9 × 106/ml) with Ewing’s sarcoma whose clinical course and laboratory findings are described later. The proportion of NK cells was low in six of nine patients (median 4.9%, min. 0.5%, max. 8.1%). The proportion of NKT-like cells among lymphocytes was predominantly low (median 2.2%), except for expanded NKT-like cells (14.8% of lymphocytes) in patient KDO-0131. γδ T-cells were low in six of nine patients (median 2.9%, min. 0.6%, max. 6.0%). Based on observed positive and negative association between particular cell-based immune markers, we constructed peripheral blood immunograms with putative anticancer effectors in upper part of an immunogram (namely, total lymphocytes, effector cytotoxic T-cells, CD56+ CD3+ NKT-like cells, γδ T-cells), and on the other hand, cancerpromoting or immunosuppressive actors (namely, NLR, MMDSC, Tregs) and related factors (activated T-cells and NK cells) in the lower part of an immunogram (Figure 3). In peripheral blood immunograms, we presented baseline values of cell-based immune markers and their level after doses 1, 3, and 6 of ITx with DCs (Figure 3). The peripheral blood immunograms revealed distinct patterns in particular patients in the study group. For instance, we observed “immune-activated” pattern with patient KDO-0101 with Ewing‘s sarcoma who started DC ITx in the second complete remission, ALC was not decreased, effector cytotoxic T-cells represented the majority of circulating T-cells, and NLR and M-MDSC count were low. On the other hand, case KDO-0114 with progressing synovial sarcoma appeared to have an “immune-suppressive pattern” with high NLR, M-MDSC count, Tregs, and low ALC, proportion of effector cytotoxic T-cells, as well as NKT-like and γδ T-cells. Regarding timedependent variations over the DC vaccination course, we did not FIGURE 2 | Association of circulating immune markers during the course (from baseline to the sixth dose) of therapy with dendritic cells (DCs) in sarcoma study group. Red—positive correlation, blue—negative correlation; strength of relationship is represented by size of the square and intensity of the color, larger squares with intensified color have stronger relationship; *p < 0.05, **p < 0.01, ***p < 0.001; ALC, absolute lymphocyte count (106 /ml); NLR, neutrophil-to-lymphocyte ratio; Ef CD8+, circulating effector cytotoxic T-cells (% of CD27− of CD8+ T-cells); Act CD8+, activated cytotoxic T-cells (% of HLA-DR+ of CD8+ T-cells); NK, circulating NK-cells (% of lymphocytes); NKT-like, circulating NKT-like cells (% of lymphocytes); GD-T, γδ T-cells (% of lymphocytes); Treg, regulatory T-cells (% of CD4+ T-cells); M-MDSC, monocytic myeloid-derived supressor cells (106 /ml). observe any consistent trend in the dose-dependent change of levels of evaluated immune system parameters. Patient T-Cells in vitro Stimulation by DCs Before and After DC Vaccination The stimulation of sarcoma patient T-cells was examined by MLR proliferation assay with DCs from manufactured IMP and autologous T-cells obtained before DC ITx (pre-DC) and after at least five doses of DCs (post-DC) (Figure 4). The level of autoMLR ranged from 0.5 to 18% (median 7.7%) with T-cells collected before DC ITx and from 4.9 to 28.4% (median 14.6%) with T-cells obtained after DC vaccination. Paired data with both pre-DC and post-DC were available for five cases, and all exhibited an increase in the T-cell stimulation after DC ITx. We observed the lowest post-DC increase in autologous T-cell stimulation by selftumor antigens in cases KDO-0114, KDO-0124, and KDO-0133 who started DC treatment in disease progression. On the other hand, the highest increase in the T-cell stimulation with postDC T-cells was exhibited by patient KDO-0101 who started DC ITx in complete remission of Ewing’s sarcoma and remained at least up to ninth dose of DCs in complete remission. This case is described in more detail. Frontiers in Oncology | www.frontiersin.org 6 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients FIGURE 3 | Peripheral blood immunograms of dendritic cell (DC)-treated sarcoma patients. Nine circulating immune parameters are radially arranged with reference ranges shown in orange. Parameters are scaled according to numbers achieved within the entire study group of nine patients. Outer circle (OC, gray dashed) represents the upper limit of the reference range for ALC, NK cells, NKT-like cells, GD T-cells, maximum number reached for the particular marker for Tregs, M-MDSC, and NLR or 100% for Ef CD8+ and Act CD8+; small inner circle (IC, gray dashed) represents zero level; middle circle (MC, pacific blue dashed) represents 50% of OC level. Particular levels are listed for each parameter as follows. ALC, absolute lymphocyte count (reference range1 10–16 years 1.4–4.2 × 106 /ml, >16 years 1.2–4.1 × 106 /ml; OC: 4.2 106 /ml); NLR, neutrophil-to-lymphocyte ratio (reference range2 1–3; OC 19.9); Ef CD8+, circulating effector cytotoxic T-cells (CD27−/CD8+; % of CD8+ T-cells) (OC: 100%); Act CD8+, activated cytotoxic T-cells (HLA-DR+/CD8+; % of CD8+ T-cells) (OC 100%), NK cells (reference range1 10–16 years 4–51% of lymphocytes, >16 years 5–49% of lymphocytes; OC: 51% of lymphocytes); NKT-like, circulating CD3+CD56+ NKT-like cells (reference range1 10–16 years 0.64–15% of lymphocytes, >16 years 1–18% of lymphocytes, OC 18% of lymphocytes); GD-T, γδ T-cells (reference range1 10–16 years 2–17% of lymphocytes, >16 years 0.8–11% of lymphocytes; OC: 17% of lymphocytes); Treg, regulatory T-cells (reference range1 10–16 years 4–20% of CD4+ T-cells, >16 years 4–17% of CD4+ T-cells; OC: 25.3% of CD4+ T-cells); M-MDSC, monocytic myeloid-derived suppressor cells (reference range3 0–0.24 × 106 /ml; OC: 0.98 × 106 /ml). Baseline levels prior to DC ITx initiation are shown in black and levels at doses d1, d3, d6 are shown in shades of blue. Clinical outcome is shown for each subject at DC ITx initiation, at dose 5, at dose 9. Clinical outcome is abbreviated as follows: CR, complete response; PD, progressive disease; SD, stable disease; NN, non-CR/non-PD; NA, not available. 1 Reference range originated from Schatorje et al. (12). 2 Estimated from reference ranges for relative differential cell blood count. 3 Our user-defined reference value, source group described in Pilatova et al. (13). DC-Based Therapy After Relapse in a Ewing’s Sarcoma Patient: Treatment Course and Outcome A girl, born 2001, was diagnosed with primary disseminated EWS/FLI-1 positive Ewing sarcoma with a primary tumor in the mandible and skull metastases in December 2011. The patient was treated by protocol EuroEwing 2008, 6x VIDE: vincristine (1.5 mg/m2/day; day 1), ifosfamide (3,000 mg/m2/day; days 1, 2, 3), doxorubicin (20 mg/m2/day; days 1, 2, 3), etoposide (15 mg/m2/day; days 1, 2, 3), 1× VAC: vincristine (1.5 mg/m2/day; day 1), actinomycin (0.75 mg/m2/day; days 1, 2), cyclophosphamide (1,500 mg/m2/day; day 1) from 12/2011 to 10/2012. Surgery was performed in June 2012 with partial resection of primary tumor. Radical resection was not possible due to mutilation. High-dose (HD) CTx treosulphan/melphalan with autologous peripheral blood stem cell transplantation (APBSC) followed in July 2012. Then, the patient underwent RTx of the mandible and parietal bone from September 2012 to November 2012 (34 Gy + 45 Gy), and CTx continued by Frontiers in Oncology | www.frontiersin.org 7 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients FIGURE 4 | AutoMLR with patients’ pre-dendritic cell (DC) and post-DC T-cells stimulated by DC-based investigational medicinal product (IMP). The stimulation is expressed as the percentage of dividing autologous T-cells after incubation with DCs. Pre-DC (blue) refers to the stimulation of patients’ T-cells obtained prior to DC-based ITx initiation. Post-DC (red) refers to the stimulation of patients’ T-cells obtained after the fifth dose of DC vaccine. The difference (post-DC)−(pre-DC) is shown in gray. The shape of symbols refers to a stage of the disease; PD, progressive disease; CR, complete remission; NN, non-CR/non-PD; PR, partial remission; SD, stable disease. Two-digit numbers refer to the last digits in patients’ number (e.g., 01 = KDO-0101, etc.). A pair of pre-DC and post-DC autoMLR in the same patient is linked by a gray line. protocol EuroEwing 2008 with 7× VAC from October 2012 to May 2013. The first complete remission was achieved and lasted until May 2015 when the first relapse occurred in the skull. The patient was enrolled in the DC clinical trial, and the surgically removed tumor from the skull was used as a source of tumor antigens. In the second-line CTx, the patient received vincristine (1.5 mg/m2/day; 5 days block), irinotecan (50 mg/m2/day; 5 days block), and pazopanib (200 mg/daily). Monocytes were harvested in January 2016, and 35 doses of DC-based medicinal product were manufactured. One week after monocyte separation, palliative RTx on lesions in the skull was started and was performed from January 2016 to February 2016 with a total dose 41 Gy. Subsequently, after recovery from HD CTx and RTx, experimental DCbased ITx (on a biweekly basis) with immunomodulation via low-dose cyclophosphamide (26 mg/m2/day) started in August 2016. The patient received 19 doses of DCs until the second relapse in 7/2017 with multiple metastases in the skull, pelvis (Figures 5A,B), and lesions in liver. FDG PET positivity without CT scan correlates was noted in the spinal column. Third-line CTx with topotecan (0.75 mg/m2; 5 days block), cyclophosphamide (250 mg/m2; 5 days block), and zoledronate (4 mg/4 weeks) with concomitant RTx was initiated. Evaluation of response showed stable disease. After three cycles, CTx was stopped due to hematological toxicity. Surprisingly, during the subsequent 4 months without treatment, substantial regression of metastases was noted both on PET/CT scan in 1/2018 (Figures 5C,D) and upon clinical examination of palpable metastases. Fourth-line maintenance metronomic CTx with lowdose vinblastine (3 mg/m2/day) and continuing zoledronate (4 mg/dose/4 weeks) was started with rechallenge with DCbased vaccines from the original manufacturing from March 2018 to August 2018. Unfortunately, the partial regression was temporary, and slow continuing progressive disease led to the death of the patient in November 2018. DC-Based Therapy After Relapse in a Ewing’s Sarcoma Patient: Ex vivo Prevaccination and Postvaccination T-Cell Response and Peripheral Blood Immunomonitoring Pre-DC treatment T-cell response evaluated by autoMLR as a part of DC quality control resulted in a mean of 5% T-cell division. Post-DC (after the fifth dose) autoMLR exhibited 28% T-cell division (Figure 6A blue). Production of cytokines (IFNγ, TNF-α, IL-17A) during auto-MLR mildly increased in postDC compared to pre-DC evaluation (Figure 6B blue). AutoMLR with T-cells collected before restart of DC treatment in February 2018 (after the third-line Ctx with topotecan, cyclophosphamide, and zoledronate with RT and an additional 4 months with no antitumor treatment) exhibited 22% T-cell division and, upon the fifth “rechallenge” dose, 40% T-cell division was observed (Figure 6A red). IFNγ production during autoMLR substantially increased after the fifth dose of DC rechallenge (Figure 6B red). The variations of circulating immune markers exhibited only minor changes at the beginning of both lines of therapy with DCs (Figure 6C). Levels of circulating immune markers at each dose of both lines of DC-based therapy are shown in Supplementary Material 4. At DC rechallenge, an increase in the proportion of circulating effector CD8+ cells and an increase in the proportion of γδ T-cells compared to the initiation of first-line DCs was observed (Figure 6C). In this patient, γδ T-cells were predominantly Vγ9-Vδ2- prior to DC ITx initiation (baseline 39%). Vγ9+Vδ2+ T-cells represented 33% of γδ T-cells, and their proportion decreased during DC Itx, and this γδ subset was almost depleted from circulation after third-line CTx (Figure 6D). In contrast to the Vγ9+Vδ2+ subset, Vγ9-Vδ2- T-cells were predominantly CD314(NKG2D)+ (Supplementary Material 4). DISCUSSION The primary endpoint of the clinical trial investigating anticancer therapy with DCs was the evaluation of treatment safety with interim result from 15 patients of no immune- or infectionrelated adverse events. Moreover, to gain more information from DC-treated patients, we performed immunomonitoring at baseline and at each DC dose. Collected data will be evaluated in the context of clinical outcomes after completion of the trial. Here we show that an ALC was positively associated with the proportion of effector CD8+ cytotoxic T-cells out of total T-cells that is reflected by an inversion of the CD4:CD8 ratio and proportion of effector cells CD8+ among total CD8+ cytotoxic T-cells. The proportion of effector CD8+ cytotoxic Frontiers in Oncology | www.frontiersin.org 8 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients FIGURE 5 | PET/CT imaging of patient KDO-0101. (A,B) Examination of patient at second relapse in July 2017 showed 18 F-FDG-positive osteolytic lesions in the skeleton (A) sacrum, sacral base with a target-to-liver ratio of 2.74 and sacral left lateral mass with a target-to-liver ratio of 2.39 (B) mandible with a target-to-liver ratio of 4.88. (C,D) Control 18 FDG-PET/CT examination in January 2018 showed a decrease or complete diminishment of 18 FDG accumulation (C) sacrum, sacral base with a target-to-liver ratio of 0.69, and sacral lateral mass with a target-to-liver ratio of 0.66 (D) mandible with a target-to-liver ratio of 1.47. T-cells among total T-cells was further correlated with the proportion of NKT-like cells and γδ T-cells. Both of these nonclassical lymphocyte subsets have been studied and described for their role in cancer surveillance (6, 14, 15). On the other hand, in the putative cancer-enhancing/immune-suppressive cluster, we observed an association between circulating MMDSC and Tregs that might be explained by increase in Tregs induced by MDSC-derived immunosuppressive cytokines (16) as described previously in non-cancer settings (17, 18). NLR associated with M-MDSC and Tregs, which may reflect “emergency” myelopoiesis induced by tumor or by hostrelated conditions, that promotes production of not only classical myeloid cells such as neutrophils and monocytes but also myeloid-derived suppressor cells (19). In line with two inversely associated clusters of immune-based circulating biomarkers, we have previously shown a negative correlation between effector CD27− cytotoxic CD8+ T-cells and number of both CD33hi PMN-MDSCs and M-MDSC in pediatric cancer patients (19). The current clinical trial was designed for patients with progressive, recurrent, or primarily metastatic high-risk tumors that are always heavily pretreated by prior multimodal anticancer therapy. Indeed, patients with measurable disease represented vast majority of cases enrolled to this clinical trial. Therefore, we may expect that patients evaluated in this clinical trial exhibit prior profound suppression of immune function. Indeed, the majority of sarcoma patients were lymphopenic. On peripheral blood immunograms, we showed distinct patterns of immune parameters such as prevailing CD8+ T-cell stimulation in patient KDO-0101 or marked immunosuppression in KDO- 0114. However, observations from immunomonitoring and clinical course in the patient KDO-0101 are worth particular attention. In comparison to the rest of the study group, patient KDO-0101 exhibited a lymphocyte count within the reference range, a high proportion of effector T-cells, and low levels of all observed parameters associated with adverse disease outcome, namely, Treg count, M-MDSC count, and neutrophilto-lymphocyte ratio. This DC-vaccinated patient experienced substantial regression of metastatic Ewing’s sarcoma after the second relapse. In comparison to the initial DC vaccination, at DC rechallenge, a proportion of effector and activated DC increased, although ALC dropped. We also observed an increase in γδ T-cells, which may be attributable to therapy with zoledronic acid that was part of the third-line therapy prior to DC rechallenge. Zoledronic acid causes accumulation of isopentenyl pyrophosphates (IPP), leading to stimulation of γδ T-cells (20). γδ T-cells responding to zoledronic acid are Vγ9+Vδ2+ T-cells that sense IPP via Vδ2 TCR (20). Frontiers in Oncology | www.frontiersin.org 9 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients FIGURE 6 | Ex vivo functional and peripheral blood immunomonitoring of subject KDO-0101 during first dendritic cell (DC) immunotherapy and its rechallenge. (A) Stimulation of T-cells by DCs, reflected by the percentage of division T-cells. (B) Production of interferon-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-17A. (A,B) Pre- and post-DC treatment T-cell response was measured i/ (blue) before start of DC administration (pre-DC) and after the fifth dose (post-DC) ii/ (red) after 4 months with no antitumor treatment, before start of DC rechallenge (pre-DC re) and after the fifth rechallenege dose (post-DC re). (C) Peripheral blood immunogram from baseline (bas) through doses 2, 4, and 6 in the course of both DC treatment (upper) and DC rechallenge (lower). The layout of immunograms is described in Figure 3. (D) Four subtypes of gamma-delta TCR (Vγ9−Vδ2−, Vγ9+Vδ2−, Vγ9+Vδ2+, Vγ9−Vδ2+) in the course of both DC treatment from baseline to dose 19 and DC rechallenge from baseline to dose 10. Interestingly, however, in this patient, we observed an increase in number of Vγ9−Vδ2− T cells and depletion of Vγ9+Vδ2+ T-cells. It is of note that only in two out of nine pediatric sarcoma patients (KDO-0118 and KDO-0139), the Vγ9+Vδ2+ subset represented a majority of circulating γδ T-cells. This is an unexpected observation in the context of reported findings (21) and of our observations in adult carcinoma patients (7) and patients treated and evaluated in the DC clinical trial with non-sarcoma cancers (data not shown). The second relapse in subject KDO-0101 occurred during maintenance therapy with DC ITx. The observed temporary regression of metastases of the Ewing’s sarcoma after second relapse may have been related to the immune response induced by previous DC treatment. Despite stable disease on the thirdline CTx topotecan/cyclophosphamide, the patient exhibited partial response after concomitant RTx and DC vaccination only. Performance status of the patient was good over a long period of time, namely, Karnofsky index over 80%, despite Frontiers in Oncology | www.frontiersin.org 10 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients heavy metastatic involvement in skull, pelvic bones, spinal column, and liver. Performance status declined after 1 year of RTx, DCs ITx, and metronomic vinblastine and zoledronic acid. This unexpected observation suggests an opportunity to deliver such treatment to more patients. We observed substantial enhancement of T-cell reactivity toward DC-presented tumor antigens upon DC vaccination in patient KDO-0101 and to a lesser extent in four other sarcoma patients vaccinated with DCs and analyzed here. Thus, we confirmed that our anticancer DC-based vaccine stimulates a preexisting immune response against self-tumor antigens. Moreover, in the case of KDO- 0101, functional ex vivo testing revealed that T-cell reactivity toward DC-presented self-tumor antigens persisted for a long period of time without DC treatment and was further boosted by DC rechallenge. In principle, the mechanism of action of anticancer DCs relies on stimulation of T-cell-mediated antitumor immune response targeting the presented cancer neoantigens. However, to date, the majority of patients treated with investigational DCs including the pediatric cancer patients in this clinical trial were end-stage or advanced cancer patients with extensive tumor mass and severely destroyed immune system. Limited clinical response achieved by DC-based ITx across numerous clinical trials can be attributed to both tumorinduced immunosuppression and, in heavily pretreated patients, also to anticancer therapy-induced immunosuppression. This is, nevertheless, supported by limited observational experience that enhancement of T-cell response to self-tumor antigens was related to the stage of the disease, that is, lower in cases with sarcomas in progression. It is thus crucial to overcome the immunosuppressive barrier to improve the efficacy of DCbased ITx as to have the antigen-presenting DC-based ITx combinable with cytokines, immune adjuvants, CTx, targeted therapy, and/or checkpoint inhibitors in order to boost T-cell effector functions and/or inhibit immune-suppressive pathways in the tumor mass (22). Ideally, selection of the right concomitant treatment to be combined with DC ITx shall be personalizable to target either particular immunosuppressive elements prevailing or particular immune effectors deficient in a particular patient, such as low-dose cyclophosphamide to deplete Tregs (23) or zoledronic acid to enhance γδ T-cells (24). In this context, immune-based biomarkers within the tumor microenvironment (if accessible) and/or systemic from peripheral blood could be exploited not only to provide an optimal ITx combination but also to select patients that would benefit from DC-based ITx. Regarding tumor-induced immunosuppression that is dependent on the tumor volume renders DC ITx less effective in patients with extensive tumor burden (25) and elicits higher tumorspecific immunologic response rates in the adjuvant compared to the metastatic setting (26). Thus, there is a rationale for the use of DC-based ITx earlier in the course of disease when tumor burden is still minimal; for example, in the adjuvant setting in patients at high risk of recurrence or in patients with minimal metastatic disease. From our perspective beyond the study, anticancer DC vaccination could be more effective if appropriately personalized not only in terms of loading DC with selftumor antigens but also in terms of (i) selection of the right patients that would benefit from ITx (such as patients with tumor with high mutational load), (ii) treatment at the right time when the disease and the level of immune suppression is minimal, and (iii) selection of right (possibly personalized) concomitant treatment that allows the optimal immunostimulation and anticancer activity of effector cells. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee, University Hospital Brno. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin or by the adult participants. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article. AUTHOR CONTRIBUTIONS LFe contributed to the study design, performed laboratory data acquisition and analysis, prepared figures, tables, supplementary material, contributed to data interpretation, and drafted the manuscript. PMu contributed to the trial design, performed patient enrollment and treatment, contributed to data interpretation, and drafted the manuscript. KP supervised IMP manufacturing, contributed to laboratory data acquisition and analyses, supplementary material preparation, and drafted the manuscript. IS performed statistical analysis, contributed to figure preparation, data interpretation, and drafted the manuscript. JM contributed to the trial design, participated in clinical and manufacturing data analysis, and drafted the manuscript. ZR performed PET/CT data acquisition, contributed to figure preparation, data interpretation, and drafted the manuscript. DV and EH contributed to the trial design, data interpretation, and revised the manuscript. DC participated in clinical data acquisition, contributed to supplementary material preparation, and revised the manuscript. LFa participated in clinical data acquisition and revised the manuscript. PMa and ZP contributed to the trial design, participated in patient treatment, and revised the manuscript. RD and JS contributed to the trial design, contributed to data interpretation, and revised the manuscript. LZ-D conceived the study design, designed and supervised laboratory data acquisition and analysis, contributed to data analysis and interpretation, and drafted and finalized the manuscript. FUNDING This work was supported by Czech Ministry of Education, Youth and Sports via Large infrastructure CZECRIN (LM2015090) Frontiers in Oncology | www.frontiersin.org 11 November 2019 | Volume 9 | Article 1169 Fedorova et al. Immunomonitoring of DC-Treated Sarcoma Patients and via National Sustainability Program I (RECAMO2020, LO1413), by Czech Ministry of Health via project No. NV18- 03-00339 and DRO 00209805, and by European Regional Development Fund–project CZECRIN_4PATIENTY (Reg. No. CZ.02.1.01/0.0/0.0/16_013/0001826). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.01169/full#supplementary-material REFERENCES 1. Mackall CL, Rhee EH, Read EJ, Khuu HM, Leitman SF, Bernstein D, et al. 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(2011) 18:e150–7. doi: 10.3747/co.v18i3.783 26. Bol KF, Aarntzen EH, Hout FE, Schreibelt G, Creemers JH, Lesterhuis WJ, et al. Favorable overall survival in stage III melanoma patients after adjuvant dendritic cell vaccination. Oncoimmunology. (2016) 5:e1057673. doi: 10.1080/2162402X.2015.1057673 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2019 Fedorova, Mudry, Pilatova, Selingerova, Merhautova, Rehak, Valik, Hlavackova, Cerna, Faberova, Mazanek, Pavelka, Demlova, Sterba and Zdrazilova-Dubska. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Oncology | www.frontiersin.org 12 November 2019 | Volume 9 | Article 1169 ORIGINAL RESEARCH published: 07 February 2020 doi: 10.3389/fonc.2019.01531 Frontiers in Oncology | www.frontiersin.org 1 February 2020 | Volume 9 | Article 1531 Edited by: George Calin, University of Texas MD Anderson Cancer Center, United States Reviewed by: Ioana Berindan Neagoe, Iuliu Ha¸tieganu University of Medicine and Pharmacy, Romania Barbara Pasculli, Casa Sollievo della Sofferenza (IRCCS), Italy *Correspondence: Kristyna Polaskova polaskova.kristyna@fnbrno.cz Specialty section: This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology Received: 18 July 2019 Accepted: 19 December 2019 Published: 07 February 2020 Citation: Polaskova K, Merta T, Martincekova A, Zapletalova D, Kyr M, Mazanek P, Krenova Z, Mudry P, Jezova M, Tuma J, Skotakova J, Cervinkova I, Valik D, Zdrazilova-Dubska L, Noskova H, Pal K, Slaby O, Fabian P, Kozakova S, Neradil J, Veselska R, Kanderova V, Hrusak O, Freiberger T, Klement GL and Sterba J (2020) Comprehensive Molecular Profiling for Relapsed/Refractory Pediatric Burkitt Lymphomas—Retrospective Analysis of Three Real-Life Clinical Cases—Addressing Issues on Randomization and Customization at the Bedside. Front. Oncol. 9:1531. doi: 10.3389/fonc.2019.01531 Comprehensive Molecular Profiling for Relapsed/Refractory Pediatric Burkitt Lymphomas—Retrospective Analysis of Three Real-Life Clinical Cases—Addressing Issues on Randomization and Customization at the Bedside Kristyna Polaskova1,2 *, Tomas Merta1,2 , Alexandra Martincekova1,2 , Danica Zapletalova1,2 , Michal Kyr1,2 , Pavel Mazanek1 , Zdenka Krenova1 , Peter Mudry1 , Marta Jezova3 , Jiri Tuma4 , Jarmila Skotakova5 , Ivana Cervinkova5 , Dalibor Valik6,7 , Lenka Zdrazilova-Dubska6,7 , Hana Noskova8 , Karol Pal8 , Ondrej Slaby8 , Pavel Fabian9 , Sarka Kozakova2,7 , Jakub Neradil2,10 , Renata Veselska2,10 , Veronika Kanderova11 , Ondrej Hrusak11 , Tomas Freiberger8,12,13 , Giannoula Lakka Klement1,14 and Jaroslav Sterba1,2,7 1 Department of Pediatric Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czechia, 2 International Clinical Research Center, St. Anne’s University Hospital, Brno, Czechia, 3 Department of Pathology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czechia, 4 Department of Pediatric Surgery, Orthopedics and Traumatology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czechia, 5 Department of Pediatric Radiology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czechia, 6 Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czechia, 7 Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czechia, 8 Central European Institute of Technology, Masaryk University, Brno, Czechia, 9 Department of Oncological Pathology, Masaryk Memorial Cancer Institute, Brno, Czechia, 10 Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czechia, 11 Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, Prague, Czechia, 12 Faculty of Medicine, Masaryk University, Brno, Czechia, 13 Centre for Cardiovascular Surgery and Transplantation, Brno, Czechia, 14 CSTS Health Care, Toronto, ON, Canada In order to identify reasons for treatment failures when using targeted therapies, we have analyzed the comprehensive molecular profiles of three relapsed, poor-prognosis Burkitt lymphoma cases. All three cases had resembling clinical presentation and histology and all three patients relapsed, but their outcomes differed significantly. The samples of their tumor tissue were analyzed using whole-exome sequencing, gene expression profiling, phosphoproteomic assays, and single-cell phosphoflow cytometry. These results explain different treatment responses of the three histologically identical but molecularly different tumors. Our findings support a personalized approach for patient with high risk, refractory, and rare diseases and may contribute to personalized and customized treatment efforts for patients with limited treatment options like relapsed/refractory Burkitt lymphoma. SUMMARY The main aim of this study is to analyze three relapsed Burkitt lymphoma patients using a comprehensive molecular profiling, in order to explain their different outcomes Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas and to propose a biomarker-based targeted treatment. In cases 1 and 3, the tumor tissue and the host were analyzed prospectively and appropriate target for the treatment was successfully implemented; however, in case 2, analyses become available only retrospectively and his empirically based rescue treatment did not hit the right target of his disease. Keywords: Burkitt lymphoma, targeted therapy, precision medicine, theranostics, pediatric oncology INTRODUCTION Burkitt lymphoma is a highly aggressive mature B-cell lymphoma commonly associated with translocation of MYC gene. The disease is classified as sporadic, endemic, or immunodeficiency related. In pediatric oncology, current standard intensive chemotherapy with anti-CD20 antibody regimens achieve longterm, disease-free survival in almost 95% of patients (1). However, a subset of patients who do not respond to the first-line chemotherapy and who experience relapse have very poor prognosis despite high-dose chemotherapy followed by stem cell transplantation (2). This subset of patients, for whom further chemotherapy-based therapies are futile, is recently often considered for therapies based on molecular analysis of their tumor tissue. We present three cases of relapsed Burkitt lymphoma. Cases 1 and 3 were treated with a therapy that reflected the molecular signature of the child’s tumor, but in case 2, the therapy “missed” the target because his molecular signature was not known at the time retrieval therapy was initiated. The findings suggest that molecular signatures are unique, and a tissue biomarker-based customized therapy may be the better approach to address these poor prognosis patients than just another biomarker agnostic randomized trial. METHODS A comprehensive molecular profiling consisted of whole-exome, gene expression profiling and a profile of phosphorylated proteins and single-cell phosphoflow cytometry of three cases of relapsed pediatric Burkitt lymphoma searching for biological rationale for different responses to the therapy and different clinical outcomes. Whole-Exome Sequencing Whole-exome sequencing (WES) using the TruSeq DNA Exome Kit, the NextSeq 500/550 Mid Output Kit v2.5, and a NextSeq 500 sequencing device (all Illumina, CA, USA) was done in all three cases. Input material was 400 ng of DNA obtained from the peripheral blood (for germline exome) and formalin-fixed, paraffin-embedded (FFPE) tumor sample with ≥20% cancer cell count measured in the surface area of tissue slides for somatic exome. WES was done with high coverage where at least 90% of targeted regions were covered 20 times. Gene Expression Profiling (Transcriptome Examination) Gene expression profiling using the Affymetrix GeneChip Human Gene 1.0. ST Array (Applied Biosystems, CA, USA) was done in all three cases. Input material was 250 ng of RNA obtained from frozen tumor tissue. Samples were prepared using the GeneChip WT PLUS Reagent Kit (Affymetrix, CA, USA) according to the manufacturer’s protocol. Subsequently, chips were hybridized using the GeneChip Hybridization Oven, washed using the GeneChip Fluidics Station, and scanned on the GeneChip Scanner (all Affymetrix, CA, USA), and CEL files were generated. Data were processed using R software version 3.3.3 (3). Gene expressions of 220 selected genes were subsequently compared to accumulated normal tissue samples as described previously (4), utilizing two comparator sets: one consisting of 408 normal tissue samples of different diagnoses (main general comparator) and one consisting of 5 samples of normal germinal center B cells (complementary-specific comparator). Samples were downloaded from Gene Expression Omnibus and ArrayExpress databases, and names of the database samples are listed in Supplementary Material 1. Expression data were calculated as Robust Multichip Average (RMA) with background correction and quantile normalization implemented in rma function in oligo package (5). Difference of expression of each gene was calculated as fold change (FC) from the mean of the comparator set and tested using a two-sided one-sample t-test, with false discovery rate (FDR) adjustment applied. An FC value of 0.5 and more was considered important. No specific p-value was considered limiting the discrimination of differently expressed genes with FC > 0.5. Utilizing the general comparator consisting of 408 samples offers highly significant results corresponding to the power of 10 to −25 for the FDRadjusted p-values for most of the evaluated genes with FC of 0.5 or more, and rising to the power of 10 to −100 for the FDR-adjusted p-values for genes with FC > 2. RNA transcription data from the tumor tissues were analyzed as well using Biogrid (http://thebiogrid.org), and http://www. genome.jp/kegg/pathway.html and mathematical simulations of protein–protein interactions as described before (6). Profile of Phosphorylated Proteins Human Phospho-RTK Array Kit (R&D Systems) was used to determine the relative levels of tyrosine phosphorylation of 49 different RTKs. Human Phospho-MAPK Array Kit (R&D Systems) was employed for the detection of phosphorylation status of 26 MAPKs, serine/threonine kinases, and other signaling proteins. Both arrays were performed as previously described (7). Single-Cell Phosphoflow Cytometry Peripheral blood mononuclear cells (PBMCs) were separated on Ficoll-Paque (GE Healthcare) according to the manufacturer’s Frontiers in Oncology | www.frontiersin.org 2 February 2020 | Volume 9 | Article 1531 Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas instructions. PBMCs were reconstituted in a culture medium consisting of RPMI 1640 with 25 mM HEPES, L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Lonza, Basel, Switzerland) to a final concentration of 2 million cells per milliliter. After a 1-h rest at 37◦C in a 5% CO2 atmosphere, the cells were stimulated on 96-well plate containing coated anti-CD3 (10 µg/ml, Exbio Praha) and free costimulatory anti-CD28/CD49d antibodies (1 µg/ml, BD Biosciences) for 5, 15, and 30 min. The cells were fixed with 4% formaldehyde for 10 min and permeabilized with ice-cold methanol for 30 min. The following fluorochrome conjugates were used for cytometric detection: phospho-Akt (Ser473)-Alexa Fluor 488, phospho-S6 (Ser235/236)-Pacific Blue (Cell Signaling Technologies), phospho-mTOR (Ser2448)-PE (eBioscience, Thermo Fisher), CD45-Pacific Orange, CD45RA-APC (Exbio), CD8-PE-Cy7 (Beckman Coulter), CD4-PerCP-Cy5.5, and CD3-APC-H7 (BD Biosciences). The samples were acquired on Canto II flow cytometer and analyzed using FlowJo software (BD Biosciences). RESULTS Case 1 A 7-year-old previously healthy boy presented with t(8;14) positive abdominal stage III Burkitt lymphoma (St. Jude staging system). The boy was initially treated as per the standard BFM B-NHL Registry 2012 protocol with the addition of rituximab according to the most recent published literature (1). He responded well to the therapy and achieved a very good partial response after two cycles. His clinical course was complicated by an episode of duodenal obstruction/intussusception requiring surgical intervention. The histology from this resection revealed sclerosing mesenteritis with no evidence of lymphoma, congruent with the conclusion of a study using 18Ffluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) that revealed a very small residual tumor with only borderline FDG PET avidity. Unfortunately, the patient had disease progression 6 weeks following the completion of protocol therapy (and 3 months from the second surgery) with a new lesion within the tumor resection margin and a new mediastinal mass. A biopsy of the abdominal lesion confirmed the recurrence of Burkitt lymphoma with persistent areas of sclerosing mesenteritis. As sclerosing mesenteritis has been associated in the literature not only with B-cell lymphomas but also with activation of the PI3K-delta pathway and immunodeficiency (8, 9), a candidate testing for this specific mutation was performed. In the tumor, there was proven disruption of MYCC and IgH in 97% of cells according to fluorescence in situ hybridization (FISH). Karyotype of the tumor showed 46 chromosomes with complex changes. A germline variant of c.935C>G (p.S312C) in the PI3K-delta subunit was found both in the child and in the father. The patient’s older sister and mother were negative for this variant. We tested the intracellular signaling downstream of PI3K using flow cytometry assessment of phosphorylation of Akt, mTOR, and S6 proteins in the patient’s peripheral blood T-lymphocytes and detected increased basal and T-cell receptor (TCR)-induced activation (Figure 1A). Similarly, increased levels of PI3K were confirmed by RNA transcriptome analysis of the tumor tissue with Affymetrix GeneChipST 1.0. This analysis also revealed an increased expression of HR23B, a predictor of response to histone deacetylase (HDAC) inhibitors. Immunohistochemistry revealed a strong expression of PD-1L. The variant p.S312C has been described previously as mutation in brain cancer cell line and prostate cancer cell line (10) but has been classified as benign for development of immunodeficiency according to the ClinVar database. The allele frequency ranges between 0.008 and 0.030 in population databases (gnomAD 0.02, ExAc 0.0217, 1000G/ALL 0.008, 1000G/EUR 0.029) and was found to be 0.018 in our cohort of 508 cord blood samples (not published). Thus, this variant cannot be considered pathogenic. However, it may predispose the PI3K pathway to be activated, if other genetic and/or non-genetic factors are present. Interestingly, even though the biopsy at the time of initial diagnosis had been tested for TP53 and no alteration of the gene was found, in the biopsy obtained from the relapse, a new TP53 R273C somatic mutation was identified in the tumor. Retrieval therapy was administered with obinutuzumab 550 mg/m2, ibrutinib 140 mg/m2, and two cycles of ifosfamide, carboplatin, and etoposide (ICE) chemotherapy. The patient had further progression on this therapy, and a more molecular biomarker-driven theranostic approach was discussed. The therapy was changed to a single-agent window using a specific inhibitor of PI3K idelalisib 200 mg/m2/d. In 2 weeks, we were able to document a markedly decreased PI3K pathway activation in the patient’s peripheral blood T-lymphocytes (Figure 1B), but the disease was still showing further radiological progression. Therapy with idelalisib was not discontinued, and ibrutinib 140 mg/m2 daily was reintroduced. Based on the transcriptome analysis, valproic acid for HDAC inhibition aiming for serum levels of 80–100 µg/ml was added, and nivolumab at 3 mg/kg every second week and metronomic cyclophosphamide at 25 mg/m2/7 days on/7 days off were introduced for immune modulation. To support local disease management and support the tumor antigen presentation, the patient received 21-Gy radiation to the site of the abdominal relapse. There was evidence of partial remission on FDG PET/CT 3 months later and stable disease 6 months later. Due to persistence of a viable tumor on FDG PET/CT and high toxicity of allogenic stem cell transplant reported in nivolumab-treated patients (11), this approach was not considered as treatment of choice. Consequently, personalized immunotherapy with dendritic cell-based vaccine was preferred to support the antitumor immunity, and treatment with dendritic cells loaded with whole tumor lysate according to phase I/II protocol (EudraCT No. 2014-003388-39) (12) was initiated. The residual tumor resected after 11 months of such therapy consisted of mainly necrotic tissue with lymphocytic infiltration with no evidence of viable tumor. Considering that the child had achieved complete remission, valproic acid, ibrutinib, and idelalisib were gradually discontinued and the patient is continuing to take biweekly intradermal applications of autologous dendritic cell vaccine and nivolumab until May 2018 when all his 37 manufactured doses of dendritic cell-based vaccine were used up. The progression-free survival (PFS) of 46 months following a customized, tumor tissue molecular analysis-guided regimen was Frontiers in Oncology | www.frontiersin.org 3 February 2020 | Volume 9 | Article 1531 Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas FIGURE 1 | Phosphorylation patterns in the PI3K pathway in peripheral blood T-lymphocytes before (A) and after (B) therapy in case 1. Case 2 patient had a germline variant of PIK3CD, which was present in the tumor as well. Peripheral blood T-lymphocytes (patient 1’s lymphocytes contained only T cells at the time of testing) were tested for activation of the PI3K signaling pathway [reflected as a phosphorylation of Akt (Ser473), mTOR (Ser2448), and S6 ribosomal protein (Ser235/236)] before and following therapy. (A) Patient T-lymphocytes showed increased basal phosphorylation of Akt as well as increased phosphorylation of Akt and S6 upon T-cell receptor (TCR) stimulation before treatment compared to an independent healthy control (the result is representative of three independent tests). (B) A week following the addition of idelalisib (a PI3K inhibitor), to the patient’s therapy, the phosphorylation of Akt, mTOR, and S6 dropped down. CD3+ T-lymphocytes are shown in basal state (tinted histograms) and 15 min upon anti-CD3/CD28/CD49d stimulation (blank histograms). Red, patient 1; black, healthy control. the longest PFS this child had achieved. The comparison of his earlier therapies reveals that he had achieved PFS1 6 months on the initial standard BFM protocol, and PFS2 only 1 month on the intensive retrieval therapy using anti-CD20 (obinutuzumab), ICE, and ibrutinib. His individualized therapy was outpatient based, associated with minimal treatment-related toxicities and allowed the child to return to school and perform all activities of daily living. Case 2 A 3-year-old boy diagnosed abroad with widely disseminated Burkitt lymphoma (abdomen, bone marrow, and both kidneys) was initially treated with the same standard BFM-based chemotherapy, but without rituximab. Before the completion of the fifth cycle, the patient had disease progression with a biopsy-positive new lesion in the right cheek. He continued with a relapse ALL protocol/ALL-REZ BFM 2002 in his home country outside the Czech Republic. As no therapeutic response was achieved, he was referred to our institution for a second opinion and management. He received two cycles of R-ICE (rituximab, ifosfamide, carboplatin, etoposide) given as per the ANHL0121 protocol achieving partial response, but the treatment was accompanied with severe life-threatening toxicities. He underwent surgery to obtain specimen for theranostic testing; however, the amount of the tumor tissue was not sufficient for all molecular studies. Based on our previous success in case 1 and as bridging to high-dose chemotherapy, he therefore continued with ibrutinib 140 mg/m2 daily, idelalisib 100 mg/m2 daily, and cyclophosphamide 1.5 mg/kg daily week on/week off for 6 weeks. Due to toxicities of intensive therapies and a clinical need for further therapy as bridging to stem cell transplant, the targeted agents were in this case based on our previous experience and a literature review. Despite a high-dose carmustine, etoposide, cytarabine, melphalan (BEAM) chemotherapy as per the AHOD0121 protocol (13) and autologous stem cell transplant being performed, he continued to do poorly. The patient had disease progression 3 weeks after BEAM conditioning and autologous stem cell transplant with a new lesion in the abdomen and continued to progress with massive L3 blast presence in the cerebrospinal fluid. He died due to disease progression 11 months from the initial diagnosis and 6 months after his first progression. Frontiers in Oncology | www.frontiersin.org 4 February 2020 | Volume 9 | Article 1531 Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas Case 3 A 12-year-old boy was diagnosed with bulky abdominal Burkitt lymphoma. The patient was initially treated as per the standard BFM B-NHL Registry 2012 protocol with the addition of rituximab, but he achieved only partial response after two cycles, and assessment after four cycles revealed residual tumor with still increased FDG PET avidity. Three months later, the FDG PET/CT showed radiological progression of the primary tumor and dissemination in the right retromandibular area and anterior mediastinum. The relapse of Burkitt lymphoma was confirmed by biopsy. However, WES from the relapsed tumor sample revealed high tumor mutation burden−31 mutations/Mb; moreover, gene expression profiling detected strong expression of PD1, and the overall expression patterns of the case 3 were very similar to case 2 patient with very high fibronectin expression. First, participation in the randomized ibrutinib retrieval trial was planned here; however, based on molecular profiling and our previous experience from case 2, we have prioritized immune therapy here. He achieved radiological partial remission after third R-ICE cycle and then continued with nivolumab single agent only. After 12 weeks of nivolumab, he achieved first complete remission. His first PFS on standard intensive protocol was 7 months, but the second PFS with using immunotherapy is 14 months. Analyses Somatic exome analysis of relapse samples revealed variants in the TP53 gene in cases 1 and 2 (p.R273C in case 1 and p.R248L in case 2, NM_000546). p.R273C and p.R248L in TP53 have been previously described as loss of function mutations based on in vitro functional analyses (14–19). Somatic exome analysis in case 1 detected a number of variants; the selected ones are shown in Supplementary Material 2. Germline exome analysis in case 1 also confirmed p.S312C (NM_005026) variant in the PIK3CD gene in the heterozygous form. Somatic exomes of cases 2 and 3 revealed a number of variants; the selected ones are also available in Supplementary Material 2. Gene expression profiles of all three cases proved to be very similar; the highest expressions showed genes involved in immune system (BTK, CD79A, CD79B, and KLHL6). In cases 1 and 2, increased expression also showed genes involved in DNA damage response (BRCA1, BRCA2, FANCA, and FANCD2). In case 1, CSF1R and PDGFRA genes were also found to be increasingly expressed, while no genes coding tyrosine kinases showed to be overexpressed in case 2. In case 3, increased expressions showed genes involved in fibroblast growth factor signaling. In comparison to other pediatric oncology patients analyzed at our institute, transcriptome analysis in cases 1 and 2 revealed significantly increased expression of the MYC proto-oncogene. In case 1, two samples of the tumor tissue were also analyzed for activity of cell signaling pathways using phosphoprotein arrays for detection of RTKs, MAPKs, serine/threonine kinases, and other signaling protein as specified above: tumor tissue sample after the first line of treatment (Figure 2: case 1a) and second sample taken during the treatment of relapsed disease (Figure 2: case 1b). Phosphorylation profiles showed high relative activities of EGFR, PDGFRβ, ROR2, CREB, ERK1/2, and HSP27 in both samples. Furthermore, a very high level of phosphorylation was detected for p53 protein on Ser46 in the second sample in comparison to the first sample from this patient. This finding is in full accordance with the previous proapoptotic treatment including etoposide administration (20). In case 2, nevertheless, phospho-RTK analysis (Figure 2: case 2) revealed high phosphorylation of EGFR and PDGFRβ, and the phosphorylation profile of MAPKs, serine/threonine kinases, and other signaling proteins showed high activities of CREB, ERK1/2, and HSP27 in ascending order of density value. Serology of Epstein–Barr virus (EBV) revealed the IgG positivity of EBV nuclear antigen (EBNA)-1 and the IgG positivity of viral capsid antigen (VCA) as well case 1 and case 2. DISCUSSION The introduction of highly intensive multiagent chemotherapy has dramatically improved the survival rates of primary childhood Burkitt lymphoma. While the initial treatment can have an over 90% success rate using standard intensive chemotherapy with rituximab, the outcome of children with relapsed Burkitt lymphoma is still very poor. The difficulties with treating chemotherapy-resistant relapsed tumors suggest an evolution of a more complex and more resistant disease (21), as could be documented by a new TP53 mutation in our case 1 at relapse, which was suggested by phosphoproteomic assay as well. The overview of our three cases reveals children with some very similar characteristics of their diseases, with alike pattern of cell signaling in tumor tissue, treated with identical agents in the first part of their relapse treatment, who experienced very dissimilar outcomes after the first relapse. It suggests that the tumors with similar histological features may harbor chemotherapy-resistant, genetically and biologically distinct subclones that become more dominant after intensive chemotherapy (21). At presentation, a fraction of these chemotherapy-resistant subpopulations may be small but, following intensive maximum tolerated dose-based chemotherapy, probably increases, and the tumor residuum is subsequently populated by resistant subclones. This evolution was furthermore evident on the evolution of molecular findings in the first patient and supports the need for a careful theranostic analysis and repeated biopsies whenever clinically indicated. Treatment of relapsed disease should be based on a detailed molecular analysis of the most recent available sample, i.e., at the time of relapse or progression rather than on original tumor biopsy only. The choice of drug combinations reflecting a broader molecular profile was based on reports that customized combinatorial therapies may produce more sustained responses (22, 23). Furthermore, as many biological agents are in fact chemotherapy sensitizers, their proper dosage should carefully be titrated to avoid severe systemic toxicity. In case 1, we have started with a single-agent idelalisib to target what was thought to be the driver mutation and gradually added additional targeted agents but at doses about 50% of those recommended in the Summary of Product Characteristics to avoid severe toxicity. Frontiers in Oncology | www.frontiersin.org 5 February 2020 | Volume 9 | Article 1531 Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas FIGURE 2 | The relative phosphorylation analysis of tumor tissue samples. Human Phospho-MAPK Array Kit (R&D Systems) was employed for the detection of phosphorylation status of 49 RTKs, 26 MAPKs, serin/threonin kinases, and other signaling proteins, which performed using phosphoprotein arrays. Frontiers in Oncology | www.frontiersin.org 6 February 2020 | Volume 9 | Article 1531 Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas To successfully apply precision oncology principles into clinical practice, a requisite testing for molecular targets for each patient needs to be completed. As pointed above, while all three patients had histologically identical disease and were given the same combination of agents in the first- and two of them as second-line treatments, in case 2, we did not have a representative tumor sample timely available and his therapy was based only on detailed literature review and not the theranostic concept (24–26). The biology of the relapsed disease of case 3 reflected by transcriptome was similar to that of case 2, so a different approach could be undertaken, and while reflecting high mutational burden and increased expression of the PD-1L detected by immunohistochemistry and transcriptome, anti-PD- 1 antibody was successfully used here. While analyzing the transcriptomic results including considerations of gene and network interactions using https:// string-db.org/ and http://www.genome.jp/kegg/pathway. html databases (6, 21), we were able to distinguish different patterns of tumor biology among our patients. Case 1 suggested neurotrophic receptor tyrosine kinase 1 (NTRK1) as a signaling protein and one of the best targets. In case 2 and case 3, in contrast, despite being clinically and histologically similar, transcriptomic results suggest an entirely different network, where fibronectin 1 (FN1) has a very complex downstream impact. Because FN1 is not a signaling protein and a druggable target, it is likely that we missed the putatively most important pathway in case 2. One may speculate that integrin inhibitors like cilengitide could be a better therapeutic option here. For case 3, FN1 seemed to be the key molecular hub as well, and it was one of the reasons for clinical decision to rely on tumor mutational burden and PD-1 ligand expression and treat the patient with immune therapies, rather than small molecules. The localization of MYC proto-oncogene on q24 of the human chromosome 8 and its translocation to chromosome 14 is considered pathogenic in most cases of Burkitt lymphoma. In our cases, the RNA transcription analyses as described above indicate the activations of different sets of genes. These patients were almost identical in their clinical presentation, histology, MYC status, and initial clinical response to standard chemotherapy. Early clinical testing initiatives are beginning to employ individual profiles/fingerprint analyses to compile patients into histologically or biologically similar series (27), and as these efforts continue, new clinical trial designs will emerge (28, 29). The research that has emerged over the last 40 years disproves the concept that cancer is a consequence of a single oncogenic change. It is widely accepted that an initiating oncogenic change such as translocation involving MYC is interpreted within the patient’s genome, and further genomic alterations lead to the oncogenic inducers hijacking host-specific physiological responses such as angiogenesis, inflammation, and immune evasion. These normal physiological responses are not detected by DNA mutational analysis because they represent reactivation of developmentally silent pathways. We advocate the use of combinations of biological agents addressing not only the DNA mutations but also the normal physiological responses of the host as they are reflected in the individual’s molecular signature reflected on transcriptomic and proteomic levels. In case 3, we successfully used immunotherapy reflecting the molecular profile of the tumor. In cases 1 and 2, we used a combination of ibrutinib (inhibitor of BCR signaling), idelalisib (direct PI3Kdelta inhibitor), valproate (HDAC inhibitor with potential to enhance responsiveness to immune therapies), and nivolumab (a host immune response modulator). Both patients were intended to receive an immune-supportive therapy using autologous dendritic cell vaccination with non-immunesuppressive maintenance agents such as checkpoint inhibitors, but only case 1 patient had achieved sufficient duration of the clinical response to live long enough to enable the preparation of his vaccine. Unfortunately, because we did not have the benefit of molecular information on genome or transcriptome in case 2, the therapy could not be customized enough to provide a more effective therapeutic combination. Our results revealing highly phosphorylated EGFR, PDGFRβ, ROR2, ERK1/2, or Hsp27 in all samples are also in accordance with previously published findings on Burkitt lymphoma (30, 31). Interestingly, activation of EGFR and ERK signaling via EBV oncoprotein LMP1 was also reported (32, 33) and our results thus concur with the latent EBV infection as suggested by serological analysis. One of the most interesting observations was the discordance between laboratory and clinical responses to biomarkerbased targeted therapy in case 1. Even though there was evidence of normalization of PI3K pathway activity, the evidence of radiological response was significantly delayed and gave an impression that the patient continued to progress. As has been frequently observed with biological therapies, the biomarker response may be more informative and preceded in this case the radiological response. While using biological therapies, we must allow sufficient time to pass before the patient is evaluated using present radiomorphological methods. As we show, in cases where individualization of treatment protocols can be based on the recent molecular information, the likelihood of successful therapy may be increased, but the use of a targeted agent without laboratory evidence of contemporary target activation may not only lack benefit— it may even be harmful. Similarly, while treating sepsis, we are not using several-month-old microbiology results to guide antimicrobial treatment. Considering that there are presently numerous initiatives intending to study the addition of idelalisib and/or ibrutinib to existing retrieval therapies for relapsed and refractory mature B-cell lymphomas, it may be of value to collect enough samples for tumor tissue analysis and enable similar retrospective comparisons of patients who either failed or responded to therapy. An attractive concept inspired by our cases may be the successful sequence of different treatment modalities, such as intensive chemotherapy to debulk the initial tumor volume, followed by targeted biomarker-based treatment and stimulation of autologous immune response later on to consolidate the response. Frontiers in Oncology | www.frontiersin.org 7 February 2020 | Volume 9 | Article 1531 Polaskova et al. Molecular Profiling of Relapsed Burkitt Lymphomas CONCLUSION Precision medicine has significantly altered the practice of clinical oncology, but no standardized approach to the choice of these therapies exists. The three cases presented here emphasize that despite similarities in the presentation, histology, age, tumor site, and initial treatment response, the biology of tumors may differ significantly between cases and may change over time. Case 2 patient had an entirely different molecular signature and thus biology, without underlying relevant germline mutation, but such differences in molecular profile could be appreciated in retrospect only. We conclude that considering the dire outcomes of relapsed Burkitt lymphoma, theranostic testing may identify the most frequent molecular profiles that lead to therapeutic resistance and may help to improve frontline therapies sufficiently to prevent relapses and 1 day to replace our decade-old and toxic drugs like anthracyclines and alkylating agents. DATA AVAILABILITY STATEMENT The datasets for this article are not publicly available because it is not in accordance with our institutional policy. We handle data of rare entities that may be at risk of identification. Requests to access the datasets should be directed to Kristyna Polaskova, polaskova.kristyna@fnbrno.cz. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee for Multicenter Clinical Trials of the University Hospital Brno. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. AUTHOR CONTRIBUTIONS KP wrote the draft of the manuscript and evaluated patient record. TM wrote the manuscript. DZ and AM evaluated patient records. MK did the statistical analyses. PMa, ZK, and PMu participated on the treatment decision and evaluated patient records. MJ performed pathological investigation. JT performed surgical procedures. JS and IC performed the radiological evaluations. DV, LZ-D, and SK participated on the manuscript. HN, KP, OS, PF, JN, RV, VK, OH, and TF performed biological samples analyses. GK supervised and wrote the manuscript. JS conceived and supervised the project and wrote the manuscript. FUNDING The study was supported by projects 16-33209A, 16-34083A from the Ministry of Healthcare of the Czech Republic, project No. MUNI/A/1586/2018 from Masaryk University, Brno, Czechia, project MH CZ - DRO (FNBr, 65269705), by projects LQ1605, LO1604, LO1413, and LQ1601 from the National Program of Sustainability II (MEYS), and by Charles University, UNCE 204012. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.01531/full#supplementary-material REFERENCES 1. Meinhardt A, Burkhardt B, Zimmermann M, Borkhardt A, Kontny U, Klingebiel T, et al. Phase II window study on rituximab in newly diagnosed pediatric mature B-cell non-hodgkin’s lymphoma and Burkitt leukemia. J Clin Oncol. (2010) 28:3115–21. doi: 10.1200/JCO.2009. 26.6791 2. Attarbaschi A, Dworzak M, Steiner M, Urban C, Fink FM, Reiter A, et al. 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Regulation of EBV LMP1-triggered EphA4 downregulation in EBV-associated B lymphoma and its impact on patients’ survival. Blood. (2016) 128:1578–89. doi: 10.1182/blood-2016-02-702530 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2020 Polaskova, Merta, Martincekova, Zapletalova, Kyr, Mazanek, Krenova, Mudry, Jezova, Tuma, Skotakova, Cervinkova, Valik, Zdrazilova-Dubska, Noskova, Pal, Slaby, Fabian, Kozakova, Neradil, Veselska, Kanderova, Hrusak, Freiberger, Klement and Sterba. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Oncology | www.frontiersin.org 9 February 2020 | Volume 9 | Article 1531 cancers Article Assessment of Tumor Mutational Burden in Pediatric Tumors by Real-Life Whole-Exome Sequencing and In Silico Simulation of Targeted Gene Panels: How the Choice of Method Could Affect the Clinical Decision? Hana Noskova 1,2, Michal Kyr 2,3,4, Karol Pal 1,5, Tomas Merta 2,3,4, Peter Mudry 2,3,4, Kristyna Polaskova 2,3,4, Tina Catela Ivkovic 1 , Sona Adamcova 1, Tekla Hornakova 1, Marta Jezova 6, Leos Kren 6, Jaroslav Sterba 2,3,4,7,* and Ondrej Slaby 1,3,6,* 1 Central European Institute of Technology, Masaryk University, 62500 Brno, Czech Republic; hana.noskova@ceitec.muni.cz (H.N.); pal@mail.muni.cz (K.P.); tina.ivkovic@ceitec.muni.cz (T.C.I.); sona.adamcova@ceitec.muni.cz (S.A.); tekla.hornakova@hotmail.com (T.H.) 2 Department of Pediatric Oncology, University Hospital Brno, 613 00 Brno, Czech Republic; kyr.michal2@fnbrno.cz (M.K.); merta.tomas@fnbrno.cz (T.M.); Mudry.Peter@fnbrno.cz (P.M.); polaskova.kristyna@fnbrno.cz (K.P.) 3 Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic 4 International Clinical Research Center, St. Anne’s University Hospital, 65691 Brno, Czech Republic 5 Department of Hematology, University Hospital Schleswig-Holstein, 24105 Kiel, Germany 6 Department of Pathology, University Hospital Brno, 62500 Brno, Czech Republic; jezova.marta@fnbrno.cz (M.J.); Kren.Leos@fnbrno.cz (L.K.) 7 Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 60200 Brno, Czech Republic * Correspondence: Sterba.Jaroslav@fnbrno.cz (J.S.); ondrej.slaby@ceitec.muni.cz (O.S.) Received: 25 December 2019; Accepted: 11 January 2020; Published: 17 January 2020 Abstract: Background: Tumor mutational burden (TMB) is an emerging genomic biomarker in cancer that has been associated with improved response to immune checkpoint inhibitors (ICIs) in adult cancers. It was described that variability in TMB assessment is introduced by different laboratory techniques and various settings of bioinformatic pipelines. In pediatric oncology, no study has been published describing this variability so far. Methods: In our study, we performed whole exome sequencing (WES, both germline and somatic) and calculated TMB in 106 patients with high-risk/recurrent pediatric solid tumors of 28 distinct cancer types. Subsequently, we used WES data for TMB calculation using an in silico approach simulating two The Food and Drug Administration (FDA)-approved/authorized comprehensive genomic panels for cancer. Results: We describe a strong correlation between WES-based and panel-based TMBs; however, we show that this high correlation is significantly affected by inclusion of only a few hypermutated cases. In the series of nine cases, we determined TMB in two sequentially collected tumor tissue specimens and observed an increase in TMB along with tumor progression. Furthermore, we evaluated the extent to which potential ICI indication could be affected by variability in techniques and bioinformatic pipelines used for TMB assessment. We confirmed that this technological variability could significantly affect ICI indication in pediatric cancer patients; however, this significance decreases with the increasing cut-off values. Conclusions: For the first time in pediatric oncology, we assessed the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels and confirmed a significant technological variability to be introduced by different laboratory techniques and various settings of bioinformatic pipelines. Cancers 2020, 12, 230; doi:10.3390/cancers12010230 www.mdpi.com/journal/cancers Cancers 2020, 12, 230 2 of 14 Keywords: pediatric tumors; tumor mutational burden; TMB; whole-exome sequencing; gene panel sequencing; immune checkpoint inhibitors 1. Introduction The cancer cell genome acquires genetic alterations differing from the germline of the host [1]. Somatic mutation rates can be affected by exposure to exogenous factors, such as ultraviolet light or tobacco smoke [2], or by compounding genetic defects, such as DNA mismatch repair deficiency, microsatellite instability, or replicative DNA polymerase mutations [1–3]. These somatic genetic alterations induce and drive carcinogenesis. The type and the number of acquired mutations varies among the cancer types but also among the affected individuals [4]. Some of these mutations lead to the formation of tumor-specific neoantigens, which could be recognized by a patient’s immune system as non-self and which are highly clinically relevant since these neoantigens can make the cancer cells sensitive to treatment with immune checkpoint inhibitors (ICIs) against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in various cancers including melanoma [5], non–small-cell lung cancer (NSCLC) [6], kidney cancer [7], bladder cancer [8] and others [9]. The genomic landscape of smoking-induced NSCLC and UV light-induced melanoma is often characterized by a high number of acquired alterations, while leukemias and pediatric tumors show the lowest mutations counts. Rapidly developing genomic methods based on next-generation sequencing (NGS) simplified the detection and quantification of these acquired changes on the level of individual cancer genomes. Tumor mutational burden (TMB) is a quantitative measure of acquired somatic mutations in the cancer cell genome. Initial exploratory analyses of TMB in cancer patients [10,11] were carried out using whole exome sequencing (WES). WES is a comprehensive research tool for assessment of genomic alterations across the entire coding region of the ~22,000 genes in the human genome, comprising of 1–2% of the genome [3,12]. Currently, WES-derived TMB values are considered to be the gold standard, but the high cost and long turnaround time limit routine diagnostic applicability of WES. Therefore, targeted NGS cancer gene panels have been promoted for TMB estimation as a feasible and cheaper alternative to WES [13]. Whereas TMB assessed by WES is typically reported as the total number of mutations per cancer cell exome, TMB assessed by gene panel assays is usually referred to as mutations per megabase (mut/Mb) because it differs in the number of genes and target region size [2,3,14]. The precise calculation of TMB may, however, vary depending on the region of tumor genome sequenced, types of mutations included, methods of subtracting germline variants and other aspects of bioinformatic analysis pipeline of the sequencing data [3,15]. Both the FDA-approved FoundationOne CDx (F1CDx) panel and the FDA-authorized Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) panel used correlation between paneland WES-based TMB to validate the reliability of panel based TMB estimation, and they claimed that these panels can assess TMB accurately (R = 0.74 for F1CDx and R = 0.76 for MSK-IMPACT) [2,13,16]. However, as Wu et al. [13] proposed in their recent work, the overall correlation between the paneland WES-based TMB could be substantially distorted by outliers (i.e., cases with relatively ultra-high TMB within each cancer type) [13], which might lead to overestimation of the reliability of panel-based TMB estimation. Therefore, additional studies are needed to evaluate the significance of correlation between the WES-based and targeted panel-based TMB values. As already mentioned, TMB is considered to be a proxy for cancer cell neo-antigenicity and therefore could potentially serve as a predictive biomarker of therapeutic response to ICI. Several studies, especially in NSCLC, retrospectively employed WES or larger NGS panels to determine TMB as a potential response predictor [17–19]. Unfortunately, the definition of cut-off values to separate “high TMB” from “low TMB” tumors is not consistent in recent NSCLC trials. For example, in the CheckMate (CM) trials CM012 (nivolumab and ipilimumab) [20], CM227 (nivolumab and Cancers 2020, 12, 230 3 of 14 ipilimumab) [17] and CM026 (nivolumab only) [21] cut-points of 158 mutations, 199 mutations and 243 somatic missense mutations (number of mutations estimated from a commercial gene panel based cut point of 10 mutations per Mbp) were used, respectively [22]. This is the first study in pediatric oncology that aims to assess the reliability of TMB estimation using real-life WES across multiple cancer types and in silico analysis of two major gene panels, which are widely used for routine diagnostics in clinical practice, where various settings of bioinformatic pipeline were employed. The performance and correlation of WES and panel-based TMB assessment methods were evaluated together with potential consequences for clinical decision making where various cut-offs for ICI indication were used. 2. Results 2.1. Comparison of TMB between Real-Life WES and In Silico Targeted Gene Panels We successfully performed germline and somatic WES and calculated TMB in 106 pediatric patients of 28 distinct cancer types. We stratified patients based on their diagnosis and expressed TMB for each group of patients as a median (min–max) or as a concrete value in cases where there was only one patient within a group (summarized in Table 1). WES-based TMB for each tumor is depicted in Figure 1. The median TMB ranged widely among diagnoses, from 0.3 mutations/Mb in myeloid sarcoma to 14.2 mutations/Mb in Burkitt lymphoma. Table 1. Comparison of TMB determined by real-life WES and in silico targeted gene panels. Diagnosis TMB WES—M1 * Real-Life (Median/Value) (Min–Max) TMB MSK—M1 * In Silico (Median/Value) (Min–Max) TMB F1CDx—M2 ** In Silico (Median/Value) (Min–Max) HGG glioma H3K27M+ 2.9 (1.6–15.7) 4.7 (2.6–17.9) 4.5 (2.6–31) Rhabdomyosarcoma 3.6 (1.7–6.4) 2.6 (1.7–4.3) 2.6 (0–5.2) Ewing sarcoma 3.1 (0.2–5.1) 2.6 (0–5.1) 2.6 (0–7.8) Ependymoma 3.1 (1.3–10.4) 1.7 (0–5.1) 3.2 (1.3–9) Neuroblastoma 3.8 (1.6–17.2) 3.0 (0.9–7.7) 4.5 (1.3–15.5) Soft tissue sarcoma 3.6 (1.7–6.7) 3.4 (0–6.8) 3.2 (0–9) Low-grade glioma 3.5 (1.6–6.8) 2.1 (0.9–4.3) 3.9 (1.3–5.2) High-grade glioma H3K27M wt 4.5 (1.4–269.8) 3.4 (0.9–294.7) 5.2 (1.3–410.9) Osteosarcoma 2.2 (1.9–7.5) 3.4 (0–5.1) 5.2 (1.3–6.5) Burkitt lymphoma 14.2 (6.1–100.7) 19.6 (6.8–46.1) 27.1 (6.5–89.2) Medulloblastoma 3.8 (3.5–63.6) 3.4 (0.9–61.5) 3.9 (1.3–89.2) Fibromatosis 6.2 (1.1–56.2) 5.1 (1.7–29) 10.3 (1.3–82.7) Wilms tumor 3.1 (2.3–3.9) 3.4 (2.6–4.3) 2.6 (1.3–3.9) Renal cell carcinoma 1.8 (1.5–2.1) 4.3 (2.6–6.0) 4.5 (1.3–7.8) Adrenocortical carcinoma 0.9 - 0.9 - 1.3 Plexus choroideus carcinoma 5.2 - 2.6 - 5.2 - Hepatocellular carcinoma 3.6 - 0.9 - 3.9 - Disseminated adenocarcinoma 2.3 - 4.3 - 6.5 Familiar infantile myofibromatosis 2.1 - 1.7 - 0.0 Myeloid sarcoma 0.3 - 0.0 - 0.0 - Undifferentiated embryonal tumor of spinal canal 3.1 - 2.6 - 2.6 - Nongerminomatous Germ Cell tumor CNS 2.3 - 1.7 - 1.3 - Epithelial hepatoblastoma 0.5 - 0.0 - 0.0 Spindle cell hemangioma 2.1 - 0.9 - 2.6 - Cancers 2020, 12, 230 4 of 14 Table 1. Cont. Fibrodysplasia ossificans progressiva 3.1 - 2.6 - 2.6 - Hepatosplenic T-lymphoma 0.4 - 0.9 - 0.0 - Multisystemic Langerhans cell histiocytosis 3.1 - 2.6 - 3.9 - Gastrointestinal stromal tumor 2.7 - 3.4 - 6.5 * M1—Method 1 for calculation of TMB excluding synonymous variants and indels; ** M2—Method 2 for calculation of TMB including synonymous variants and indels. Figure 1. Tumor mutational burden (TMB) values determined in our pediatric cancer patient cohort (WES—Method1) stratified by cancer type. Hypothetical TMB cut-off values are shown as dashed lines (green, TMB ≥ 5; blue, TMB ≥ 10, red, TMB ≥ 20). Furthermore, we determined, by an in silico approach, whether TMB, as measured by WES, correlates with TMB calculated by the gene sets and bioinformatic approaches used by two commercially available targeted gene panels. Panel-based TMB (MSK-IMPACT and F1CDx) for each group of patients expressed as a median (min–max) or as a concrete value in cases where there was only one patient in a group are summarized in Table 2. We confirmed a strong Pearson correlation of the panel TMB with the WES-based TMB characterized by R = 0.993 (F1CDx), and R = 0.974 (MSK-IMPACT), respectively (Figure 2A,C). Correlation between MSK-IMPACT and F1CDx panels was R = 0.993 (Figure 2B). The TMB assessment method was adapted for each panel accordingly (MSK-IMPACT—Method 1; F1CDx—Method 2). However, when the few hypermutated cases were excluded and only samples with TMB <10 mut/Mb were considered for analysis, the correlation decreased significantly: R = 0.514 (F1CDx), and R = 0.560 (MSK-IMPACT). Correlation between TMBs determined by the two panels remained remarkably higher (R = 0.726). Cancers 2020, 12, 230 5 of 14 Table 2. Comparison of TMB determined by real-life WES and the FMI laboratory testing service FoundationOne Heme (F1Heme). Gender Age at Diagnosis Diagnosis TMB F1Heme Real-Life (Mut/Mb) TMB WES—M1 * Real-Life (Mut/Mb) Same Sample (Yes/No) F 9 Renal cell carcinoma 1.63 1.45 yes F 7 Diffuse intrinsic pontine glioma H3K27M+ 2.44 1.60 yes M 13 Desmoid fibromatosis 0.81 1.14 yes M 6 Spindle cell hemangioma 0.81 2.05 yes F 14 Gastrointestinal stromal tumor 4.07 2.71 yes F 14 Osteosarcoma 2.44 1.91 yes M 2 Langerhans cell histiocytosis 2.44 3.11 yes M 11 Wilms tumor 1.63 2.34 yes M 11 Ewing sarcoma 1.63 2.57 yes F 7 Ependymoma 2.44 3.48 yes M 18 Embryonal rhabdomyosarcoma 4.89 2.82 yes F 14 Ewing sarcoma 1.63 3.57 yes F 6 Wilms tumor 0.81 3.91 yes F 18 Ewing sarcoma 0.81 2.97 yes M 9 Alveolar rhabdomyosarcoma 3.26 3.62 yes F 5 Diffuse intrinsic pontine glioma 2.44 2.85 yes M 10 Ewing sarcoma 1.63 0.17 yes F 1 Neuroblastoma 1.63 7.53 yes F 10 Ewing sarcoma 7.33 4.82 yes M 20 Glioblastoma H3G34R+ 7.33 8.02 yes F 2 Neuroblastoma 5.70 6.33 yes F 1 Embryonal rhabdomyosarcoma 1.63 6.39 yes M 3 Burkitt lymphoma 10.59 6.08 yes M 7 Burkitt lymphoma 19.55 14.18 yes M 18 Glioblastoma 265.56 269.75 yes F 10 Low-grade astroblastoma 1.63 1.83 no M 4 Adrenocortical carcinoma 0.00 0.88 no M 15 Hepatocellular carcinoma 2.44 3.59 no M 3 Epithelial hepatoblastoma 2.44 0.46 no M 5 Embryonal rhabdomyosarcoma 6.52 3.68 no M 3 Embryonal rhabdomyosarcoma 4.07 5.71 no F 7 Glioblastoma 0.81 4.48 no M 1 Anaplastic ependymoma 1.63 6.65 no F 4 Diffuse intrinsic pontine glioma H3K27M+ 9.78 5.39 no * M1—Method 1 for calculation of TMB excluding synonymous variants and indels. Cancers 2020, 12, 230 6 of 14 Figure 2. Correlation of tumor mutational burden (TMB) determined by real-life WES and targeted gene panels: real-life WES vs. in silico MSK-IMPACT (A), in silico F1CDx vs. MSK-IMPACT (B), real-life WES vs. in silico F1CDx (C), real-life WES vs. real-life laboratory service F1Heme (D). 2.2. Comparison of TMB between Real-Life WES and the Foundation Medicine Inc. (FMI) Testing Service (Subcohort of Patients) In the subgroup of 34 patients (randomly selected from the patients where a Formalin-Fixed Paraffin-Embedded (FFPE) block with tumor tissue was available), comparative study of real-life WES-based TMB assessment and the FMI testing service was performed. For the WES samples, tumor and normal tissue were each sequenced in order to distinguish germline polymorphisms from somatic mutations. For the targeted FMI testing, no matched normal material was sequenced; rather, genomic variants were stringently filtered to eliminate germline polymorphisms, as declared by the vendor. For TMB determination from WES data, we used Method 1 (excluding indels and synonymous mutations). The FMI testing services are done using Method 2 (including indels and synonymous mutations). In nine cases, different samples from one resection or biopsy collection were used. This is summarized in Table 2. However, the Pearson correlation between TMBs determined by these two real-life approaches was comparable to the correlation of real-life WES and in silico F1CDx panel (R = 0.998 vs. R = 0.993) indicating the relevance of the in silico approach for TMB assessment comparative studies. When hypermutated cases were excluded, correlation decreased to R = 0.488 (Figure 2D), which is similar to the decrease observed in the in silico approach (R = 0.514). Cancers 2020, 12, 230 7 of 14 2.3. WES-Based TMB Values during Tumor Progression In nine cases, we determined the TMB by WES in sequential tumor biopsies or tumor tissues from surgical resection. In five cases, we used tumor tissue from a primary tumor and its relapse. In the remaining four cases, tumor tissue was collected from two consequent local or metastatic relapses. TMB values are summarized in Table 3. In seven out of nine cases, an increase in TMB in the second tumor tissue was observed, with the average increase being 1.6 ± 1.3 mut/Mb. Table 3. WES-based TMB values during tumor progression in nine patient case cohorts. Gender Age at Diagnosis Diagnosis Diagnosis/Relapse Year of Biopsy TMB (WES M1 *) Real-Life F 9 Supratentorial ependymoma local relapse 2016 2.31 local relapse 2018 3.88 F 1 Neuroblastoma metastatic relapse 2017 7.53 metastatic relapse 2018 3.17 M 11 Ewing sarcoma primary tumor 2017 2.57 local relapse 2018 4.19 M 5 DIPG primary tumor 2015 2.51 local relapse 2018 6.68 F 10 LG astroblastoma primary tumor 2017 1.83 local relapse 2018 3.05 M 3 Epithelial hepatoblastoma primary tumor 2016 0.46 local relapse 2018 2.48 F 2 Ependymoma primary tumor 2014 10.38 metastatic relapse 2018 10.53 M 18 Osteosarcoma metastatic relapse 2018 7.47 metastatic relapse 2018 8.10 M 1 Infantile myofibromatosis metastatic relapse 2015 2.08 metastatic relapse 2018 1.88 * M1—Method 1 for calculation of TMB excluding synonymous variants and indels. 2.4. Consequence of TMB Assessment Method for ICI Indication TMB as a predictive biomarker is currently the focus of several clinical trials with ICI. We have evaluated how the sequencing region (WES vs. the gene set used in MSK-IMPACT vs. the gene set used in F1CDx) and method for TMB calculation affect the final TMB and potential ICI indication when various hypothetical cut-off values are applied. Results of this analysis are summarized in Table 4. As expected, the number of patients above a cut-off is always higher with WES-based TMB assessment (compared to panel-based) and when TMB is assessed by Method 2 (including indels and synonymous mutations). Number of patients above a cut-off differs significantly when low TMB cut-off value is applied (cut-off ≥ 5). With the increasing cut-off values, the significance of technological variability introduced by sequencing various genome regions and different TMB calculating methods decreases. However, even with a relatively high cut-off value (cut-off ≥ 20), the number of pediatric patients hypothetically indicated for ICI therapy differs between TMB groups calculated with Method 1 and Method 2 (e.g., four vs. seven pediatric patients with WES). Cancers 2020, 12, 230 8 of 14 Table 4. WES-based TMB values during tumor progression in nine patient case cohorts. TMB—M1 * In Silico (Number of Cases Above Cut-Off) TMB—M2 ** In Silico (Number of Cases Above Cut-Off) Cut-off for ICIs Indication (mut/Mb) ≥5 ≥10 ≥20 ≥5 ≥10 ≥20 WES 30 8 4 75 25 7 MSK-IMPACT 23 6 4 61 12 6 F1CDx 24 7 5 42 11 6 * M1—Method 1 for calculation of TMB excluding synonymous variants and indels; ** M2—Method 2 for calculation of TMB including synonymous variants and indels; ICIs—immune checkpoint inhibitors. 3. Discussion The predictive power of TMB as a biomarker for response to ICI is currently being investigated in many clinical trials across various cancer types. Patients with a higher TMB are more likely to respond to ICI in various settings, including PD-(L)1 blockade in NSCLC [10], CTLA-4 blockade in malignant melanoma [11], and combined PD(L)-1 and CTLA-4 blockade in NSCLC [17]. Studies have shown that TMB is to a large extent independent of the PD-L1 status and might thereby identify additional subgroups of patients who benefit from ICI [17,20,22]. Based on these clinical observations, TMB became an emerging predictive biomarker for ICI in various cancer types, and an urgent need occurred to answer the questions concerning the technological aspects affecting TMB detection by WES and targeted panel sequencing to ensure implementation of lab developed tests that guarantee optimal reference standard quality for patient stratification [19]. In initial studies, WES was widely used to determine TMB and is still considered to be the gold standard; however, targeted sequencing panels are more readily interpretable and are a more pragmatic and potentially cost-effective approach to TMB testing in clinical diagnostics [3]. While in the context of clinical trial, TMB testing is mainly carried out by commercial vendors, many clinical laboratories depending on the regulatory approval context may eventually use in-house designed panels to determine TMB scores [22]. Endris and others have already investigated the minimum required size of a gene panel by comprehensive in silico analyses of available WES data sets and have shown that at least 1 Mbp of exonic and/or intronic region should be sequenced to achieve a similar power in discriminating ICI responders from non-responders comparable to WES [19]. Furthermore, Buchhalter at al. showed that “size does matter”, with an optimal panel size being between 1.5 and 3 Mbp, considering the benefit–cost ratio, and that the inclusion of all point mutations (instead of only missense mutations) in the TMB calculation is possible and recommendable to enhance precision [9]. In our study, we focused on the potential technological variability introduced to TMB scoring by the usage of various platforms and bioinformatic pipelines for their assessment in pediatric tumors. As a reference method, we performed WES and subsequently in silico simulated two most frequently used sequencing panels, MSK-IMPACT and F1CDx. We confirmed a strong Pearson correlation of the panel-based TMB with the WES-based TMB; however, when the few hypermutated cases were excluded and only samples with TMB < 10 mut/Mb were considered for analysis, the correlation decreased significantly (Figure 2). This indicates a significant bias introduced to correlation analysis by only a few hypermutated cases included in the study. Correlation between samples with TMB < 10 mut/Mb was not satisfactory and probably lead to significant clinical misclassifications in the routine diagnostic scenario based on the usage of a cut-off value in the range of 5 to 15 mut/Mb. Similar observations were also provided by other authors describing adult tumors [9,19]. In a subgroup of patients, we performed a comparative study of real-life WES-based TMB assessment and the FMI testing service where we observed a similar effect of the hypermutated cases on the correlation significance. In agreement with others [9,19], we observed that the identification of high TMB tumors can be reliably achieved by any of the tested methods (cases with ultra-hypermutated tumors). However, the vast majority of tumors have intermediate TMB values; in these cases, Cancers 2020, 12, 230 9 of 14 a technological variability interferes with the reliable differentiation between TMB-high and low tumors [9,19]. In nine cases, we determined the TMB by WES in sequential tumor biopsies or tumor tissues from surgical resection. As expected, in seven out of nine cases, there was an increase in TMB in the second tumor with the average increase being approx. 2 mut/Mb. Surprisingly, in two cases, we observed a decrease in TMB, which could be explained mainly by the quality of the tumor tissue specimen and a low content of tumor cells in the second tumor which could decrease detectable mutations used for TMB assessment. It is important to mention that tumor content in the tissue specimens is an important factor affecting TMB scoring and is often not considered in TMB studies. Finally, we evaluated how the sequencing region (WES vs. the gene set used in MSK-IMPACT vs. the gene set used in F1CDx) and the bioinformatic pipeline used for TMB calculation affect the final TMB and potential ICI indication when various hypothetical cut-off values are applied. In general, as expected, the number of patients above a cut-off is always higher in WES-based TMB assessment (compared to panel-based) and when the TMB is assessed by Method 2 (including indels and synonymous mutations). We also found that with the increasing cut-off values, the significance of technological variability and consequent clinical misclassification decreases. However, certain combinations of settings of TMB assessment methods (e.g., WES-M2 vs. F1CDx-M1), compounded by the use of a cut-off value of 10 mut/Mb, yield extremely different results. While the first approach predicts 25 patients to be good responders to ICI, the second approach predicts only seven patients. This indicates a potentially very strong misclassification issue for routine diagnostics. Based on the currently available results from clinical trials, it is very difficult to judge whether TMB assessed by Method 1 or Method 2 is a more accurate predictive biomarker of response to ICI therapy. Unfortunately, this in silico modeling has not been performed in the context of clinical outcomes from ICI trials. 4. Materials and Methods 4.1. Patients and Biological Specimens We reviewed tumor mutational burden (TMB) results from 106 patients with pediatric high-risk/recurrent solid tumors (both newly diagnosed and relapsed) who had undergone laboratory WES at Central European Institute of Technology (CEITEC, Masaryk University, Brno, Czech Republic). Informed consent was obtained from all patients and all experiments using clinical samples were performed in accordance with the approved international guidelines. After surgical resection of the tumor or collection of the tumor biopsies, tissue samples were evaluated by an experienced surgical pathologist for the tumor cell content, and only specimens with more than 20% of the tumor cells were included. In addition, peripheral blood was collected to obtain DNA for germline WES. Number of patients stratified according to their diagnoses and related clinical data are summarized in Table 5. In nine cases, we collected two consequent tissue specimens (diagnosis/relapse or two relapses) and both were used for WES and TMB assessment. 4.2. DNA Isolation Tumor DNA was extracted from the FFPE samples or fresh frozen tissues using QIAmp DNA FFPE Tissue Kit (Qiagen, Venlo, The Netherland) or QIAamp DNA Micro Kit (Qiagen). Germline DNA was extracted from peripheral blood leukocytes using QIAamp DNA Micro Kit (Qiagen). The purified DNA was quantified using Qubit 2.0 Fluorometer and NanoDrop 2000c spectrophotometer (both Thermo Fisher Scientific, MA, USA). Cancers 2020, 12, 230 10 of 14 Table 5. Number of patients stratified according to their diagnoses and baseline clinical data. Diagnosis Number of Patients Gender Ratio (F/M) Age Median Age (Min–Max) Type of Sample Ratio (Primary Tumor/Local or Metastatic Relapse) High-grade glioma H3K27M+ 12 8/2 9 4–20 12/0 Rhabdomyosarcoma 11 7/4 5 0–18 6/5 Ewing sarcoma 11 6/5 11 8–18 2/9 Neuroblastoma 10 6/4 2 1–8 1/9 Ependymoma 10 6/4 5.5 1–16 4/6 Non-rhabdomyosarcoma soft-tissue sarcomas 8 2/6 12 8–19 0/8 High-grade glioma H3K27M wt 6 0/6 16 8–23 5/1 Low-grade glioma 6 1/5 9.5 3–19 1/5 Osteosarcoma 5 4/1 18 14–28 0/5 Burkitt lymphoma 3 0/3 7 3–12 0/3 Medulloblastoma 3 0/3 4 2–5 1/2 Fibromatosis 3 1/2 17 13–20 1/2 Wilms tumor 2 1/1 8.5 6–11 1/1 Renal cell carcinoma 2 1/1 13.5 9–18 1/0 Adrenocortical carcinoma 1 F 4 - primary tumor Choroid plexus carcinoma 1 M 1 - primary tumor Hepatocellular carcinoma 1 M 15 - primary tumor Lung adenocarcinoma 1 F 15 - metastatic relapse Familiar infantile myofibromatosis 1 M 1 - primary tumor Myeloid sarcoma 1 F 5 - primary tumor Undifferentiated embryonal tumor of spinal canal 1 M 2 - primary tumor CNS germ cell tumor 1 M 11 - local relapse Epithelial hepatoblastoma 1 M 3 - primary tumor Spindle cell hemangioendothelioma 1 M 6 - primary vascular malformation Fibrodysplasia ossificans progressiva 1 F 1 - primary tumor Hepatosplenic T-lymphoma 1 M 17 - diagnostic aspiration/bone marrow Multiple system Langerhans cell histiocytosis 1 M 2 - metastasis Gastrointestinal stromal tumor 1 F 14 - metastatic relapse Cancers 2020, 12, 230 11 of 14 4.3. Whole Exome Sequencing Libraries for whole exome capture and sequencing were prepared using TruSeq Exome Kit (Illumina, CA, USA) according to manufacturer´s recommendations. Quantity and quality of the exome libraries were checked using Qubit 2.0 Fluorometer and NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). Prepared libraries were loaded onto NextSeq 500/550 Mid Output Kit (150 cycles) and sequenced on the NextSeq 500 instrument (both Illumina). Sequencing coverage for both exomes was >20 × at >90% of captured regions. 4.4. Bioinformatic Analysis Sequencing reads in FASTQ format were mapped to the human reference genome hg19 with the BWA-MEM algorithm [23] for both the tumor and the healthy control sample. The resulting alignments in BAM format were postprocessed with the SAMBLASTER program [24] for marking PCR duplicates. The final alignment file of the control sample was used to assess single nucleotide variants (SNVs) and short insertions/deletions (indels). Two variant callers were used for germline variant calling; the GATK HaplotypeCaller [25] and VarDict [26]. Reported variants were annotated with Annovar [27] and Oncotator [28] annotation programs. Tumor specific variants were assessed by somatic (paired; tumor vs. control) variant calling. For this purpose, we used GATK MuTect2 (SNVs), Scalpel [29] (Indels), and VarDict (SNVs and Indels) variant callers. The annotation of somatic variants was performed with the addition of the COSMIC database [30]. Overview of the bioinformatic pipeline is depicted in Figure 3. Figure 3. Workflow for tumor mutational burden (TMB) assessment by WES in this study. 4.5. Tumor Mutational Burden Estimation An annotated list of somatic variants from the previous step was used to assess the TMB. We chose to compare two methods of TMB estimation, both based on publicly available approaches. Method 1 (M1)—In our laboratory, we only consider somatic single nucleotide variants (SNVs) for TMB calculation from WES data, since indels (short insertions and deletions) tend to be called with high false positive rates and could potentially skew the outcome. Additionally, two bases before and Cancers 2020, 12, 230 12 of 14 after each exon are considered as splicing mutations. Synonymous variants are filtered out, as they do not fit the definition of TMB. Finally, variants with variant allele frequency (VAF) of less than 5% are also filtered out. This approach is also used by MSK-IMPACT NGS panel. Method 2 (M2)—This approach, used by the Foundation Medicine Inc. (FMI) targeted panels (e.g., F1CDx [2] as well as F1Heme), defines TMB as the number of SNVs (including synonymous variants) and indels in the coding regions of targeted genes. However, splicing variants are not included. A 5% cut-off for the VAF was also applied. For the final TMB calculation, in both methods, the sum of variants remaining after application of the all filters, is then divided by the size (in megabases) of the target region from which the variants have been assessed. The target regions together with their sizes are listed below. Both methods were applied to the three target regions (as shown in Table 5): 1. All coding sequences (whole exome; 35 Mb; using M1 for TMB calculation); 2. The coding sequences of genes analyzed by the FMI (F1CDx panel; 324 cancer-related genes; 0,8 Mbl using M2 for TMB calculation); 3. The coding sequences of genes analyzed by the Memorial Sloan Kettering Cancer Center (MSK-IMPACT; 468 cancer-related genes; 1.22 Mb; using M1 for TMB calculation) The coding region locations on the hg19 genome were downloaded from the UCSC web site. 4.6. Comparative Study with the Foundation Medicine Inc. (FMI) Sequencing Service FFPE tumor tissue samples of 34 patients who were previously examined by WES in our laboratory and were sent to the FMI for the FoundationOne Heme (F1Heme) test, which is recommended by vendor for pediatric tumors. In the nine cases, WES was performed using fresh frozen tissue, while different FFPE samples were sent for the F1Heme test. These specimens are indicated in the summarizing tables (Table 3) with the TMB results. 5. Conclusions We present a study, where, for the first time in the context of pediatric tumors, the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels was assessed. We confirmed a significant technological variability introduced by different laboratory technologies and various settings of bioinformatic pipelines. These results may provide valuable information for improving the accuracy of TMB estimation based on targeted gene panel sequencing in a diagnostic setting. Our study confirmed previous observations from adult tumors and thus supports the incentive to establish concordance between assay platforms used across different clinical trials in order to achieve a successful real-world implementation of TMB testing. To this end, worldwide efforts to ensure the harmonization of TMB assessment are ongoing [31–33]. Author Contributions: Conceptualization, H.N., K.P., T.H., J.S. and O.S.; data curation, M.K., K.P., T.M., P.M., K.P., T.C.I., S.A., M.J. and L.K.; formal analysis, M.K., K.P. and O.S.; funding acquisition, J.S. and O.S.; Investigation, H.N., M.K., T.M., P.M., K.P., T.C.I., S.A., M.J., L.K. and J.S.; methodology, S.A. and T.H.; project administration, H.N. and O.S.; resources, L.K. and J.S.; supervision, J.S. and O.S.; validation, T.M., P.M., K.P., T.C.I., S.A. and L.K.; visualization, M.J.; writing—original draft, H.N. and O.S.; writing—review and editing, H.N., K.P., T.H., J.S. and O.S. All authors have read and agreed to the published version of the manuscript. Funding: This work has been supported by Roche supplying FoundationOne Heme tests and by the Czech Ministry of Health, grant no 16-33209A. Roche did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat. Med. 2017, 23, 703–713. [CrossRef] 17. Hellmann, M.D.; Ciuleanu, T.E.; Pluzanski, A.; Lee, J.S.; Otterson, G.A.; Audigier Valette, C.; Minenza, E.; Linardou, H.; Burgers, S.; Salman, P.; et al. Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden. N. Engl. J. Med. 2018, 378, 2093–2104. [CrossRef] 18. Yarchoan, M.; Hopkins, A.; Jaffee, E.M. Tumor Mutational Burden and Response Rate to PD-1 Inhibition. N. Engl. J. Med. 2017, 377, 2500–2501. [CrossRef] 19. Endris, V.; Buchhalter, I.; Allgauer, M.; Rempel, E.; Lier, A.; Volckmar, A.L.; Kirchner, M.; von Winterfeld, M.; Leichsenring, J.; Neumann, O.; et al. Measurement of tumor mutational. burden (TMB) in routine molecular diagnostics: In silico and real-life analysis of three larger gene panels. Int. J. Cancer 2019, 144, 2303–2312. [CrossRef] Cancers 2020, 12, 230 14 of 14 20. Hellmann, M.D.; Nathanson, T.; Rizvi, H.; Creelan, B.C.; Sanchez-Vega, F.; Ahuja, A.; Ni, A.; Novik, J.B.; Mangarin, L.M.B.; Abu-Akeel, M.; et al. Genomic Features of Response to Combination Immunotherapy in Patients with Advanced Non-Small-Cell Lung Cancer. Cancer Cell 2018, 33, 843–852. [CrossRef] 21. Carbone, D.P.; Reck, M.; Paz-Ares, L.; Creelan, B.; Horn, L.; Steins, M.; Felip, E.; van den Heuvel, M.M.; Ciuleanu, T.E.; Badin, F.; et al. First-Line Nivolumab in Stage IV or Recurrent Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2017, 376, 2415–2426. [CrossRef] [PubMed] 22. Budczies, J.; Allgauer, M.; Litchfield, K.; Rempel, E.; Christopoulos, P.; Kazdal, D.; Endris, V.; Thomas, M.; Frohling, S.; Peters, S.; et al. Optimizing panel-based tumor mutational burden (TMB) measurement. Ann. Oncol. 2019, 30, 1496–1506. [CrossRef] [PubMed] 23. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv 2013, arXiv:1303.3997. 24. Faust, G.G.; Hall, I.M. SAMBLASTER: Fast duplicate marking and structural variant read extraction. Bioinformatics 2014, 30, 2503–2505. [CrossRef] 25. Poplin, R.; Ruano-Rubio, V.; DePristo, M.A.; Fennell, T.J.; Carneiro, M.O.; Van der Auwera, G.A.; Kling, D.E.; Gauthier, L.D.; Levy-Moonshine, A.; Roazen, D.; et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv 2017, 201178. 26. Lai, Z.; Markovets, A.; Ahdesmaki, M.; Chapman, B.; Hofmann, O.; McEwen, R.; Johnson, J.; Dougherty, B.; Barrett, J.C.; Dry, J.R. VarDict: A novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res. 2016, 44, e108. [CrossRef] 27. Wang, K.; Li, M.; Hakonarson, H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010, 38, e164. [CrossRef] 28. Ramos, A.H.; Lichtenstein, L.; Gupta, M.; Lawrence, M.S.; Pugh, T.J.; Saksena, G.; Meyerson, M.; Getz, G. Oncotator: Cancer variant annotation tool. Hum. 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Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch. 2017, 470, 5–20. [CrossRef] [PubMed] 33. Van Krieken, H.; Deans, S.; Hall, J.A.; Normanno, N.; Ciardiello, F.; Douillard, J.Y. Quality to rely on: Meeting report of the 5th Meeting of External Quality Assessment,c Naples 2016. ESMO Open 2016, 1, e000114. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Oncotarget46813www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 29 Future paradigms for precision oncology Giannoula Lakka Klement1,2 , Knarik Arkun3 , Dalibor Valik4,5 , Tina Roffidal1 , Ali Hashemi6 , Christos Klement6 , Paolo Carmassi6 , Edward Rietman6,7 , Ondrej Slaby4,8 , Pavel Mazanek4,5 , Peter Mudry4,5 , Gabor Kovacs9 , Csongor Kiss10 , Koen Norga11 , Dobrin Konstantinov12 , Nicolas André13,14 , Irene Slavc15 , Henk van Den Berg16 , Alexandra Kolenova17 , Leos Kren18,19 , Jiri Tuma19,20 , Jarmila Skotakova8,19 and Jaroslav Sterba4,19,21 1 Department of Pediatric Hematology/Oncology, Floating Hospital for Children at Tufts Medical Center, Boston, MA, USA 2 Department of Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, USA 3 Department of Pathology, Tufts Medical Center, Boston, MA, US 4 Department of Paediatric Oncology, University Hospital Brno, Brno, Czech Republic 5 Regional Center for Applied Molecular Biology, RECAMO, Brno, Czech Republic 6 CSTS Health Care® , Toronto, Canada 7 Computer Science Department, University of Massachusetts, Amherst, MA, USA 8 Central European Institute of Technology, Masaryk University, Brno, Czech Republic 9 2nd Department of Pediatrics, Semmelweis University, Budapest, Hungary 10 Department of Pediatric Hematology-Oncology, Institute of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary 11 Antwerp University Hospital, Edegem, Belgium 12 Specialized Children’s Oncohematology Hospital, Sofia, Bulgaria 13 Department of Pediatric Hematology and Oncology, AP-HM, Marseille, France 14 UMR S_911 CRO2 Aix Marseille Université, Marseille, France 15 Department of Pediatrics, Medical University of Vienna, Vienna, Austria 16 Department of Pediatric Oncology, Emma Children Hospital Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 17 Department of Pediatric Oncology, Comenius University, Bratislava, Slovakia 18 Department of Pathology, University Hospital Brno, Brno, Czech Republic 19 Faculty of Medicine, Masaryk University, Brno, Czech Republic 20 Department of Pediatric Surgery, University Hospital Brno, Brno, Czech Republic 21 ICRC St. Anna University Hospital Brno, Brno, Czech Republic Correspondence to: Giannoula Lakka Klement, email: glakkaklement@tuftsmedicalcenter.org Keywords: precision medicine, targeted therapy, genomics, metronomic chemotherapy Received: December 07, 2015 Accepted: March 31, 2016 Published: May 19, 2016 ABSTRACT Research has exposed cancer to be a heterogeneous disease with a high degree of inter-tumoral and intra-tumoral variability. Individual tumors have unique profiles, and these molecular signatures make the use of traditional histology-based treatments problematic. The conventional diagnostic categories, while necessary for care, thwart the use of molecular information for treatment as molecular characteristics cross tissue types. This is compounded by the struggle to keep abreast the scientific advances made in all fields of science, and by the enormous challenge to organize, cross-reference, Review Oncotarget46814www.impactjournals.com/oncotarget INTRODUCTION The conventional approaches to cancer therapy have been until very recently based on eradicating cancer cells by three modalities - surgery, radiation and chemotherapy. While this approach improved outcomes for children with acute lymphoblastic leukemia where survival rose from 20% in the 1950’s to about 95% now, it was much less effective in solid tumors and adult leukemias. In these more genetically complex cancers, some modest initial improvements in survival rates were achieved, but even those modest gains have been stagnating since the late 90’s. Many different reasons contribute to the treatment resistance of solid tumors and adult leukemias, but chiefly among those are: 1. the genomic complexity and heterogeneity of these entities, and 2. the protective effect of the host / tumor microenvironment.[1, 2] Novel, molecularly-based treatment modalities target not only tumor cells, but also the tumor cell-induced changes in the tumor microenvironment. In addition to those agents directed against tumor cell epitopes and receptor tyrosine kinases, there are monoclonal antibodies directed against endothelial growth factors and receptors, inflammatory cells and immune surveillance cells. All of those can be combined to correct the tumor/ microenvironment interaction, and not only sensitize to existing therapies but to effectively target the developmental end-stage characteristics of tumorigenesis. The term biologic agent is therefore quite broad. It should be considered synonymous with “biological response modifiers”, “targeted agents” or “molecularlyguided therapies”, as well as with other terms used in the broader scientific literature to describe agents that target an otherwise physiological biological events “hijacked” by the tumor for growth benefit. The physiological mechanisms used by tumor cells for survival, i.e. inflammation, angiogenesis, immune system and regenerative pathways, have not been considered as targets in the past, even though wide-ranging spectrum of agents exists for their modulation. They include inhibitors of growth factor pathways, angiogenesis inhibitors, enhancers of pro-apoptotic signals, immune response modifiers, adhesion inhibitors, proteasome inhibitors, signal transduction inhibitors and any other agents targeting a defined biological process in the cancer tissues. Unfortunately, while all these new insights have come to the forefront of cancer science, their implementation to clinical practice has been quite slow. The understanding that cancer-specific biology may be less dependent on the tissue of origin, and more dependent on a genomic (molecular) signatures, represents a paradigm shift in thinking. This new definition accepts cancer not as foreign tissue, but rather as a natural consequence of lifelong accumulation of molecular alterations, lending credence to therapeutic approach that considers cancer a chronic disease. Unlike the present goal of cancer eradication in a manner similar to antibacterial therapy; scientists now accept that cancer may be managed as a lingering chronic illness influenced by the inflammatory, immune and angiogenesis phenotype of the host. Scientists continue to identify the many molecular lesions that can lead to cancer progression and recognize that each tumor harbors its own genomic signature.[3] The basic question that remains to be answered is which part(s) of the molecular signature are related to the primary oncogenic event, and which are secondary. The traditional picture of a linear evolution of a cancer through clonal expansion driven by accumulation of sequential mutations inherent to the cancer clone has now been nuanced by the influence of tumor microenvironment. Most cancers are a mixture of cancer cells and normal host cells that have been recruited to the site, or that have been induced to action by oncogenic changes occurring in cancer cells during malignant and apply molecular data for patient benefit. In order to supplement the site-specific, histology-driven diagnosis with genomic, proteomic and metabolomics information, a paradigm shift in diagnosis and treatment of patients is required. While most physicians are open and keen to use the emerging data for therapy, even those versed in molecular therapeutics are overwhelmed with the amount of available data. It is not surprising that even though The Human Genome Project was completed thirteen years ago, our patients have not benefited from the information. Physicians cannot, and should not be asked to process the gigabytes of genomic and proteomic information on their own in order to provide patients with safe therapies. The following consensus summary identifies the needed for practice changes, proposes potential solutions to the present crisis of informational overload, suggests ways of providing physicians with the tools necessary for interpreting patient specific molecular profiles, and facilitates the implementation of quantitative precision medicine. It also provides two case studies where this approach has been used. Oncotarget46815www.impactjournals.com/oncotarget transformation. In genetically complex forms of cancers, it is difficult to define a specific “driver gene” within the multiplicity of gene alterations, unless one can evaluate the quorum of signals within the tumor microenvironment. A vastly improved ability to establish the hierarchy of genomic alterations present in the tumors of individual patients will be needed for a correct analysis and interpretation of biological information. Despite the incomplete and continuously amended molecular information, and notwithstanding the fragmented understanding of its usefulness for effective anti-cancer therapies, many molecularly-based therapies have been implemented with spectacular success. Yet, as the example of imatinib demonstrates, the deployment of targeted therapy - from its discovery to standard of practice clinical use - can take more than thirty years in the present clinical climate.[4] Even in the case of CML, a cancer with a single therapeutic target, the traditional route to clinical implementation of bcr/abl complex inhibitors was uncomfortably slow. The process may be streamlined in rare diseases - the use of denosumab (inhibitor of RANKL) for the treatment of giant cell tumor of the bone - but the implementation of even a single agent therapy is filled with trepidations and insurance denials. It is therefore not surprising that for those diseases with activation of more than one molecular pathway, the implementation of molecularly-guided therapy remains challenging. Therapeutic strategies incorporating inhibition of multiple molecular pathways will need to address the considerable differences in tumors between individuals, the heterogeneity within a single tumor, as well as the differences between the primary tumor and its metastatic lesions. Numerous and quite comprehensive catalogues of somatic mutations obtained by comparing a patient’s tumor DNA/RNA sequences to his/her germline DNA/ RNA[5, 6] indicate a great deal of heterogeneity in cancer genome evolution across different tumor types, across individual patients with the same tumor type, and even within a tumor.[7, 8] Considering this heterogeneity, the present appeal of enhancing the traditional siteand histology-specific treatment protocols with a more personalized approach (ie. precision medicine), can be more easily understood. Scientists[9, 10] and leading politicians[11] have recognized that supporting progress toward precision medicine and increasing the use of biological therapies holds a strong promise of not only improving health outcomes,[12] but also of potentially improving cost effectiveness of cancer therapies.[13] The concept of precision medicine, as heretical as it may have initially sounded in cancer therapy, is not foreign in medicine. We test for antibiotic sensitivity, and we match blood for HLA subtypes in transfusion and transplantation medicine, and it is not surprising that our cancer patients are beginning to demand the same.[14] Ultimately, effective, precise, target-tailored medicines may abolish the use of oldfashioned cytotoxic treatments, or at least eliminate the need for maximum tolerated doses of radiation and chemotherapy. The implementation of these new treatment modalities will, require a number of necessary changes to the oncological practice and research in oncology. We will need to: 1. change clinical trial design in order to obtain efficacy data from n - 1 trials 2. provide and interpret large data while maintaining excellent data integrity 3. develop novel mathematical approaches for establishing hierarchy of genomic alterations in individual tumor samples 4. provide combination therapies based on pathway analyses 5. avoid combinations with maximum tolerated doses of chemotherapy: the argument for low dose (metronomic) chemotherapy backbone THE NEED TO CHANGE CLINICAL TRIAL DESIGN IN ORDER TO OBTAIN EFFICACY DATA FROM N - 1 TRIALS Medical practice is a conservative vocation, and one of the most often repeated quotation in medical lore is: Primum non nocere (“first do no harm”). As such, in order to facilitate the translation of precision medicine to practice, sufficient evidence about precision medicine being as good or better than present therapies is requisite for the larger scientific and medical community to use the therapy. Unfortunately, over the last 40 years various regulations, were instituted in order to protect the public from unfounded claims of cure. While these were initially created for the benefit of the patient, they have led to a very inflexible structure of clinical trials - one that is no longer optimal for testing of new biological agents. Present clinical trials involve the addition of a single new agent to standard, established, maximum tolerated dose of therapy. To arrive at such a trial, the new agent must first go through a dose finding (dose escalating) trial (Phase I), which determines its maximum tolerated dose (MTD). The need to know the MTD is based on the well ingrained notion that the relationship between dose and cancer cell kill is linear[15] and more must be better. The notion, even though disavowed by the same scientist that first introduced it[16, 17], continues to be very dominant in oncology, even though some oncologists have begun using lower doses of chemotherapy in combination with targeted therapies.[18-21] Once the MTD is defined in Phase I trial, the agent is put through an early efficacy trial (Phase II), before proceeding to a randomized, double blind, placebocontrolled (Phase III) trial to validate its efficacy, and to post-marketing surveillance studies (Phase IV). While Phase I-IV trials were informative for evaluation of the Oncotarget46816www.impactjournals.com/oncotarget conventional surgery/chemotherapy/radiation approach, it is not optimal for biological agents where optimal dose is not the MTD and where toxicities are minimal.[22] This particular point is further discussed in section 5.1, and represented graphically in the Figure 1. Phase I-IV clinical trial design may not only be unsuitable for testing biological agents, they may be detrimental to the testing of biologically based therapies because most biologic agents sensitize to chemotherapy and radiation, and thus heighten the toxicity in the combination arms.[23, 24] A body of pre-clinical and clinical evidence indeed suggests that the relationship between the dose of a biologic agent and its effect is NOT linear.[25, 26] It is most commonly U-shaped. One of the earliest publications suggesting this phenomenon showed that the effect of interferon alpha 2B differed at low, medium and high doses[27] (see Figure 1A). This was subsequently found to be true for most biologic agents, especially those that depend on receptor/ligand interaction. Once all receptors are engaged, and the full effect achieved, any further increase in dose leads to off-target effects rather than further receptor inhibition. The excess of drug therefore intensifies toxicities. For example, while the effect of TGF beta1 at low doses is anti-tumorigenic, its effect at higher levels is pro-tumorigenic, creating a U-shaped response curve (see Figure 1B).[28] This characteristic u-shaped response curve of biological agents, termed hormesis,[26] further illustrates that levels and function of biological agents influence the equipoise of several pathways, and can be tumor suppressive or tumor promoting. The doses of biological agents should therefore be determined by the optimal biologically effective Figure 1: The U-shaped curve associate with the effect of biological therapies. Unlike the linear relationship between dose and cell kill assumed in the early work of Skipper and Schabel15 - the effect of a biologic agent may differ at low and high doses. Panel A is an adaptation of figure first published by Slaton23 in 1999. The optimum biologically effective dose is often a medium rather than maximum dose. This U-SHAPED CURVE may facilitate the initial up and the subsequent down-regulation during physiological biological processes. In a stress response a linear increase of interleukins is desired during the initial stress, but a relaxation needs to follow in presence of excess ligand. Oncotarget46817www.impactjournals.com/oncotarget dose, rather than by a maximum tolerated dose, and the Phase I/II trials are not suitable for the introduction of a biological agent to clinic. In the case of biologic agents more is not necessarily better, and dose escalations using the traditional Phase I trial may not only be inappropriate, they can be detrimental, because the effect of the biological agent at high doses may be opposite to the desired effect.[25, 26] The change in pharmacodynamics of metronomically dosed vinblastine vs MTD vinblastine provides a very good example. The dose of vinblastine used for inhibition of angiogenesis is many folds lower than the anti-proliferative dose of vinblastine (~6mg/m2 ). [29] The fact that Phase I trials are in general meant to establish dose-limiting toxicities rather than offer therapy is something most patients may not be able to appreciate when a Phase I trial is presented to them as the “last option”. The chance of cure or even of a positive response is very small, especially in situations where the intended target is not tested for and may not even be present. While some early efficacy trials of targeted agents for relapsed cancers may show some effectiveness,[30] the response is rarely sustained. The role of a randomized, double-blind placebo controlled trial (RCT) is similarly questionable in an era where precision medicine is available. An RCT is in principle a comparison of two populations, one with and the other without the tested agent. Its goal is to find an agent that would be effective for the largest percentage of the general population, rather than optimize therapy for an individual. Because identifying the best treatment for an individual is so fundamentally different from a treatment that performs best at the population level, it is highly unlikely that Phase III approaches will be able to capture the outcomes of targeted therapies in precision medicine. There is an early level of recognition of the need to revise the present model of clinical trials. Timely changes to clinical practice have been suggested by the recent National Cancer Institute Precision Medicine Initiatives for the new National Clinical Trials Network,[31] but most molecular testing continues to be used only as means to streamline the enrollment in clinical trials. In order to accommodate the n = 1 trial model, early discussions have been initiated about creation of a “cancer knowledge network”,[10] where information from the numerous case studies of truly individualized cancer treatments could be shared and evaluated. A case in point is the early effort to collect data from patients using targeted therapies in the NCI-Molecular Analysis for Therapy Choice (NCI-MATCH) Trial. In this trial, which opened in August 2015, analyzes patients’ tumors to determine whether they contain genetic abnormalities for which a targeted drug exists (that is, “actionable mutations”) and assigns the patient to a clinical trial based on one of the detected abnormalities. While the trial will make some data available, its limitation lies in its traditional trial design. The trial suffers from two shortcomings; one, it is likely that of the hundreds of patients tested, only very few will find a matching clinical trial, and two, even though the tumor tissues will be analyzed for more than 4,000 different variants across 143 genes, patients with more than one genomic abnormality will still be enrolled on a single agent therapy trial, ignoring the actual tumor biology. This approach does not change the paradigm, as it does not address the complexity of tumor biology, heterogeneity and especially not the need for pathway analysis in cancer therapy. A special problem in clinical studies is the current practice to include at first instance only relapsed and refractory patients. As mentioned, malignant cell proliferation is under control of the primary oncogenic event, but secondary (acquired) changes may independently control further malignant cell proliferation. The chance that analysis of tumors in newly diagnosed patients may elucidate the basic oncogenic driver(s) and the respective pathway(s) is much more likely. In this respect, newly diagnosed patients with cancers where the prognosis is poor should be considered for individualized therapies before resorting to the present standards. In children with poor prognosis disease, a well designed up-front window therapy, would clarify response to biological agent(s) more clearly. Examples where these studies should be considered are children with metastatic sarcoma, brain tumors or neuroblastoma where up 80% of children die despite elaborate standard chemotherapy and radiation protocols. To identify the basic oncogenic driver(s), all newly diagnosed malignancies would need additional molecular analysis as mentioned below. A POTENTIAL SOLUTION To remedy the difficulty of collecting individual case study data we propose formation of consortium(s) of pediatric and adult institutions providing a standardized approach to selection of targets aided by computer assisted information processing and facilitated through an online tumor board review. The outcomes of the individual cases within the consortium(s) can then be pooled, evaluated, and used to inform selection of targets for future patients in real time (Figure 2). It is unlikely that all collaborative groups will be able to use the same tissue biomarker analysis outside a collaborative clinical trial. Only a collaborative, synchronized evaluation can lead to the meticulous collection and sharing of the DNA/ RNA/Protein tissue analysis, that can lead to standardized selection of targets and therapeutic agent combinations, and where meticulous collection of the respective outcomes can be done. The approach of this consortium has some similarities to the efforts extended by the ECOG-ACRIN Oncotarget46818www.impactjournals.com/oncotarget Cancer Research Group, NMTRC, SWOG, Alliance for Clinical Trials in Oncology, NRG Oncology Group and the multiple sites participating in the NCI National Clinical Trial Network for establishing the MATCH trial. But it differs, in its use of using bio-marker driven, molecularly-targeted metronomic combination therapy. The consortium(s) stresses the use of a multi-target, multi-modality approach rather than enrollment on single agent trials. The hope is that sufficient amount of data will be accumulated to provide the necessary evidence to inspire other organizations to extend the examination of tumor tissue to include genomic, proteomic and metabolomics examination of the host as well as of the tumor, and promote individualized cancer therapies. Because only a very small number of patients is going to have overlapping molecular alterations and as such require the same combination of agents, traditional populationbased statistical approaches comparing two disparately treated groups may not be applicable, and novel statistical approaches using predictive models of cancer growth are going to be needed. The data from all individual patients treated by a precision medicine approach will be stored in a single de-identified database to be shared not only with the consortium members but also with other clinicians and researchers interested in using targeted approaches. The additional benefit of sharing information of these N = 1 trials is going to be learning about the changed pharmacokinetics as combinations of different agents are being used. Pharmacokinetic studies are an integral part of present PhaseI/IV clinical trial structure. If we remove this resource, alternative experimental procedures that would allow for establishing clearance and biodistribution of these biologic agents will be needed. We will need to provide the clinicians with means to be able to quickly identify the key factors that govern absorption, distribution, metabolism, and excretion of the individual biologics, [32] the pharmacogenomics, [33], as well as the effect of using combinations of agents. Consideration will need to be given to developing new intelligence-enabled tools for quick dose adjustments if more than one cyp3a4 or other members of the cytochrome P450 family involved in drug metabolism, are being used in the therapeutic regimen. The information collected would, in addition to traditional outcome measures such as survival, response, and toxicities, include information about quality of life and health care costs. The outcome database could thus be used to not only inform future selection of therapeutic agents and their combinations based on response, survival and toxicities, but also aid in formulating fiscally responsible clinical strategies based on cost-effectiveness models.[13] THE NEED TO PROVIDE AND INTERPRET LARGE AMOUNTS OF DATA WHILE MAINTAINING EXCELLENT DATA INTEGRITY However brilliant the physician may be, there is no way he/she is going to remember the millions of possible genetic variants and what each of those variants may mean for the individual patient. Moreover, given our continuously evolving understanding of the genomics, proteomics, metabolomics and other characteristics of tumor growth, it is unrealistic to expect any individual to remain current and on top of new discoveries. Invariably, in order for physicians to access and make use of the vast and constantly emerging information, she/he will need to use a variety of computational tools, and have access to a well-maintained computational support infrastructure. While initially, the focus of this computational infrastructure may be on tumor genomic signatures, and on genomic backgrounds of the hosts, it should eventually incorporate for a true personalized medicine application all of the patient’s medical history, family history, dietary history, and exercise/activity information. To implement precision medicine – and incorporate individual differences in genomic make-up and individual biological characteristics into treatment decisions – we will require the development and easy access to largescale genomic, proteomic, biologic and health information databases. While some protein-protein interaction (PPI) networks are already publicly available on the Internet, Figure 2: Pathway to combination targeted therapy design.Theabilitytoevaluateoutcomesofcombinationtargeted therapies is dependent on the ability to standardize selection of therapeutic targets and low-dose metronomic backbones. The diagnosis of patient’s molecular profile should be based not only on the genomic analysis of the patient’s and the patient, but also on detecting the target proteins and their activation in the tissues. In order to incorporate, and consolidate the vast amount of information computer-assisted complex sociotechnical systems need to be employed to provide tumor boards with upto-date information about the best molecular targets. Finally, to continuously improve the quality of the information provided to tumor boards, AI should be used to inform future decisions. Oncotarget46819www.impactjournals.com/oncotarget they are, at least at present, mostly complex interaction maps developed by academic biologists over the last 50 years. Of concern is that because they are maintained by academic institutions with varied levels of funding, they may be of varied levels of information integrity, and of different ability to integrate emerging information or to provide for any corrections/additions driven by new information. Due to the clear and potentially immediate impact precision medicine can exert on cancer therapies much of the information in these databases are dedicated to oncology. However, the long term goal should be to generate a broad ranging source of information about diseased and physiologic states that would be useable for general medical purposes. A POTENTIAL SOLUTION To address the difficulty accessing, curating and interpreting large data, a clinician-relevant computer assisted search of available information of the publicly available databases needs to be created. While more information than ever is available to the clinician, the information is not only overwhelming, it is also dispersed across varied and copious sources, few of which are geared to clinical applications. Automated systems that can trawl, collect and align available relevant information and provide assistive interpretations for clinicians would significantly alleviate this problem. We can begin by accessing available information in publicly available academically or National Institute of Health curated databases and incorporate cancer knowledge networks as they become available. Such augmented human intelligence can improve the ability of an institutional tumor board to understand and interpret all of the available gamut of molecular information and remaining current on published medical information. The computerized system, containing a variety of artificial intelligence technologies can integrate a wide variety of information and apply an “understanding” of cancer biology in order to guide a tumor board in designing the most effective therapy for each of its unique patients. The system can do so by incorporating and cross-referencing information from multiple modalities, integrating this information in a clinical oncology context, and providing mathematical analysis of molecular pathways relevant to the patient’s specific (identified) molecular changes. The information incorporated into this stream can come not only from traditional academically curated databases, but also from medical and popular scientific literature sources, public media as well as health/ fitness tracking databases as recovered through social media. The information relevant to the individual patient can therefore superimposed onto a consolidated and highly cross-referenced informational stream providing the safest avenue for using the most up-to-date and continuously extended by emerging information. THE NEED TO DEVELOP NOVEL MATHEMATICAL APPROACHES FOR ESTABLISHING HIERARCHY OF GENOMIC ALTERATIONS IN INDIVIDUAL TUMOR SAMPLES While the advent of genomic testing - whether by a panel of genes or the entire genome - offers tremendous potential in clinical decision-making. There is presently a dearth of choices in ways to interpret and apply the information to the clinic. Scientists and clinicians are besieged with methods for differentiating between driver genes and passenger genes, realizing that not all gene alterations detected in cancer tissues are of equal importance. The conservative approach has been to use an expert-approved panel of candidate oncogenes and tumor suppressor genes in clinical testing. However, most candidate gene panels test only for gene alterations well documented in the literature and other authoritative sources. Those targets are ‘assumed’ by experts to be necessary for cancer progression based on the fact that some of these candidate genes have been around for decades. They may be considered universal driver genes just by virtue of our familiarity with them and their commonness. While these candidate approaches help alleviate the information glut, they are based on insufficient information given our relative paucity and incomplete knowledge about the role genetic mutations may play in the host, in tumor specific host tissues, and/ or in cancer biology. While BRAFV600E and BRAFV600K mutations are established driver genes for neuroectodermal tumors such as melanoma, the use of BRAF fusions, and non BRAFV600E or non BRAFV600K gene alterations in gliomas will have to be established.[24, 34] To use and organize the continuously emerging and heterogeneous information being deposited into genomic (The Cancer Genome Atlas, TCGA; Gene Expression Omnibus, GEO; the NCI’s Database of Genomic Structural Variation; dbVar etc), proteomic (UniProt, Swiss-Prot end may others), and metabolomics (Kyoto Encyclopedia of Genes and Genomes, KEGG; and other) databases, as well as the concerted effort to identify and catalog genomic vulnerabilities across hundreds of cancer cell lines (Broad Institute’s Project Achilles), new computational tools for repeated and potentially automated analysis of large data sets need to be developed. A POTENTIAL SOLUTION The impetus lies in improving the ability to select the most appropriate therapeutic target(s) for a particular patient. This necessitates development of novel approaches for large genomic or proteomic data analysis through multidisciplinary collaborations between mathematicians, physicists, statisticians, pharmacists, physicians, Oncotarget46820www.impactjournals.com/oncotarget bioinformaticians, artificial intelligence developers, biologists and software developers. The trans disciplinary process is mandatory in order to cover the end-to-end process, from cancer diagnosis, to testing for genomic alterations, to selecting appropriate targets, to analyzing pathways involved in cancer progression, to the design and administration of therapies. The motivation should be improving the ability to select the most appropriate therapeutic target(s) for a particular patient. There are two approaches to this. The first is more established and uses high-throughput statistical analysis (bioinformatics) of genomic data such as mRNA transcriptomes or RNA Seq from tumors of a population of patients with the same disease.[35-39] This approach provides the means to identify the most frequent genetic alterations in a population. The alternative approach applies novel mathematical and physical methods to determine how the individual patient compares to the genomic information derived from the population studies. [40, 41] While it is expected that both approaches will merge in the not too distant future, they remain distinct at present and exist in two separate solitudes. Yet, in order to base a treatment decision on the unique molecular signature of the patient’s tumor, an a priori resolution of the detected molecular alterations using both methods is an absolute starting point for the process. One previously described novel physical method for prioritization of targets applies a thermodynamic interpretation to gene expression, and then uses a topological filter to identify a set of potential therapeutic targets by their predicted effect on survival.[42, 43] The method makes use of publically available proteinprotein interaction networks (PPI networks). These PPIs are online repositories of interaction datasets compiled by international teams of academicians and researchers, and comprehensively curated into networks akin to telecommunication or social network maps. The thermodynamic entropy method considers these PPI networks a closed system where all interactions tend to equilibrium, and where entropy is a measure of the PPI network disorder. Because degree entropy of PPI networks for different cancers, correlates with likelihood of survival of patients with this cancer,[43] one can calculate the effect of eliminating a specific target (or eliminating multiple targets). This approach has demonstrated promising results, and points to the benefits arising from incorporating multidisciplinary perspectives to cancer models. Another previously described method performs a pan-cancer analysis of mutated networks.[44] This unbiased and open-ended analysis had revealed 16 significantly mutated subnetworks that were not previously thought to play significant role in cancer, and demonstrated that rare combinations of mutations, across multiple PPI networks may provide new insights and new opportunities for diagnostics and therapeutics across cancer types. The PPI approach can be used in a number of ways. For instance, one can overlay transcriptional data from a single patient onto a PPI network, or a data set from The Cancer Genome Atlas (TCGA). As an example of the later, TCGA transcription data from a population of patients with glioblastoma multiforme (GBM) was overlaid on the BioGrid PPI network. The current Biogrid Index[45, 46] version 3.3.124 (http://thebiogrid.org/), holds more than 820,000 protein interactions derived from highthroughput datasets, individual focused experiments, and from over 44,000 publications. The types of proteinprotein interactions include actual chemical bonding, or temporary bonds known as secondary bonding, and the concentration of the specific proteins dictates the degree of interaction. If a protein is in limited supply, it is said to have low chemical potential, and if it is abundant, it is said to have high chemical potential. Thus, using protein concentration, we can calculate the chemical potential of each protein in the network (i.e. Gibbs free energy), compute a topological measure known as filtration threshold (an energy threshold), and “filter out” the most energetic subnetworks from the larger network and try to reduce complexity of these subnetworks by inhibiting each protein in turn. Using this strategy, the “best therapeutic targets” are those that, when inhibited, most effectively reduce the complexity of a PPI network. As an alternative, one can superimpose patientspecific tumor mRNA transcription data (a surrogate for protein concentration) onto BioGrid, calculate Gibbs free energy for all proteins in the network, and identify those nodes with most effect on entropy. Many of these nodes may not have been identified in the specific tumor type. For example, BRACA1, an accepted therapeutic target in breast or ovarian cancer, was identified as best therapeutic target for 41 out of 342 glioblastoma multiforme (GBM) patients in TCGA,[47] even though the importance of its overexpression in GBM is unknown. Similarly SIN3 was important in 38 of the 342 GBM patients in TCGA, and SIN3 turns out to be a member of a regulatory complex in the biology of glioblastoma.[48] A total of 46 unique targets were identified using GBM transcription data from 342 patients with glioma available in TCGA. The complex sociotechnical system[49] considered here should be designed to work with as much genetic, proteomic and biologic information as available, and involve as many fields of expertise as possible. It should be noted, that even though it is being designed for maximum efficacy in cancer (both solid tumors and leukemias/lymphomas), it can be broadened to cardiology, inflammatory bowel disease and other medical specialties as genomic information in these fields emerges. It is able to use full transcription information from the tumor tissue; subtractive transcription information of tumor tissue and patient normal tissue; proteomic analysis of the same; phosphorylation maps, methylation arrays etc. At Oncotarget46821www.impactjournals.com/oncotarget a minimum, it requires genetic information in the form of gene expression (transcription) microarrays or a panel of genes. Its strength lies in being able to continuously incorporate new information, as well as new mathematical and thermodynamic methods for therapeutic target prediction. THE NEED TO PROVIDE COMBINATION THERAPIES BASED ON PATHWAY ANALYSIS Treatment decisions are, at least in present oncology practice, made on the basis of histological diagnosis, site of tumor origin (breast, lung, prostate etc), and the familiarity of the oncologist with a therapeutic agent. Despite the documented genetic and biological differences in even histologically identical site-specific cancer types,[8] most first line therapies do not diverge from the National Comprehensive Cancer Network (NCCN) Guidelines for Treatment of Cancer and national guidelines in other countries by site. They do not incorporate RNA/DNA sequence, transcription or protein expression information. Despite the evidence that molecular signatures of seemingly diverse and distinct cancers (lung squamous, head and neck, and a subset of bladder cancers) can coalesce into a common, site-independent molecular subtype,[50] most patients are still treated according to cancer site specific protocols. If considered, new treatment modalities are used only in second or later line of therapy, when additional molecular changes may have been added to the cancer initiating event adding to the complexity of controlling cancer growth. It is encouraging, however, that more and more oncologists are looking for safe and rational ways to incorporate genomic and biological information into first line therapies and individualize treatment protocols. This is especially true for oncologists treating patients with poor prognoses cancers such as sarcomas or brain tumors. But the approaches differ widely. The phrase “precision medicine” or “targeted therapies” are employed to describe a wide range of approaches in clinical oncology such as: 1. Targeted therapies used because a specific, single molecule is presumed to be present on the basis of previously published data (populational approach). 2. Therapies where, based on the histology of the tumor, a specific molecular target is looked for, identified and, if the mutation is present, treated as part of a single agent trial (a candidate target approach). 3. Targeted therapies that test for a panel of candidate molecules (usually an expert established panel of genes), but where a single target, selected either on the basis of its availability in a clinical trial, or on the availability of an FDA approved drug, is used (a panel of candidate targets approach). 4. Therapies that test the entire genome or transcriptome of the tumor and/or of the patient, but where a single molecular target is selected and treated. 5. Therapies that test the entire transcriptome and/or proteome and/or exome (note that the candidate approach is a subset of the full exome), a combination of molecular targets according to the ‘pathway activation strategy’ is selected, and all targets contributing to tumor progression are treated (the position of the authors). It should be stressed, that using targeted agents in absence of testing for molecular alterations may be detrimental.[12] A recent comparison of outcomes of patients treated with targeted agents without testing the tumor tissues for targets (i.e. non-personalized targeted therapies) was associated with significantly poorer outcomes than even traditional cytotoxic agents approaches.[12]. The same comprehensive analysis of phase II, single-agent arms revealed that, across malignancies, a personalized strategy was an independent predictor of better outcomes and fewer toxic deaths[12] Similarly, using strategies that do not use combination therapies and thus do not inhibit the majority of molecular pathways contributing to tumor progression (the single agent approach) also provide no benefit.[51] The SHIVA prospective randomized trial[51-53] compared a personalized approach with conventional therapy in relapsed refractory adult solid tumors. This was a singleagent treatment enrolling patients on the basis of limited molecular profiling of known targetable pathways, and it was not surprising that there was no difference in progression-free survival between the molecular alteration based therapy and conventional treatment. There may be more than one reason for the reduced efficacy of a single agent approach. There is a high likelihood of missing some important targets due to limited molecular profiling, and there is a high likelihood of treatment resistance due to alternative pathways with single agent approach. The use of several molecularly targeted agents in combination with low dose chemotherapy based on comprehensive analysis of individual tumor biology is an appealing way to counteract this type of treatment resistance. The incorporation of tumor molecular signatures information into clinical practice has not been easy, and for most physicians the most acceptable manner of using tumor molecular signature information is to screen for commonly occurring alterations and to enroll the patient on a clinical trial using the particular inhibitor. While this may be a practical and rational solution, the approach is inadequate for patients with complex genomic signatures consisting of more than one gene alteration. With the exception of chronic myeloid leukemia (CML), gastrointestinal stromal tumor (GIST), dermatofibrosarcoma protruberans (DFSP), or other similarly rare cancers, single mutations rarely account for the complexity of cancer biology, or for the secondary gene activation(s) caused by alterations within the tumor microenvironment. The protection of cells from xenobiotic such as cytotoxic agents do not Oncotarget46822www.impactjournals.com/oncotarget require a mutation, commonly an increased expression (or activation) of molecular pathways already encoded in the genome is sufficient for emergence of resistant clone. As such targeting a single gene alterations is unlikely to be effective in most tumors. As one pathway is inhibited, an alternate pathway is activated or additional genomic alterations are acquired. A good example is provided in targeted treatment of melanoma using monotherapy. Treatment with either vemurafenib (BRAF inhibitor) or trametinib (MEK inhibitor) alone can lead to excellent, but invariably shortlasting responses [54, 55] due to feedback activation of other pathways.[56-58] Because most oncogenic changes tend to hijack physiologic host responses such as inflammation, nullify other host defense mechanisms such as immune surveillance, and/or re-activate dormant developmental pathways for angiogenesis, immune evasion, and growth – the feedback loops are endless. Because oncogenic BRAFV600E can lead to melanoma cancer cell immune evasion,[59] and the reversal of this evasion by addition of PD1 or CTLA4 immunologic therapies has been shown to provide additional benefit to BRAF inhibition alone. The combination of immune checkpoint inhibitors and BRAF-targeted agents in melanoma suggests a synergistic action of these otherwise independent therapeutic modalities,[60, 61] and a much longer response duration. While there may be a specific genomic signature that corresponds to immune evasion,[62] the use of combination therapy using inhibitors of BRAF, MEK and immune checkpoint inhibitors has caused 2-year survival rates of patients with metastatic melanoma to rise to 79%.[63] A POTENTIAL SOLUTION A potential solution to managing the information glut and helping the oncologist to provide patients with the right combination of targeted agents and chemotherapy, is to enable them to use all of the available information. While producing complete genomic, proteomic and metabolomics datasets for each patient is not feasible at present, it has been possible in some well-funded research units to access the entire tumor and host transcriptomic information. The more complete the information provided for the analysis of the involved pathway(s), the more complete the therapeutic coverage. Unfortunately, for most physicians practicing clinical oncology today, the most feasible option is using a panel of candidate genes, because this may be covered by the patient’s insurance. At lease in the US, clinical ‘omic’ testing is restricted to genomic panels through CLIA certified laboratories. Even though this approach carries the inherent risk that some driver genes may not be identified, and thus not included in therapy, it is a good initiating step towards the future. The complex sociotechnical system being deployed by the authors of this manuscript maps the available molecular information from patients’ tumors onto an oncology interpretation knowledge base pooled and cross-referenced from multiple sources, and weighted in PPI networks according to the unique composition of the patient’s distinctive molecular signature. The combination of genetic alterations and mutational variants are matched to a series of filtered (see above) PPI subnetworks corresponding to biologic pathways relevant to cancer growth and progression, thus identifying molecular lesions that can be targeted with therapeutic intent. This complex sociotechnical system then searches the available literature and other reliable resources to find therapeutic agents targeting the identified molecular lesion(s), and minimize the number of drugs needed to inhibit all pathways within the identified PPI subnetwork. The system also considers the topology and interaction of each of the identified anomalous pathways in order to use the minimum possible drugs, and still achieve the same therapeutic result. in situations where specific genomic alterations may confer an a priori resistance to a therapeutic agent,[64, 65] the agent is eliminated. Roughly similar to the current use of Artificial Intelligence technologies deployed in recommending movies on the basis of our previous choices, likes or dislikes, one of the AI components in this system records and documents the selection of targets, the treatment protocols and the respective outcomes in order to inform future therapeutic selections. More specifically, as oncologists and other experts on the tumor board introduce novel evidence for, or arguments against a therapeutic choice provided by the system, the information is recorded and used to refine future pathway analyses. The hope is that genomic/proteomic information will become affordable and we will include the genomic/proteomic analysis as a standard component of the electronic medical record. In turn, as more information from patient’s medical record is incorporated, we will be able to consider any co-morbid conditions of the host and filter out harmful or ineffective drugs from the therapy recommendations further, resulting in improvement of the safety of our treatments. THE NEED TO AVOID COMBINATIONS WITH MAXIMUM TOLERATED DOSES OF CHEMOTHERAPY: THE ARGUMENT FOR LOW DOSE (METRONOMIC) CHEMOTHERAPY BACKBONE A commonly employed approach for enhancing the ability chemotherapy to fight cancer is to use chemotherapy in combination with a biological agent. An assumption is made that the inhibitory effect of the biological agent would be additive to the effect achieved by traditional chemotherapy or radiation. However, the use of biologic agents, especially those inhibiting Oncotarget46823www.impactjournals.com/oncotarget host responses (such as angiogenesis or inflammation), strip the anomalous cells (but also the patient’s normal cells) of its defense mechanisms such as growth factors and inflammatory cytokines and lead to sensitization of all cells to DNA damaging agents such as radiation or chemotherapy. Because most mechanisms used to protect cells from xenobiota such as chemotherapy or radiation tend to activate developmental pathways already encoded in the genome, inhibition of these pathways increases toxicities whenever standard (maximum tolerated) doses of chemotherapy or radiation are used with biological agents.[66] In a standard clinical trial, where a standard arm is compared to standard arm with the biological agent, the approach greatly disadvantages the intervention arm. The combination of the biologic agent and high dose chemotherapy, makes an already maximally toxic regimen lethal. As a result, the benefit of any tumor response will be concealed by these increased toxicities, and no overall survival benefit will be seen.[66] An example of this is the case of combining bevacizumab with standard MTD chemotherapy. While the RIBBON2 trial showed an improved progression-free survival compared to patients treated only with chemotherapy alone [PPS 7.2 months in the experimental group compared to 5.1 months in the chemotherapy only arm (p - .0072)]. The 10% improvement in overall survival rate was not statistically significant.[67] Based on this finding, the US Food and Drug Administration (FDA) revoked the approval of bevacizumab as a first line treatment for breast cancer, even though the majority of women had responded, and some remain well controlled on the drug to date. The concept of “metronomic chemotherapy” was initially introduced in the year 2000,[29, 68, 69] and constituted a marked departure from the classic model of maximum tolerated dose (MTD) strategy. It emerged in the face of early clinical and pre-clinical evidence supporting its ability to suppress tumor growth even in cases where the cancer cell was resistant to the MTD of the used chemotherapeutic agent.[29, 68, 70, 71] Unfortunately, the concepts were poorly understood and underused. It has gained momentum however and at present it is being adopted with increasing frequency around the world,[72-74] and the website www. clinicaltrials.org now lists over 150 trials that use the word “metronomic” in their title. The mechanism of action of metronomic chemotherapy has been subject to excellent recent reviews,[75] and its value to implementation of precision medicine well documented.[22] To summarize briefly, because of the side effects induced by maximally dosed chemotherapy, the duration of the therapy has to be limited and breaks for bone marrow recovery incorporated. Furthermore, because conventional chemotherapy targets only proliferating malignant cells, a large portion of malignant cells is not affected. Only once these cells are re-engaged in the cell cycle process cytotoxic drugs are able to corrode them. Metronomic therapy implies that the use of low, continuous doses of chemotherapy in combination with biologic response modifiers not only avoid toxic side effects, but also preferentially target the host biological responses such as stromal induction,[76] angiogenesis,[68, 77, 78] immune surveillance,[75, 79, 80] and inflammation.[76] Angiogenesis and inflammation represent a physiological repair mechanisms hijacked by the proliferating tumor and actively contributing to tumor cell re-growth. The enormous success in the treatment of pediatric acute lymphoblastic leukemia, is at least partially due to the one and a half year long maintenance low dose metronomic chemotherapy. Thus, it should be stated that in cases where upfront eradication of the cancer is not possible with MTDs, the MTD-induced up regulation of host inflammatory responses, rather than defending us from cancer, contributes to subsequent cancer progression. Because MTD chemotherapy kills only chemo-sensitive cells with each cycle, the chance of selection of a chemotherapy resistant subpopulation and recurrence is very high. Metronomic chemotherapy, with its goal of longterm “tumor control”, lower toxicity, and prevention of tumor progression (rather than immediate reduction in tumor size), may represent a more realistic strategy for cancer therapy. This is especially true for cancers not amenable to upfront cancer eradication. While slower in its onset of action (see Figure 3), metronomic dosing has demonstrated better long term tumor control, even for cancers rendered resistant to the same drug under MTD,[29, 68, 78] because the low-dose chemotherapy approach avoids selection of a resistant cancer cell population. A very strong argument for the use of a metronomic chemotherapeutic backbone in combination with targeted therapies is the risk of metastatic growth.[81, 82] This risk of exacerbating metastases has however, only been documented with single agent therapy and only in preclinical murine models. It remains theoretical in clinical practice where it is usually prevented by the synergistic action of biologic agents and low dose chemotherapy. The same is true for avoiding emergence of therapeutic resistance with targeted agents alone.[57] A POTENTIAL SOLUTION In the coming decade(s) a background for the combination therapies will be applied for any patients with chemotherapy resistant cancer or for patients with very poor prognosis. As much information as possible should be gathered about the patient’s tumor molecular signature, about the host specific germline gene alterations, and about the host phenotype as soon as possible, so as to avoid unnecessary toxicities and delays with standard therapies whenever success cannot be reasonable expectation. The hope is that data from each of these cases will be collected Oncotarget46824www.impactjournals.com/oncotarget and each of the individual outcomes will inform any future therapeutic decision. CASE 1 A previously healthy 11 year old girl with neurofibromatosis type 1 was diagnosed in 2011 with a large right parietal glioblastoma multiforme following an episode of left sided weakness. She was found to have a hemorrhagic stroke, and despite a partial resection of the tumor, her hemiparesis never resolved. She was started on COG ACNS0822, randomized to Arm A, and she completed the 6 weeks of radiation and vorinostat. In November 2011 she started maintenance chemotherapy with Avastin 10mg/kg Day 1 and 14/ Temozolomide 200mg/m2 Days 1-5 for 28 day cycle. She completed 11 out of 12 cycles before coming off protocol for disease progression in October 2012. She was started on melatonin, metformin, cyclophosphamide and erlotinib based on a proteomic analysis done at Texas Children’s. She progressed again within 2 months with leptomeningeal spread to the spine, and was changed to VP-16, vincristine, crizotinib, erlotinib, vorinostat. The regimen resulted in unacceptable toxicities with myelosupression, severe mucositis, and QTc prolongation with cardiac compromise. She was taken off any disease directed therapy in March 2013 and referred to us for molecular analysis and individualized therapy. The characteristics of the tumor at diagnosis showed activation of a number of pathways associated with cancer growth and progression. The findings and initial pathology are summarized in Figure 4. The genomic analysis revealed NF1 R1968*, BRCA1 N1355fs*10, CDK4 amplification, TP53 R175H, SOX2 amplification. Because loss of neurofibromin function leads to increase in signaling through the RasRaf-MAPK and mTOR pathways, [83] she was started on sirolimus 2mg daily and sorafenib to inhibit growth factors downstream from these pathways in addition to metronomic (50 mg/m2) etoposide daily. She remained stable on this regimen until December 2015 (3 years) when she had a radiological progression. She underwent an excisional biopsy and the molecular analysis of this relapse was consistent with a radiologically, histologically and genetically more aggressive phenotype (Figure 4). In addition to the original gene alterations, she now had BRCA2 splice site 67+1G > A, ERBB3 S1074N, TSC1 splice site 364-1G > A, GLI1 amplification, STAG2 Q1167*. Her therapy was therefore changed to everolimus (Ras-Raf-MAPK and mTOR pathways), ceritinib (GLI1/sonic Hedgehog pathway), and trametinib on a metronomic chemotherapy backbone of temozolomide 25 mg/m2, and remains stable. The case provides a good illustration about the need for multi-agent therapy based on molecular signature. It also stresses the need to consider re-biopsy with relapse as the eco-evolutionary forces within the tumor microenvironment may cause therapeutic resistance and escape from tumor dormancy.[84] CASE 2 A 7-y old previously healthy boy with no family history of cancer was diagnosed with stage III abdominal Burkitt lymphoma in December 2014. He was initially treated standard BFM B-NHL 04 therapy, which Figure 3: Comparison of Metronomic and Standard dose strategies. The onset of action of metronomic chemotherapy is slower, but because of its ability to suppress biological processes such as angiogenesis or inflammation which are often “hijacked” by the tumor for growth, and because it avoids selection of the resistant population of cells, its effects are more sustained. However if comparison of these two therapies is made before 6 months, the wrong conclusion about the effectiveness of metronomic chemotherapy may be made. Oncotarget46825www.impactjournals.com/oncotarget included a single initial dose of 375mg/m2 Rituximab followed with 5 cycles of BNHL 04 chemotherapy consisting of dexamethasone, methotrexate, ifosfamide, cyclophosfamide, cytarabine, etoposide, doxorubicine, vincristine as well as intrathecal therapy. After 2 cycles, he had a very good partial response reaching < 5% of the initial tumor volume. An episode of the intestinal obstruction in February 2015 led to excision, and the histology confirmed sclerosing mesenteritis, without histological or rtPCR evidence of lymphoma (the original tissue was positive for cMYC translocation). The FDG PET was borderline positive, but this was thought to be due to inflammation. Unfortunately the child was found to have an isolated radiological progression in the same region in which the intestinal obstruction had occurred two months after completing chemotherapy. The biopsy in June 2015 confirmed relapsed Burkitt lymphoma, this time with marked areas of sclerosing mesenteritis and mesenteric panniculitis. Mutational analysis of PI3K delta subunit proved germinal mutation/variant outside the classical Activated PI3K-delta syndrome (APDS) 1 or 2 variants. The mutational activation was confirmed by testing the patient’s T- lymphocytes, and the S6 (Ser235/236) phosphorylation was found to be 33 fold that of a healthy control. While undergoing the genomic testing, the boy was started on retrieval therapy with ibrutinib, obinutuzumab and ICE chemotherapy. Unfortunately, after a transient response and disease stabilization, he had an early progression following the first cycle. Based on the finding of germline mutation in PI3K delta subunit, he got 2 weeks of idelalisib (a phosphoinositide 3-kinase inhibitor, which blocks P110δ, the delta isoform of the enzyme phosphoinositide 3-kinase). The single agent therapy led to normalization of the S6 (Ser235/236) phosphorylation in patients peripheral T lymphocytes, but he had further disease progression. It was only when the combination of high dose cytarabine/ etoposide (CyVe) with idelalisib and obinutuzumab was used that the disease was stabilized. A biopsy on 9/2015 showed a CD20 positive tumor, with high degree of proliferation and strong expression of PD- 1L. The second biopsy was analyzed using Affy GeneChip ST 1.0 and the whole transcriptome analysis confirmed increased levels of PI3K and revealed additional HR23B. Because HR23B can be used as a good predictor of response to HDAC inhibitors, valproic acid was being considered. Additional tumor specific (somatic) gene alterations in R273C and p53 were also shown. The child, who had continued on oral ibrutinib + idelalisib and low dose cyclophosphamide since 9/2015, received palliative 21Gy local radiation. In 10/2015, based on the second biopsy findings, the nivolumab, a human IgG4 anti-PD-1 monoclonal antibody, and valproic acid, and HDAC inhibitor, were added. As of March 2016 the boy is doing very well. He has had partial response of the single residual abdominal tumor disease, and remains clinically well with Lansky score 100 and OS > 15 months. He comes to clinic biweekly for nivolumab infusions and assessments, but remains outpatient otherwise. He started his personalized therapy after his second relapse, and this 3rd EFS (7 months) is already the longest EFS, compared to 6 months post his initial standard BFM protocol and just 1 month post ibrutinib, obinutuzumab and ICE chemotherapy. The case may illustrate a new variant of Activated PI3K-delta syndrome (APDS). At least at present the disease is not tested for and generally not recognized in children with Burkitt’s lymphoma. Even if this child had a family history supporting testing for the autosomal dominant form of APDS, he would not have been found. Yet, he had an atypical germinal mutation in the gene that leads to lymphoid hyperplasia, and increases the risk of malignant transformation to B-cell lymphoma. The p110δ protein is a crucial subunit of the PI3K enzyme, and regulates activation of proliferative pathways in B-cells. As such, unless this constitutional activation can be blocked, it will be unlikely that 5 cycles of conventional chemotherapy could successfully prevent a relapse. It may be prudent, in cases where a mutational activation of an important proliferative pathway is found, to use maintenance biological therapy. This could be similar to the 2 years maintenance therapy used in childhood Acute Lymphoblastic Leukemia, which has cure rates of about 90%. It is our hope that this case illustrates a potential for keeping even children with poor prognosis due to genetically complex cancers at home. While not able to eradicate the cancer or it causative mutation, we may be able to keep them well, in school and active by prescribing a combination of low-dose metronomic chemotherapy, an immune checkpoint inhibitor, and a direct inhibitor of the activated pathway(s). SUMMARY Many oncologists treating recurrent, chemotherapy resistant or poor prognosis cancers have begun repurposing anti-inflammatory agents or immune modulators. Similarly, many oncologist use direct anticancer agents in an off-label setting to target specific genomic mutations regardless of the cancer subtype. An equal number of oncologists however, due to the time required for researching the vast amount of molecular information, continue treating children with conventional therapies. But for those cancers where the present chemotherapeutic, surgical and radiation strategies fail – the option of targeted strategies should be strongly considered. The difficulty is that physicians using targeted therapies today do so without the benefit of computational infrastructure. While we use complex sociotechnical Oncotarget46826www.impactjournals.com/oncotarget Figure 4: Histology of case 1, glioblastoma progression. The original right temporal mass resected in 2011 showed glial neoplasm with vascular proliferation, necrosis, mitosis, and numerous pleomorphic cells, including rare giant cells. At this time, there was strong and diffuse immunopositivity for GFAP, and markedly elevated Ki-67 proliferative index, consistent with Glioblastoma WHO grade IV/IV. The original lesion regressed after the initial targeted therapy with sirolimus, sorafenib and metronomic VP16, but relapsed with a new extra axial lesion. The relapsed tissue in 2015 showed glial neoplasm with numerous tumor giant cell and atypical mitoe. The tumor cells were immunonegative for NEU-N, IDH-1(R132H) and BRAFv600E, but the molecular signature had obviously evolved, adding further genomic alterations. At this time, the tumor was negative for GFAP, and the ganglional component was no longer present. Both the 2011 and 2015 specimens had shown increased lymphocytic component and myxoid background, along with tumor giant cells, but the number of giant cells was increased significantly in the 2015 specimen. Oncotarget46827www.impactjournals.com/oncotarget systems to manage nuclear plants and airports, we have not developed similar systems for the analysis and application of omics information. We need an efficient complex sociotechnical system that would allow us to analyze a molecular signature of the patient’s cancer in minutes and select the appropriate molecular agent(s) in time for effective therapy. We also need to abandon the present model of drug development. The present process often takes decades for each of the new therapeutic agents. Millions are spent testing each of the agents in individual Phase I-IV trials before its introduction to the clinic resulting in cost-prohibitive therapies. Most importantly however, thousands of patient lives are lost as we struggle to determine whether an agent “is clinically active” in the incorrectly designed clinical trials. A wealth of bioinformatics resources exists that can help narrow the choice of therapeutic combinations from the wide selection of already available molecular agents, and provide a treatment for a wide range of difficult to treat cancers TODAY. ACKNOWLEDGMENTS The authors are grateful for the many informal discussions with pediatric oncologists attending the October 2015 CEPOETA meeting in Brno, Czech Republic. GLK would like to acknowledge the philanthropic support from the Campanelli Foundation, CSTS Health Care, and institutional support. ER, AH, CK and PC have received salary support from CSTS Health Care. JS and OS were supported by Czech Republic Ministry of Health grant 16-33209A and 16-34083A. 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CASE REPORT Open Access Case report: rapid and durable response to PDGFR targeted therapy in a child with refractory multiple infantile myofibromatosis and a heterozygous germline mutation of the PDGFRB gene Peter Mudry1* , Ondrej Slaby2 , Jakub Neradil3,1,7 , Jana Soukalova4 , Kristyna Melicharkova1,7 , Ondrej Rohleder1 , Marta Jezova5 , Anna Seehofnerova6 , Elleni Michu2 , Renata Veselska3,1,7 and Jaroslav Sterba1,7 Abstract Background: Infantile myofibromatosis belongs to a family of soft tissue tumors. The majority of these tumors have benign behavior but resistant and malignant courses are known, namely in tumors with visceral involvement. The standard of care is surgical resection. Observations suggest that low dose chemotherapy is beneficial. The treatment of resistant or relapsed patients with multifocal disease remains challenging. Patients that harbor an actionable mutation in the kinase domain are potential subjects for targeted tyrosine kinase inhibitor therapy. Case presentation: An infant boy with inborn generalized infantile myofibromatosis that included bone, intracranial, soft tissue and visceral involvement was treated according to recent recommendations with low dose chemotherapy. The presence of a partial but temporary response led to a second line of treatment with six cycles of chemotherapy, which achieved a partial response again but was followed by severe toxicity. The generalized progression of the disease was observed later. Genetic analyses were performed and revealed a PDGFRB gene c. 1681C>A missense heterozygous germline mutation, high PDGFRβ phosphokinase activity within the tumor and the heterozygous germline Slavic Nijmegen breakage syndrome 657del5 mutation in the NBN gene. Targeted treatment with sunitinib, the PDGFRβ inhibitor, plus low dose vinblastine led to an unexpected and durable response without toxicities or limitations to daily life activities. The presence of the Slavic NBN gene mutation limited standard chemotherapy dosing due to severe toxicities. Sister of the patient suffred from skull base tumor with same genotype and histology. The same targeted therapy led to similar quick and durable response. Conclusion: Progressive and resistant incurable infantile myofibromatosis can be successfully treated with the new approach described herein. Detailed insights into the biology of the patient’s tumor and genome are necessary to understand the mechanisms of activity of less toxic and effective drugs except for up to date population-based chemotherapy regimens. Keywords: Infantile myofibromatosis, Tyrosine kinase inhibitor, PDGFR, Chemotherapy, Theranostics, Case report * Correspondence: pmudry@fnbrno.cz 1 Department of Pediatric Oncology, University Hospital Brno and School of Medicine, Masaryk University, Cernopolni 9, Brno 613 00, Czech Republic Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mudry et al. BMC Cancer (2017) 17:119 DOI 10.1186/s12885-017-3115-x Background The family of fibroblastic-myofibroblastic tumors consists of more than 30 distinguished entities, such as inflammatory myofibroblastic tumor (IMT), aggressive fibromatosis and infantile myofibromatosis (IM). These tumors have uncertain biologic behaviors that range from low grade, locally aggressive and rarely metastasizing to a highly aggressive course that eventually evolves to a true high-grade sarcoma after recurrences. IM is a rare tumor that affects infants with a median age of 3 months; approximately 100 solitary lesion cases have been published in the literature during the past decade [1]. Soft tissue lesions of IM can arise at any time during life and, intriguingly, can regress spontaneously. However, visceral lesions are associated with high morbidity and mortality. The standard of care is the surgical resection of a single lesion. Multiple lesions and surgically unresectable lesions could be treated with anti-inflammatory drugs, interferon alpha, or distinct chemotherapeutic regimens that are based on low dose metronomic or maximum tolerated doses (MTD) of chemotherapeutics, such as the vinca alkaloids vincristine, vinorelbine and vinblastine; the alkylating agents cyclophosphamide and ifosfamide; or others, such as actinomycine D, doxorubicin or methotrexate [2–4]. The results of such treatments are under investigation in ongoing observational clinical trials of cooperative groups, such as European Soft Tissue Sarcoma Study Group (EpSSG) or Children’s Oncology Group (COG). Several studies of desmoid-type fibromatosis with response rates of 33–49% were reviewed elsewhere [4]. Nevertheless, the treatment of resistant patients, particularly those with visceral involvement, remains challenging. For patients with progressive disease after MTD based chemotherapy, there are no established standards of care, and these patients are, thus, subjected to experimental treatments. One of the most promising agents with proven activity for IMT is the ALK tyrosine kinase inhibitor crizotinib [5]. Patients with ALK rearrangement are reportedly rapidly responding to crizotinib, but those without the detected fusion are not [5]. A recent work by Lovly et al. on IMTs revealed multiple fusion partners of ALK, and newly reported ROS1 and PDGFRβ fusions with projected TKI sensitivity were demonstrated in a patient with an ROS1 fusion [6]. Similar to IMTs, IMs may harbor missense mutations in the PDGFRβ kinase that constitutively alter PDGFR activity. Moreover, in several families, the c.1681C>T (p.Arg561Cys) mutation in the PDGFRB gene was found to cause familial infantile myofibromatosis [7]. A phase II study of sunitinib in 19 patients with aggressive fibromatosis has been published and described a 26.3% overall response, but the analysis of the kinase pathway was lacking [8]. A case report of aggressive fibromatosis that favored the PDGFRβ inhibitor sunitinib against imatinib was published that described a good response with sunitinib which was interrupted after 13 months and substituted by imatinib. But reactivation of painful lesions occurred within several days and re-growth of aggressive fibromatosis led to successful re-treatment with sunitinib [9]. Herein, we report the case of a patient with refractory multiple infantile myofibromatosis who was confirmed to harbor the PDGFRB germline mutation and who responded well to treatment with the PDGFRβ tyrosine kinase inhibitor sunitinib. Case presentation The newborn boy with microtia and meatal atresia and with family history of two spontaneous missed abortions and myofibroblastic lesions with spontaneous regression in his older sister and father, was diagnosed with generalized myofibromatosis that affected the calva and radius bones, the spleen and subcutaneous tissue of face, the head, inguina and arm. Histopathology, with regard to the family history, revealed the presence of infantile familial myofibromatosis. Immunohistochemistry (ICH) and FISH did not reveal any pathological staining for ALK. The patient was treated according to the EpSSG 2005 observational trial recommendation with the metronomic vinblastine/methotrexate combination, which was expected to be less toxic than MTD based regimens. Despite this, severe neutropenia had been observed; therefore, a dose reduction was necessary down to 10%/30% of the original doses of vinblastine/methotrexate, respectively. The therapy was stopped after 8 weeks due to clearly progressive disease in the soft tissues and in the spleen and with the appearance of new FDG PET positive lesions in the bones. Thereafter, the standard MTD based therapy with vincristine/actinomycine D/cyclophosphamide – the “VAC” regimen with doses based on body weight (vincristine 0.05 mg/kg, actinomycine D 0.05 mg/kg, cyclophosphamide 50 mg/kg) had been initiated. Such treatment after the second course (the first course was given with a 75% reduction of cyclophosphamide) had led to severe febrile neutropenia, gastrointestinal toxicity with gastric palsy, subileus and bilateral bronchopneumonia. However, a reassessment after those 2 cycles revealed a partial response. Due to the previous toxicity, we decided to substitute vincristine with vinblastine at 10% of the recommended dose and cyclophosphamide at 75% of the recommended dose. The patient received the treatment without dose limiting toxicities up to six cycles and continued to respond. The patient was still in partial remission according to CT and MRI images and the FDG PET of the remaining measurable lesions was negative. Unfortunately, the first follow-up re-assessment confirmed the presence of progressive disease just 3 months Mudry et al. BMC Cancer (2017) 17:119 Page 2 of 7 after the last chemotherapy dose and several new lesions were detected in the humerus, head, lungs and skin, and all were FDG-PET positive. A new biopsy was carried out to obtain tumor tissue for phosphoproteomic analysis of the new lesion. The Human Phospho-RTK Array Kit was used to determine the relative levels of tyrosine phosphorylation of 49 different RTKs. The analysis was performed as previously described [10]. In addition to the antibodies (spotted in duplicate) against individual RTKs, each membrane contained three positive reference double spots and one negative control that was also spotted in duplicate and contained phosphate-buffered saline only. Furthermore, we also performed the following negative control experiment in each run: the membrane treated with lysis buffer only (without protein lysate) to ensure the specificity of the spotted antibodies. In such a design, a healthy control sample is not necessary for the determination of the RTK phosphorylation profile of the examined tumor tissue [11–13]. The phosphorylation profile of receptor tyrosine kinases showed that PDGFRβ kinase exhibited the highest level of activity and less intense positivity was observed for EGFR, M-SCFR, Axl and PDGFRα (Fig. 1). Targeted DNA analysis of the PDGFRB gene and next generation sequencing (NGS) were performed on genomic DNA from peripheral blood samples. We performed Sanger sequencing of the two PDGFRB regions to detect the presence of the c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys) mutations [6] and uncovered a germ-line heterozygous c.1681C>A missense mutation that had previously been shown to be an IM causing mutation [14, 15]. To obtain the complex picture of the genetic background of the case we performed DNA analysis from peripheral blood with the Illumina TruSight Cancer panel, which enabled the sequencing of the hotspots in 94 predisposition cancer genes, according to the standard Illumina protocol (Illumina Inc., USA) and identified the heterozygous Slavic mutation 657del5 in the NBN gene of the NBS. In the meantime, and based on parental request, the patient was observed for the next 4 months. He was doing very well clinically, with a Lansky performance status of 90% and with respect to his treatment history with toxicities after chemotherapy; we did not initiate another chemotherapy regimen but were awaiting the results of genetic analyses, which have revealed potential therapeutic targets. Further follow-up confirmed that the disease continued to progress; several new lesions were detected within the head and the left orbit, a new one was detected in the spine, and the spleen lesion had increased in size. Due to clear clinical and radiologic progression and new molecular genetic findings, and with respect to the history of the disease, we initiated the single agent offlabel treatment with sunitinib 12.5 mg once a day. This dose corresponded to 2/3 of the recommended adult dose. An unexpected and dramatic reduction of the palpable soft tissue and bony lesions on the head was observed during the 4 weeks of treatment with the single agent sunitinib. An MR scan confirmed the regression of intracranial and intraorbital lesions as well (Figs. 2, 3 and 4). However, this dosing schedule led to grade 3–4 neutropenia, and the drug was stopped for 4 days. After only 4 days, we could observe the reactivation of the skin and soft tissue lesions; therefore, the sunitinib was given at the same dose every other day. Reactivated reddish swollen and painful sentinel lesions responded again to lower doses of sunitinib, but three more weeks of reduced doses of the single agent sunitinib did not lead to any further regression of the regressed but still palpable skin lesions. A low dose of vinblastine was added to the sunitinib. The starting vinblastine dose was 2 mg/m2 ; however, based on the further hematological Fig. 1 The relative phosphorylation of kinases in the tumor tissue sample Mudry et al. BMC Cancer (2017) 17:119 Page 3 of 7 toxicity, the dose was tapered down to a 0.4 mg/m2 dose once weekly. An unexpected toxicity of sunitinib occurred after 4 months of treatment when accidental hypoglycemia led to a coma and the patient had to be admitted for glycemia corrections. Thereafter, the parents were educated on regular feeding before sunitinib administration. Further episodes of hypoglycemia were not noted. The patient remained on the treatment paradigm with a marked continuing response with no disease activity 1 year after the initiation of the treatment and without any dose limiting toxicities. Interestingly, the 8 year old sister of the patient, who had a history of spontaneous regression of subcutaneous lesions, suffered from the symptomatic re-activation of the disease when the patient was receiving treatment. She presented with tumor size of 29 × 24 × 16 mm on the skull base with night pain. Histopathological and detailed mutation analyses found the same IM histopathology and the same genotype in the PDGFB and NBN genes. As with the index case, the sister is doing well on sunitinib and vinblastine treatment and has exhibited a rapid response. The nigh pain relieved after 2 weeks on sunitinib + vinblastine. Initial tumor volume shrinked by 44% after 97 days of combined treatment without any adverse events requiring reduction of doses. Timeline of both cases is shown on Additional file 1. Discussion and conclusions Despite the finding that the patient exhibited a partial response to systemic VAC treatment, the disease continued to progress; moreover, the patient experienced severe, life threatening dose-limiting toxicities. Inflammatory myofibroblastic tumors that harbor an ALK/ROS1 or PDGFRβ kinase fusion are potentially targetable with TKIs due to the presence of a constitutively active kinase domain that drives cellular proliferation [6, 16]. A response to the ALK inhibitor crizotinib is reported in tumors that harbor any of the ALK kinase fusions. Patients with IMT and ALK negative rearrangements are unlikely to respond to such targeted treatment. PDGFRB mutations are reported to be involved in the pathogenesis of infantile myofibromatosis in a proposed autosomal dominant pattern with incomplete penetrance and variable expressivity [7]. The missense PDGFRB c.1681C>T (R681C) mutation is located in exon 12 and is predicted to decrease the autoinhibition of the JM domain (an autoinhibitory domain that masks the catalytic cleft when the receptor is not bound by its ligand) at baseline, which leads to increased kinase firing and promotes the formation of myofibromas in tissues with high PDGFRβ signaling activity. More recently, it was demonstrated in a cell culture model that the R561C mutation activates signaling pathways that are normally A B C Fig. 2 MRI Frontal view (seq. eFLAIR_long_TR_CLEAR). Two lesions of the left orbit and the skull in the fronto-parietal region (bars). a Before sunitinib treatment. b Day + 56 of sunitinib. c Day + 156 of sunitinib A B C Fig. 3 MRI Axial view (seq. esT1W_3S_FFE post-contrast). Intracranial lesions of the right temporal and right parieto-occipital regions (bars). a Before sunitinib treatment. b Day + 56 of sunitinib. c Day + 156 of sunitinib Mudry et al. BMC Cancer (2017) 17:119 Page 4 of 7 activated by the stimulated wild-type PDGFRβ receptor in the absence of PDGF [14]. PDGFR is the immediate NOTCH3 target gene [17]. If these two signaling pathways are linked and the IM disease-causing mutations in either PDGFRB or NOTCH3 are demonstrated to be activating, theoretically, the inhibition of PDGFRB or NOTCH3 would result in a targeted therapeutic strategy [7]. Our case report shows the clinical efficacy of such an approach. Targeted therapy against altered PDGFRβ with a TKIs inhibitor can overcome tumor growth and can lead to tumor shrinkage. Compared to the toxicity of conventional chemotherapy, treatment with sunitinib was tolerated well except for the occurrence of asymptomatic granulocytopenia and one episode of symptomatic hypoglycemia. However, the cessation of the drug lead to increased tumor activity and a decreased drug dose of the single agent sunitinib led to a stable disease only. The analysis of tumor tissue or a patient’s samples and the use of a subsequent results driven treatment provide a new opportunity for personalized medicine as opposed to a population based study. Such treatments are supported by new insights into the molecular pathology of rare diseases, such as IM. A similar strategy would at least justify the off-label use of new drugs when the individual tumor biology and data about the safety of such drugs is well defined. TKIs could be an example, as these drugs are not available to orphan disease patients because of the absence of appropriate clinical trials. The careful management and regular observation of the patient is mandatory, however, in situations where standard approaches are either exploited or ineffective or absent, the prudent use of targeted agents based on the mechanism of action might lead to impressive results. The rapid tumor re-growth that occurred when the patient was off of the sunitinib during the induction treatment indicates that metronomic dosing should be maintained at a lower dose with limited toxicity rather than being interrupted. The successful use of low dose vinblastine that is described here, together with the use of sunitinib at a dose of approximately 1/3 of the usually recommended dose per kg or m2 in adults, could be at least in part explained by the fact that targeted agents could act as biology response modifiers and lower doses of biological agents and chemotherapy could be nontoxic and advantageous [18, 19]. This theory is supported by our observation of the clear disease progression when sunitinib therapy was interrupted. Regular observations of the patient and preemptive measures such as the after-feeding dosing of sunitinib should be considered during treatment. The finding of the Slavic mutation of the NBS was noted as accidental during NGS sequencing and the relevance for the disease course is unknown. The toxicity of chemotherapy might be at least in part conditioned by the NBS mutation As known, the intensity of chemotherapy in NBS patients must be adapted to individual risk factors and tolerance. The use of radiomimetics, alkylating agents, and epipodophyllotoxins should be avoided, and the dose of methotrexate should be limited [20]. However, the overall duration of such clinically effective treatment remains speculative, especially in patients with germline mutations. Different approaches that consider cancer to be a chronic disease, such as diabetes, should be considered in instances in which pathogenic germline mutations are in place. Should such targeted agents be maintained for a very long time, e.g., maintenance therapies in childhood acute leukemia, where other mechanisms of action, not only the cytostatic effect are in place? [21]. Should some pulses of targeted agents be considered? These are only a few of the new questions that arose by the increased availability of diagnostic methods, such as NGS and functional proteomics. The patients with an orphan disease like IM could benefit from detailed insights into the biology of their tumor and genome. Such approach is necessary to better understand the molecular pattern of disease and mechanisms of action of less toxic and effective drugs except for up to date population-based chemotherapy regimens. Morover, an unexpected finding of germline mutation can be important for treatment decisions. Progressive and resistant incurable infantile myofibromatosis can be successfully treated with the new approach described herein. A B C Fig. 4 MRI Sagittal view (seq. esT1W_3S_FFE post-contrast). Frontal and parieto-occipital lesion (bars). a Before sunitinib treatment. b Day + 56 of sunitinib. c Day + 156 of sunitinib Mudry et al. BMC Cancer (2017) 17:119 Page 5 of 7 Additional file Additional file 1: Timeline. This file shows timeline of both described cases. (PDF 466 kb) Abbreviations ALK: Anaplastic lymphoma kinase; COG: Children’s oncology group; EpSSG: European Soft Tissue Sarcoma Study Group; FDG PET: Fluorodeoxyglucose positron emission tomography; FISH: Fluorescent in situ hybridization; IHC: Immunohistochemistry; IM: Infantile myofibromatosis; IMT: Inflammatory myofibroblastic tumor; IVA: Ifosfamide/vincristine/ actinomycine D; MRI: Magnetic resonance imaging; MTD: Maximum tolerated doses; MTX: Methotrexate; NBS: Nijmegen breakage syndrome; NGS: Next generation sequencing; PDGFR: Platelet derived growth factor receptor; PDGFRB: Platelet derived growth factor receptor gene B; PDGFRβ: Platelet derived growth factor receptor beta; TKI: Tyrosine kinase inhibitor; VAC: Vincristine/actinomycine D/cyclophosphamide; VBL: Vinblastine Acknowledgements The authors thank Drs. Eva Machackova and Lenka Foretova from Masaryk Memorial Cancer Institute for helpful comments and NGS gene analysis. Martina Svobodova has substantially contributed to the resolution of administrative issues of the treatments including insurance coverage. Funding This study was supported by projects No. 16-34083A and No. 16-33209A from the Ministry of Healthcare of the Czech Republic, by project No. LQ1605 from the National Program of Sustainability II (MEYS CR). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials The datasets and/or the analyzed current case report are available from the corresponding author upon reasonable request. Authors’ contributions PM performed the review of the literature and wrote the draft of the manuscript. OS and EM performed the DNA analysis of the PDGRFB gene. JN and RV designed and performed the phosphoproteomic analysis. JSo proposed to perform the NGS analysis and participated as clinical geneticist. KM took care of the patient and participated in the writing of the manuscript. OR took care of the patient and participated in the writing of the manuscript. MJ performed the histopathological analysis. AS performed the radiological evaluation and managed the MRI images. JSt proposed the study of molecular biology details of the case with a theranostic aim. All of the authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Written informed consent for the publication of their clinical details and/or clinical images was obtained from the parents of the patient. A copy of the consent form is available for review by the Editor of this journal. Ethics approval and consent to participate The study was approved by both the Ethics Committee of the University Hospital Brno on 9.6.2015 and the Ethics Committee of the School of Medicine Masaryk University on 23.6.2015, reference number 30/2015. All of the research described herein was conducted according to the Declaration of Helsinki. Written informed consent for the tissue and blood analysis and the off-label treatment of the child with the tyrosine kinase inhibitor was obtained from parents. Author details 1 Department of Pediatric Oncology, University Hospital Brno and School of Medicine, Masaryk University, Cernopolni 9, Brno 613 00, Czech Republic. 2 Central European Institute of Technology, Masaryk University, Kamenice 753/ 5, Brno 625 00, Czech Republic. 3 Laboratory of Tumor Biology, Department of Experimental Biology, School of Science, Masaryk University, Kotlarska 2, Brno 611 37, Czech Republic. 4 Division of Medical Genetics, Department of Biology, University Hospital Brno and School of Medicine, Masaryk University, Cernopolni 9, Brno 613 00, Czech Republic. 5 Department of Pathology, University Hospital Brno and School of Medicine, Masaryk University, Cernopolni 9, Brno 613 00, Czech Republic. 6 Department of Pediatric Radiology, University Hospital Brno and School of Medicine, Masaryk University, Cernopolni 9, Brno 613 00, Czech Republic. 7 International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, Brno 656 91, Czech Republic. Received: 2 August 2016 Accepted: 4 February 2017 References 1. Levine E, Fréneaux P, Schleiermacher G, Brisse H, Pannier S, Teissier N, et al. Risk-adapted therapy for infantile myofibromatosis in children. Pediatr Blood Cancer. 2012;59:115–20. 2. Johnson K, Notrica DM, Carpentieri D, Jaroszewski D, Henry MM. Successful treatment of recurrent pediatric inflammatory myofibroblastic tumor in a single patient with a novel chemotherapeutic regimen containing celecoxib. J Pediatr Hematol Oncol. 2013;35:414–6. 3. Auriti C, Kieran MW, Deb G, Devito R, Pasquini L, Danhaive O. Remission of infantile generalized myofibromatosis after interferon alpha therapy. J Pediatr Hematol Oncol. 2008;30:179–81. 4. Ferrari A, Alaggio R, Meazza C, Chiaravalli S, de Pava MV, Casanova M, et al. Fibroblastic tumors of intermediate malignancy in childhood. Expert Rev Anticancer Ther. 2013;13:225–36. 5. Butrynski JE, D’Adamo DR, Hornick JL, Dal Cin P, Antonescu CR, Jhanwar SC, et al. 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BMC Cancer (2017) 17:119 Page 7 of 7 International Journal of Molecular Sciences Article Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis Martin Sramek 1,2,3, Jakub Neradil 1,2,3, Petra Macigova 1,2 ID , Peter Mudry 2, Kristyna Polaskova 2,3, Ondrej Slaby 4, Hana Noskova 4, Jaroslav Sterba 2,3 and Renata Veselska 1,2,3,* 1 Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, 61137 Brno, Czech Republic; martin.sramek@mail.muni.cz (M.S.); jneradil@sci.muni.cz (J.N.); macigova@med.muni.cz (P.M.) 2 Department of Pediatric Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, 66263 Brno, Czech Republic; mudry.peter@fnbrno.cz (P.M.); polaskova.kristyna@fnbrno.cz (K.P.); sterba.jaroslav@fnbrno.cz (J.S.) 3 International Clinical Research Center, St. Anne’s University Hospital, 65691 Brno, Czech Republic 4 Central European Institute of Technology, Masaryk University, 62500 Brno, Czech Republic; ondrej.slaby@ceitec.muni.cz (O.S.); hana.noskova@ceitec.muni.cz (H.N.) * Correspondence: veselska@sci.muni.cz; Tel.: +420-549-49-7905 Received: 1 August 2018; Accepted: 29 August 2018; Published: 1 September 2018 Abstract: Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta. Keywords: infantile myofibromatosis; receptor tyrosine kinases; platelet-derived growth factor receptor; protein kinase inhibitors; sunitinib; erlotinib; FR180204; U0126; targeted therapy 1. Introduction Infantile myofibromatosis (IM; [MIM#228550]) is a disorder of mesenchymal proliferation characterized by the development of nonmetastatic tumors [1] that present as firm, flesh-colored to purple nodules usually located in the skin, subcutaneous tissues, bone, muscle or visceral organs [2,3]. This disease was described under different names, the name “infantile myofibromatosis” was first used in 1981 [4]. Although rare, with an incidence of 1 in 400,000 children, IM represents the most common proliferative fibrous tumor of infancy [5,6]. Myofibromas are usually present at birth or Int. J. Mol. Sci. 2018, 19, 2599; doi:10.3390/ijms19092599 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 2599 2 of 16 develop shortly thereafter, and almost 90% of the tumors are diagnosed before the age of two years, with a median age of three months [6–8]. A male predominance has been reported, and the ratio of male to female patients varies from 1.5:1 to 1.8:1 [5]. IM clinically presents in three main forms: (1) Solitary, (2) multicentric without visceral involvement, and (3) multicentric with visceral involvement [6]. The prognosis is excellent in solitary or multicentric nonvisceral forms with a possibility of spontaneous regression of the lesions but is poor when detected in the viscera [9]. Surgical excision of a single lesion is the standard of care [8]. Multiple lesions or surgically unresectable lesions are treated using various therapeutics, such as anti-inflammatory drugs, interferon-alpha, vinblastine, vincristine, dactinomycin, cyclophosphamide and methotrexate [6,8]. The molecular pathogenesis of IM is not completely understood. Familial forms exhibiting autosomal dominant and recessive transmission have been reported over the past two decades [10]. In 2013, several point mutations in the platelet-derived growth factor receptor beta (PDGFR-beta) gene (PDGFRB) were identified to be associated with familial IM. A study of nine unrelated families diagnosed with IM revealed two disease-causing mutations in PDGFRB: c.1978C>A (p.P660T) and c.1681C>T (p.R561C) [1]. Interestingly, one family did not have either of these PDGFRB mutations, but all affected individuals had a c.4556T>C (p.L1519P) mutation in NOTCH3. The germline mutation c.1681C>T (p.R561C) in PDGFRB was also detected in 11 individuals with familial IM [7]. In addition, one individual harbored a c.1998C>A (p.N666K) somatic mutation. Very recently, a novel PDGFRB mutation (c.1679C>T; p.P560L) was identified in a 3-generation family with multicentric IM [11]. Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) have important functions in the regulation of cell growth and survival [12]. The PDGF family consists of four structurally related single polypeptide units that constitute five functional homo- or heterodimers: PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD [13]. PDGFs act via two receptor tyrosine kinases (RTKs), PDGFR-alpha and PDGFR-beta [14]. Both receptors can activate many major signal transduction pathways, including the Ras/MAPK, PI3K/Akt and phospholipase C-gamma pathways [15]. Moreover, other genes were associated with IM etiology, which demonstrates the possible genetic heterogeneity of this disease. As mentioned above, a connection between a c.4556T>C (p.L1519P) mutation in NOTCH3 and IM was described in one study [1]. Human cells express four different Notch receptors, Notch 1–4, each encoded by a different gene [16]. The expression of PDGFRB can be regulated by Notch activity, as PDGFR-beta expression can be robustly upregulated by Notch 1 and Notch 3 signaling [17]. Another example is a c.511G>C (p.V171L) mutation in the potential tumor suppressor NDRG4 that was associated with IM in one case [18]. In the same year, it was demonstrated that the c.1276G>A (p.V426M) mutation in PTPRG (protein tyrosine phosphatase, receptor type G) was able to substantially influence the penetrance of a c.1681C>T (p.R561C) mutation in PDGFRB [19]. PTPRG encodes an enzyme that could dephosphorylate PDGFR-beta and thus reduce PDGFR-beta activity [19,20]. A recent work revealed that two IM-associated mutations in PDGFRB, c.1681C>T (p.R561C) and c.1998C>A (p.N666K), constitutively activate PDGFR-beta and can induce cancer development in vivo [21]. The same study showed that cells harboring p.R561C and p.N666K mutations are sensitive to specific tyrosine kinase inhibitors, which were able to decrease PDGFR-beta phosphorylation and downstream signaling. These results suggested that blocking PDGFR-beta activity would offer a therapeutic option for IM treatment. Indeed, in a recently published study, targeted treatment with sunitinib and low-dose vinblastine led to a robust response in a child with refractory multiple IM and a c.1681C>T (p.R561C) mutation in PDGFRB [8]. In this work, we demonstrate for the first time the efficacy of sunitinib, erlotinib, U0126 and FR180204 on the cell line harboring a c.1681C>T (p.R561C) PDGFRB mutation found in patients with IM. Sunitinib is known as an inhibitor of several kinases, including PDGFR-beta [22], erlotinib is an inhibitor of epidermal growth factor receptor (EGFR) [23], U0126 inhibits MEK1/2 phosphorylation [24], and FR180204 inhibits ERK1/2 phosphorylation. These inhibitors were chosen Int. J. Mol. Sci. 2018, 19, 2599 3 of 16 on the basis of our previous findings [8] as well as on the results of subsequent phosphoprotein profiling of the NSTS-47 cell line. 2. Results 2.1. Germline Mutations in PDGFRB Were Identified in Both Children, and the Same Mutation in PDGFRB Was Confirmed in NSTS-47 Cells Genetic analyses revealed that both siblings harbor a heterozygous germline c.1681C>T (p.R561C) mutation in the PDGFRB gene (Table 1). It was also confirmed that NSTS-47 cell line harbors the same heterozygous germline mutation c.1681C>T (p.R561C) in PDGFRB. Table 1. Germline mutations identified in patients. Gender Age PDGFRB Mutation Male 3.5 months c.1681C>T (p.R561C) Female 8 years c.1681C>T (p.R561C) 2.2. PDGFR-Beta, EGFR and ERK1/2 Kinases Are Highly Phosphorylated in Cells Harboring c.1681C>T (p.R561C) Mutation in PDGFRB Given that both siblings and NSTS-47 cells harbor the c.1681C>T (p.R561C) mutation in PDGFRB and that PDGFR-beta c.1681C>T (p.R561C) mutants are constitutively phosphorylated and can activate various signaling pathways [21], we assessed the phosphorylation level of 49 RTKs and 26 other signaling proteins in tumor samples as well as in NSTS-47 cells. NSTS-47 cells were harvested, and phosphorylation levels were analyzed after cultivation for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) without fetal calf serum (FCS) to eliminate the effects of various serum growth factors on the phosphorylation of the studied proteins. The screening of all 75 proteins showed that PDGFR-beta, EGFR (Figure 1) and ERK1/2 (Figure 2) kinases exhibited very high levels of phosphorylation in all samples. High levels of phosphorylation were also observed for ROR2, AXL (Figure 1), HSP27 and p38-gamma (Figure 2). These results confirmed that some kinases (namely, PDGFR-beta, EGFR and ERK1/2) were constitutively activated, as the high phosphorylation levels of these proteins were easily detectable in both tumor samples and in NSTS-47 cells after cultivation under serum-free conditions for 24 h. Int. J. Mol. Sci. 2018, 19, 2599 4 of 16 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 16 Figure 1. Phospho-receptor tyrosine kinases (RTK) array analysis. The relative phosphorylation of 49 RTKs was analyzed in tumor tissue obtained from the boy when he was 3.5 months old (Tumor sample 1), in the NSTS-47 cell line (derived from a tumor tissue of the boy obtained when he was 1 year and 7 months old) and in the tumor tissue of his 8-year-old sister (Tumor sample 2). platelet-derived growth factor receptor beta (PDGFR-beta) and epidermal growth factor receptor (EGFR) exhibited high levels of phosphorylation in all cases. Phosphorylation in NSTS-47 cells was measured after 24 h of serum-free cultivation. The array images captured using X-ray film are shown for each sample, and the five most phosphorylated receptor tyrosine kinases (RTKs) are marked. The upper part of the figure (Tumor sample 1) was already published in our previous case report [8] under the Creative Commons Attribution 4.0 International License. Figure 1. Phospho-receptor tyrosine kinases (RTK) array analysis. The relative phosphorylation of 49 RTKs was analyzed in tumor tissue obtained from the boy when he was 3.5 months old (Tumor sample 1), in the NSTS-47 cell line (derived from a tumor tissue of the boy obtained when he was 1 year and 7 months old) and in the tumor tissue of his 8-year-old sister (Tumor sample 2). platelet-derived growth factor receptor beta (PDGFR-beta) and epidermal growth factor receptor (EGFR) exhibited high levels of phosphorylation in all cases. Phosphorylation in NSTS-47 cells was measured after 24 h of serum-free cultivation. The array images captured using X-ray film are shown for each sample, and the five most phosphorylated receptor tyrosine kinases (RTKs) are marked. The upper part of the figure (Tumor sample 1) was already published in our previous case report [8] under the Creative Commons Attribution 4.0 International License. Int. J. Mol. Sci. 2018, 19, 2599 5 of 16 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 16 Figure 2. Phospho-mitogen-activated protein kinase (MAPK) array analysis. The relative phosphorylation of 26 signaling proteins, including 9 MAPKs, was detected in tumor tissue obtained from the boy when he was 3.5 months old (Tumor sample 1), in the NSTS-47 cell line (derived from a tumor tissue of the boy obtained when he was 1 year and 7 months old) and in the tumor tissue of his 8-year-old sister (Tumor sample 2). ERK1/2 exhibited high levels of phosphorylation in all cases. Figure 2. Phospho-mitogen-activated protein kinase (MAPK) array analysis. The relative phosphorylation of 26 signaling proteins, including 9 MAPKs, was detected in tumor tissue obtained from the boy when he was 3.5 months old (Tumor sample 1), in the NSTS-47 cell line (derived from a tumor tissue of the boy obtained when he was 1 year and 7 months old) and in the tumor tissue of his 8-year-old sister (Tumor sample 2). ERK1/2 exhibited high levels of phosphorylation in all cases. Phosphorylation levels in NSTS-47 cells was measured after 24 h of serum-free cultivation. The array images captured using X-ray film are shown for each sample, and the five most phosphorylated proteins are marked. Int. J. Mol. Sci. 2018, 19, 2599 6 of 16 2.3. NSTS-47 Cells Are Sensitive to Sunitinib and Erlotinib It was confirmed that cells with the mutation c.1681C>T (p.R561C) in PDGFRB are sensitive to the tyrosine kinase inhibitors imatinib, nilotinib and ponatinib [21]. Given the phosphorylation profile in the NSTS-47 cell line, whether specific tyrosine kinase inhibitors could affect the proliferation of this cell line was assessed. NSTS-47 cells were first treated with sunitinib. Sunitinib was chosen for several reasons: (1) The NSTS-47 cell line harbors a c.1681C>T (p.R561C) mutation in PDGFRB, and PDGFR-beta was substantially phosphorylated in these cells; (2) sunitinib treatment inhibits PDGFR-beta phosphorylation [25]; and (3) sunitinib was successfully used to treat the boy with IM whose tumor tissue was used to generate the NSTS-47 cell line [8]. Cells were treated for six days with various concentrations of sunitinib, and after incubation, the proliferative activity was determined using the MTT assay. At sunitinib concentrations of 50 and 100 nM, which can be achieved in the plasma of children treated with sunitinib [26], the proliferative activity of NSTS-47 cells was significantly decreased (Figure 3A). In addition, 50 nM and 100 nM sunitinib decreased the proliferative activity of NSTS-47 cells to 75% and 73%, respectively, after six days. To verify whether the observed effect of sunitinib is robust, NSTS-47 cells were cultivated with sunitinib in medium supplemented with PDGF-BB. A significant decrease in proliferative activity was observed after sunitinib treatment even when the cells grew in medium supplemented with PDGF-BB at a high concentration of 10 ng/mL (Figure 3B). In some experiments, the cultivation medium was changed every 24 h, and new medium with fresh inhibitor and fresh PDGF-BB was added (at medium changes) to prevent the potential degradation of sunitinib and PDGF-BB (Figure 3C). Int. J. Mol. Sci. 2018, 19, 2599 7 of 16 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 16 Figure 3. Proliferative activity of NSTS-47 cells after various experimental treatments. Proliferative activity was measured using an MTT assay after 6 days of incubation. The data represent the mean ± SD. Experiments were repeated three times in hexaplicate (A,D–H) or in triplicate (B,C). * p < 0.05 indicates a significant difference compared to control cells. (A) Sunitinib significantly decreased the proliferative activity of NSTS-47 cells. (B) NSTS-47 cells were sensitive to sunitinib, and this effect was not influenced by the presence of PDGF-BB at a high concentration (10 ng/mL). (C) Medium containing inhibitor and PDGF-BB was changed every 24 h during cultivation, which had no significant effect on the efficacy of the inhibitor. (D) NSTS-47 cells were also sensitive to erlotinib, as this inhibitor significantly affected cell proliferation. (E) No significant effect was observed after U0126 treatment. (F) FR180204 also did not significantly affect proliferative activity. (G) The combination of erlotinib and sunitinib significantly decreased the proliferative activity of NSTS-47 cells. (H) The combination of U0126 and FR180204 did not have a significant effect on NSTS-47 cell proliferation. Figure 3. Proliferative activity of NSTS-47 cells after various experimental treatments. Proliferative activity was measured using an MTT assay after 6 days of incubation. The data represent the mean ± SD. Experiments were repeated three times in hexaplicate (A,D–H) or in triplicate (B,C). * p < 0.05 indicates a significant difference compared to control cells. (A) Sunitinib significantly decreased the proliferative activity of NSTS-47 cells. (B) NSTS-47 cells were sensitive to sunitinib, and this effect was not influenced by the presence of PDGF-BB at a high concentration (10 ng/mL). (C) Medium containing inhibitor and PDGF-BB was changed every 24 h during cultivation, which had no significant effect on the efficacy of the inhibitor. (D) NSTS-47 cells were also sensitive to erlotinib, as this inhibitor significantly affected cell proliferation. (E) No significant effect was observed after U0126 treatment. (F) FR180204 also did not significantly affect proliferative activity. (G) The combination of erlotinib and sunitinib significantly decreased the proliferative activity of NSTS-47 cells. (H) The combination of U0126 and FR180204 did not have a significant effect on NSTS-47 cell proliferation. Int. J. Mol. Sci. 2018, 19, 2599 8 of 16 Next, NSTS-47 cells were treated with erlotinib, U0126 and FR180204. These three inhibitors were chosen based on EGFR and ERK1/2 phosphorylation in NSTS-47 cells (Figures 1 and 2). The ability of the combination of sunitinib and erlotinib to block both highly phosphorylated RTKs was tested, and a combination of U0126 and FR180204 was used to block the MEK/ERK signaling pathway. At an erlotinib concentration of 1 µM, which can be achieved in the plasma of children treated with erlotinib [27], the proliferative activity of the NSTS-47 cell line was significantly decreased to 75% after 6 days of cultivation (Figure 3D). In contrast, NSTS-47 cells were not sensitive to U0126 and FR180204 because treatment of the NSTS-47 cell line with these inhibitors did not induce a significant decrease in proliferative activity (Figure 3E,F). The combination of erlotinib and sunitinib also significantly decreased the proliferative activity of NSTS-47 cells (Figure 3G), but the effect of this combined treatment was similar to the effects of sunitinib or erlotinib alone. For instance, 100 nM sunitinib and 100 nM erlotinib decreased the proliferative activity to 70% (Figure 3G), but 100 nM sunitinib alone decreased the proliferative activity to 73% (Figure 3A). Another example is the combination of 1 µM erlotinib and 1 µM sunitinib; this treatment decreased the proliferative activity to 61% (Figure 3G), but 1 µM erlotinib alone decreased the proliferative activity to 75% (Figure 3D), and 1 µM sunitinib decreased the proliferative activity to 76% after six days (Figure 3A). Therefore, the combination of sunitinib and erlotinib did not have a significant additional effect on the reduction of NSTS-47 cell proliferation. In addition, the combination of U0126 and FR180204 did not show any significant effect on proliferative activity (Figure 3H). Taken together, our results demonstrate that sunitinib and erlotinib can significantly decrease the proliferative activity of NSTS-47 cells, which harbor a c.1681C>T (p.R561C) mutation in PDGFRB, at concentrations that are achievable for these inhibitors in children plasma. However, the combination of sunitinib and erlotinib did not show an additional significant effect on cell proliferation. The inhibitors FR180204 and U0126 also did not have a significant effect on NSTS-47 cell proliferation. 2.4. PDGFR-Beta and EGFR Exhibited Ligand-Dependent Tyrosine Phosphorylation Considering that only some kinase inhibitors significantly decreased the proliferative activity of the NSTS-47 cell line, detailed analyses of target kinases that should be affected by previously used inhibitors were performed using Western blotting. First, it was observed that the constitutively phosphorylated receptors PDGFR-beta and EGFR in NSTS-47 cells can respond to their ligands: Our results show that phosphorylation of both receptors was considerably increased in response to PDGF-BB or EGF (Figure 4A,B). Cell populations were serum starved for 24 h and then stimulated for 15, 30 or 60 min using two different concentrations of PDGF-BB or EGF. The cells that were serum starved for only 24 h and cells that were cultivated with FCS were used as negative controls. Receptor phosphorylation was significantly increased after 15 min, and then decreased in a time-dependent manner. Surprisingly, serum-starved cells that were not stimulated with PDGF-BB or EGF also exhibited an increase in receptor phosphorylation, in comparison to serum-cultivated cells. These experiments demonstrated that both receptors were functional and were able to activate downstream signaling molecules. Int. J. Mol. Sci. 2018, 19, 2599 9 of 16 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 16 Figure 4. Analysis of protein phosphorylation. (A) PDGFR-beta phosphorylation is increased in response to PDGF-BB. Cells were stimulated for 15, 30 or 60 min using two different concentrations (10 ng/mL and 30 ng/mL) of PDGF-BB. (B) EGFR phosphorylation is increased in response to epidermal growth factor (EGF). Cells were stimulated for 15, 30 or 60 min using two different concentrations (40 ng/mL and 100 ng/mL) of EGF. (C) Sunitinib was able to decrease PDGFR-beta and Akt phosphorylation but not MEK1/2 and ERK1/2 phosphorylation. (D) Erlotinib decreased EGFR and Akt phosphorylation but had no effect on MEK1/2 and ERK1/2 phosphorylation. (E) U0126 treatment did not decrease MEK1/2 phosphorylation. (F) FR180204 treatment did not cause any changes in ERK1/2 phosphorylation. (G) The combination of sunitinib and erlotinib decreased PDGFR-beta, EGFR and Akt phosphorylation, but MEK1/2 and ERK1/2 phosphorylation was not affected. 2.5. Detailed Analysis of Signaling Pathways Revealed Constitutive Phosphorylation of MEK1/2 and ERK1/2 Proteins In the next step, we analyzed the phosphorylation of PDGFR-beta, EGFR and downstream kinases, which can be activated by these RTKs after treatment with kinase inhibitors. In all experiments, cells were cultivated for 24 h in medium containing an inhibitor but not FCS. After 24 h, some cells were stimulated with PDGF-BB or/and EGF for 15 min to observe the effects of inhibitors on ligand-stimulated cells. Cells that were serum starved for only 24 h, and cells that were cultivated with FCS were used as negative controls. Sunitinib alone decreased the phosphorylation of PDGFR-beta (Figure 4C). Akt phosphorylation was also decreased after sunitinib treatment, but a substantial decrease in MEK1/2 and ERK1/2 phosphorylation was not observed. Erlotinib decreased the phosphorylation of EGFR, but only at higher concentrations, and Akt phosphorylation was also slightly decreased (Figure 4D). No effect of erlotinib on MEK1/2 and ERK1/2 phosphorylation was observed. Surprisingly, U0126 did not decrease the phosphorylation of MEK1/2 (Figure 4E). Similarly, FR180204 treatment had no effect on Figure 4. Analysis of protein phosphorylation. (A) PDGFR-beta phosphorylation is increased in response to PDGF-BB. Cells were stimulated for 15, 30 or 60 min using two different concentrations (10 ng/mL and 30 ng/mL) of PDGF-BB. (B) EGFR phosphorylation is increased in response to epidermal growth factor (EGF). Cells were stimulated for 15, 30 or 60 min using two different concentrations (40 ng/mL and 100 ng/mL) of EGF. (C) Sunitinib was able to decrease PDGFR-beta and Akt phosphorylation but not MEK1/2 and ERK1/2 phosphorylation. (D) Erlotinib decreased EGFR and Akt phosphorylation but had no effect on MEK1/2 and ERK1/2 phosphorylation. (E) U0126 treatment did not decrease MEK1/2 phosphorylation. (F) FR180204 treatment did not cause any changes in ERK1/2 phosphorylation. (G) The combination of sunitinib and erlotinib decreased PDGFR-beta, EGFR and Akt phosphorylation, but MEK1/2 and ERK1/2 phosphorylation was not affected. 2.5. Detailed Analysis of Signaling Pathways Revealed Constitutive Phosphorylation of MEK1/2 and ERK1/2 Proteins In the next step, we analyzed the phosphorylation of PDGFR-beta, EGFR and downstream kinases, which can be activated by these RTKs after treatment with kinase inhibitors. In all experiments, cells were cultivated for 24 h in medium containing an inhibitor but not FCS. After 24 h, some cells were stimulated with PDGF-BB or/and EGF for 15 min to observe the effects of inhibitors on ligand-stimulated cells. Cells that were serum starved for only 24 h, and cells that were cultivated with FCS were used as negative controls. Sunitinib alone decreased the phosphorylation of PDGFR-beta (Figure 4C). Akt phosphorylation was also decreased after sunitinib treatment, but a substantial decrease in MEK1/2 and ERK1/2 phosphorylation was not observed. Erlotinib decreased the phosphorylation of EGFR, but only at higher concentrations, and Akt phosphorylation was also slightly decreased (Figure 4D). No effect of erlotinib on MEK1/2 and ERK1/2 phosphorylation was observed. Surprisingly, U0126 did not decrease the phosphorylation of MEK1/2 (Figure 4E). Similarly, FR180204 treatment had no effect on ERK1/2 phosphorylation (Figure 4F). As expected, the combination of sunitinib and erlotinib markedly decreased the phosphorylation of PDGFR-beta, EGFR and Akt, but no effect was observed on MEK1/2 and ERK1/2 phosphorylation (Figure 4G). Int. J. Mol. Sci. 2018, 19, 2599 10 of 16 Altogether, sunitinib and erlotinib showed inhibitory effects on RTKs and Akt. Interestingly, no substantial changes in MEK1/2 and ERK1/2 phosphorylation were observed after treatment with any inhibitor. 2.6. Serum Starvation of NSTS-47 Cells Induces an Increase in PDGFA Expression In some cases, our data indicated higher phosphorylation of PDGFR-beta and EGFR in serum-starved cells than in cells cultivated in DMEM supplemented with FCS (Figure 4A,B). Therefore, the expression of selected EGFR and PDGFR-beta ligands was measured to investigate whether there is a possible autocrine PDGF/PDGFR or EGF (TGF-alpha)/EGFR signaling loop that could contribute to the higher phosphorylation of RTKs. Expression of EGF, PDGFA, PDGFB and TGFA was analyzed under normal serum conditions (DMEM supplemented with 20% FCS) and under serum starvation conditions using qPCR. Substantial differences were observed in the transcriptional response of serum-starved cells (Figure 5). qPCR analyses also showed increased levels of PDGFA expression, while EGF and PDGFB mRNA levels were not significantly influenced by serum starvation, and TGFA expression was considerably decreased. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 16 ERK1/2 phosphorylation (Figure 4F). As expected, the combination of sunitinib and erlotinib markedly decreased the phosphorylation of PDGFR-beta, EGFR and Akt, but no effect was observed on MEK1/2 and ERK1/2 phosphorylation (Figure 4G). Altogether, sunitinib and erlotinib showed inhibitory effects on RTKs and Akt. Interestingly, no substantial changes in MEK1/2 and ERK1/2 phosphorylation were observed after treatment with any inhibitor. 2.6. Serum Starvation of NSTS-47 Cells Induces an Increase in PDGFA Expression In some cases, our data indicated higher phosphorylation of PDGFR-beta and EGFR in serum-starved cells than in cells cultivated in DMEM supplemented with FCS (Figure 4A,B). Therefore, the expression of selected EGFR and PDGFR-beta ligands was measured to investigate whether there is a possible autocrine PDGF/PDGFR or EGF (TGF-alpha)/EGFR signaling loop that could contribute to the higher phosphorylation of RTKs. Expression of EGF, PDGFA, PDGFB and TGFA was analyzed under normal serum conditions (DMEM supplemented with 20% FCS) and under serum starvation conditions using qPCR. Substantial differences were observed in the transcriptional response of serum-starved cells (Figure 5). qPCR analyses also showed increased levels of PDGFA expression, while EGF and PDGFB mRNA levels were not significantly influenced by serum starvation, and TGFA expression was considerably decreased. Figure 5. Effect of serum starvation on EGF, PDGFA, PDGFB and TGFA expression in the NSTS-47 cell line. Cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% FCS or in DMEM without FCS. After 24 h, cells were harvested, and the expression of selected genes was analyzed using qPCR. The results represent the mean ± SD of nine (six in case of PDGFB) independent experiments. * p < 0.05 indicates statistically significant differences. 3. Discussion IM is a rare disorder of mesenchymal proliferation that is characterized by the development of nonmetastatic tumors [1]. Several studies have confirmed that specific point mutations in the PDGFRB gene are involved in the pathogenesis of IM [1,7,11]. However, mutations in the PDGFRB gene presumably show incomplete penetrance and variable expressivity, and other genes may be involved in the pathogenesis of IM [1,18,19]. The main goal of this study was to analyze the effects of various protein kinase inhibitors (PKIs) on the NSTS-47 cell line, which harbors the IM-associated c.1681C>T (p.R561C) mutation in PDGFRB. The results showed that sunitinib, a potent inhibitor of PDGFR-beta phosphorylation, can significantly decrease the proliferation of NSTS-47 cells. Previously published results [21] show that PDGFR-beta p.R561C mutant cells have constitutively phosphorylated PDGFR-beta and are able to induce the phosphorylation of ERK1/2, PLC-gamma, STAT3, STAT5 and Akt in the absence of PDGF. These results are in accordance with our observations. We found that PDGFR-beta and ERK1/2 kinases were highly phosphorylated in Figure 5. Effect of serum starvation on EGF, PDGFA, PDGFB and TGFA expression in the NSTS-47 cell line. Cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% FCS or in DMEM without FCS. After 24 h, cells were harvested, and the expression of selected genes was analyzed using qPCR. The results represent the mean ± SD of nine (six in case of PDGFB) independent experiments. * p < 0.05 indicates statistically significant differences. 3. Discussion IM is a rare disorder of mesenchymal proliferation that is characterized by the development of nonmetastatic tumors [1]. Several studies have confirmed that specific point mutations in the PDGFRB gene are involved in the pathogenesis of IM [1,7,11]. However, mutations in the PDGFRB gene presumably show incomplete penetrance and variable expressivity, and other genes may be involved in the pathogenesis of IM [1,18,19]. The main goal of this study was to analyze the effects of various protein kinase inhibitors (PKIs) on the NSTS-47 cell line, which harbors the IM-associated c.1681C>T (p.R561C) mutation in PDGFRB. The results showed that sunitinib, a potent inhibitor of PDGFR-beta phosphorylation, can significantly decrease the proliferation of NSTS-47 cells. Previously published results [21] show that PDGFR-beta p.R561C mutant cells have constitutively phosphorylated PDGFR-beta and are able to induce the phosphorylation of ERK1/2, PLC-gamma, STAT3, STAT5 and Akt in the absence of PDGF. These results are in accordance with our observations. We found that PDGFR-beta and ERK1/2 kinases were highly phosphorylated in both s and even in NSTS-47 cells that were serum starved for 24 h. We also detected increased phosphorylation of Akt2 in Tumor Sample 1. The same study that revealed a role for the p.R561C mutation in PDGFR-beta [21] showed that imatinib, nilotinib, and ponatinib can decrease PDGFR-beta phosphorylation and inhibit cell Int. J. Mol. Sci. 2018, 19, 2599 11 of 16 proliferation. We studied the effects of sunitinib, a multi-tyrosine kinase inhibitor that is able to target PDGFR-beta. Sunitinib was chosen because siblings from whom tumor tissue samples were obtained responded very well to treatment with this inhibitor [8]. Sunitinib alone significantly decreased the proliferative activity of the NSTS-47 cell line, and this finding could explain the response of the siblings to the targeted therapy. Western blot analyses showed that sunitinib is able to decrease the phosphorylation of mutant PDGFR-beta even in the presence of high PDGF-BB levels and can also decrease the phosphorylation of Akt. Because activated Akt is a well-established survival factor [28], these effects of sunitinib on PDGFR-beta and Akt phosphorylation can explain why sunitinib reduced the proliferative activity of NSTS-47 cells. A similar inhibitory effect was observed for EGFR and erlotinib (the inhibitor of EGFR phosphorylation). Erlotinib also decreased NSTS-47 cell proliferation, and Western blot analysis showed that it was able to decrease EGFR and Akt phosphorylation. However, neither sunitinib nor erlotinib inhibited the phosphorylation of the corresponding receptor completely, and some receptor molecules remained phosphorylated even when high doses of those inhibitors were used. Surprisingly, phosphorylation of MEK1/2 and ERK1/2 proteins was not significantly influenced by any inhibitor. This observation could explain why sunitinib and erlotinib incompletely decreased proliferative activity and why U0126 and FR180204 did not influence proliferative activity. MEK1/2 and ERK1/2 belong to the Ras/MAPK signaling cascade, which transmits signals from receptors and participate in regulating the cell cycle, apoptosis and differentiation [29]. All tyrosine kinase inhibitors have been previously shown to be able to simultaneously decrease PDGFR-beta and ERK1/2 phosphorylation, which resulted in the inhibition of proliferative activity [21]. In NSTS-47 cells, sunitinib and erlotinib decreased the phosphorylation of PDGFR-beta, EGFR and Akt, but for yet unknown reasons, MEK1/2 and ERK1/2 kinases remained phosphorylated at levels that were comparable with those detected in untreated cells. Interestingly, incomplete penetrance of the c.1681C>T (p.R561C) mutation was found in a family with two children suffering from IM [19]. Genetic analyses revealed a c.1681C>T (p.R561C) mutation in PDGFRB in both siblings and, surprisingly, also in their healthy mother. However, both siblings had inherited a heterozygous c.1276G>A (p.V426M) mutation in PTPRG from their healthy father. The PTPRG gene encodes a protein called receptor-type tyrosine-protein phosphatase gamma that can dephosphorylate PDGFR-beta [19,20]. Therefore, the mutation in PTPRG could probably decrease the efficiency of the phosphatase to dephosphorylate its substrates and thus positively influence the phosphorylation of PDGFR-beta and the penetrance of mutant PDGFRB [19]. Finally, our analyses of gene expression showed that the phosphorylation status of PDGFRs in NSTS-47 cells was not influenced by only mutations in PDGFR-beta. We analyzed the gene expression levels of EGF, PDGFA, PDGFB and TGFA in NSTS-47 cells that were serum starved for 24 h. The expression of TGFA decreased, but no difference was observed in the expression of EGF and PDGFB; however, PDGFA gene expression was significantly increased. The increase in PDGFA expression was unexpected and could result in the stimulation of cells via an autocrine mechanism, an increase in PDGFR-alpha phosphorylation and improved survival of NSTS-47 cells in the absence of serum. 4. Materials and Methods 4.1. Tumor Samples Two tumor samples and one tumor-derived cell line were used in this study. Tumor Sample 1 was obtained from a 3.5-month-old infant boy suffering from inborn generalized IM, and Tumor Sample 2 was obtained from his 8-year-old sister who was suffering from a skull base tumor and had a history of spontaneous regression of subcutaneous lesions. The Research Ethics Committee of the School of Medicine (Masaryk University, Brno, Czech Republic) approved the study protocol, and written informed consent was obtained from legal guardians of the siblings. A case report concerning these siblings was published recently [8]. Int. J. Mol. Sci. 2018, 19, 2599 12 of 16 4.2. Cell Line and Cell Culture The NSTS-47 cell line was established in our laboratory with the procedure previously described [30]. A tumor sample was obtained from the same boy mentioned in the previous paragraph during curative surgical procedure when he was 1 year and 7 months old. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% fetal calf serum (FCS), 2 mM glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin (all purchased from GE Healthcare Europe GmbH, Freiburg, Germany). The cell line was maintained under standard conditions at 37 ◦C in a humidified atmosphere containing 5% CO2 and subcultured one or two times per week. Cells from passage number 8 to 19 were used for experiments. 4.3. Genetic Analyses The mutation in PDGFRB was identified by Sanger sequencing using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and confirmed by whole exome sequencing (WES). In all cases, WES was performed using the TruSeq Exome Kit, NextSeq® 500/550 Mid Output Kit v2 and NextSeq 500 (all Illumina, San Diego, CA, USA). 4.4. Chemicals Sunitinib, erlotinib, U0126 (all purchased from Cell Signaling Technology, Danvers, MA, USA) and FR180204 (Sigma-Aldrich, St. Louis, MO, USA) were prepared as a 20 mM stock solution in dimethyl sulfoxide (DMSO) and stored at −20 ◦C. PDGF-BB (Cell Signaling Technology) was prepared at a concentration of 100 µg/mL in 20 mM citric acid (pH 3.0) supplemented with 0.8% BSA (bovine serum albumin) and stored at 4 ◦C. EGF (Sigma-Aldrich) was prepared at a concentration of 100 µg/mL in 10 mM HCl and stored at 4 ◦C. For the determination of proliferative activity, concentrations of protein kinase inhibitors (PKIs) ranging from 0.001 to 10 µM and PDGF-BB concentrations of 0.25 and 10 ng/mL were tested. For Western blot analyses, PKI concentrations ranging from 0.05 to 10 µM, PDGF-BB concentrations of 10 and 30 ng/mL and EGF concentrations of 40 and 100 ng/mL were used. 4.5. Phospho-RTK and Phospho-MAPK Array Analysis The relative phosphorylation levels of 49 RTKs were analyzed using the Human Phospho-RTK Array kit (R&D Systems, Minneapolis, MN, USA), and the relative phosphorylation levels of 26 proteins, including 9 MAPKs, were determined using the Human Phospho-MAPK Array kit (R&D Systems) according to the manufacturer’s protocol. The levels of phosphorylation were quantified using ImageJ software [31] and normalized to control spots and the background. The analysis was performed as described in previous studies [8,32]. 4.6. MTT Assay The MTT assay was used to determine the proliferative activity of the NSTS-47 cell line. A total of 103 cells were seeded in 200 µL of culture medium into each well of 96-well microplates, and cells were allowed to adhere overnight. The next day, the medium was carefully removed, and fresh medium containing various concentrations of chemicals described above or control medium was added. The microplates were incubated under standard conditions. To evaluate changes in cell proliferation, the medium was removed and replaced with 200 µL of fresh DMEM containing 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at a concentration of 0.5 mg per mL. The microplates were then incubated at 37 ◦C for 3.5 h. The medium was carefully removed, and the formazan crystals were dissolved in 200 µL of DMSO. The absorbance was measured at 570 nm using a Sunrise Absorbance Reader (Tecan, Männedorf, Switzerland), with a reference absorbance at 620 nm. Int. J. Mol. Sci. 2018, 19, 2599 13 of 16 4.7. Western Blotting and Immunodetection Whole-cell extracts were loaded onto 10% polyacrylamide gels, electrophoresed, and blotted on polyvinylidene difluoride membranes (Bio-Rad Laboratories, Munich, Germany). The membranes were blocked with 5% nonfat dry milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 and incubated overnight with the corresponding primary antibody. The primary and secondary antibodies used in this study are shown in Table 2. Membranes were incubated with corresponding secondary antibodies for 1 h. ECL-Plus detection was performed according to the manufacturer’s instructions (GE Healthcare, Little Chalfont, UK). Table 2. Primary and secondary antibodies. Primary Antibodies Antigen Manufacturer Catalog No. Dilution Beta-actin Sigma-Aldrich A5441 1:20,000 Akt (pan) Cell Signaling Technology 4691 1:1000 Phospho-Akt (Ser473) Cell Signaling Technology 4060 1:2000 ERK1/2 Cell Signaling Technology 4695 1:1000 Phospho-ERK1/2 (Thr202/Tyr204) Cell Signaling Technology 4370 1:2000 MEK1/2 Cell Signaling Technology 9122 1:1000 Phospho-MEK1/2 (Ser217/221) Cell Signaling Technology 9121 1:1000 EGFR Cell Signaling Technology 2646 1:1000 Phospho-EGFR (Tyr1068) Cell Signaling Technology 2236 1:1000 PDGFR-beta Cell Signaling Technology 3169 1:1000 Phospho-PDGFR-beta (Tyr751) Cell Signaling Technology 4549 1:1000 Secondary antibodies Specificity Conjugate Manufacturer Catalog No. Dilution Anti-Mouse IgG horseradish peroxidase Cell Signaling Technology 7076 1:2000–1:20,000 Anti-Rabbit IgG horseradish peroxidase Cell Signaling Technology 7074 1:2000 4.8. RT-qPCR The relative expression levels of selected genes were studied using RT-qPCR. Total RNA was extracted using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich), and RNA concentration and purity were determined spectrophotometrically. For all samples, equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase (Top-Bio, Prague, Czech Republic). RT-qPCR was carried out in 10 µL reaction volumes using the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and analyzed using the 7500 Fast Real-Time PCR System and 7500 Software v. 2.0.6 (both Life Technologies, Carlsbad, CA, USA). Changes in the transcript levels were determined using the 2−∆∆CT method [33]. The housekeeping gene HSP90AB1 was used as an endogenous reference control. The primers used in this study are listed in Table 3. Table 3. Primers. Gene Gene Symbol Primer Sequence Epidermal growth factor EGF F: 5′-AGGATTGACACAGAAGGAACCAA-3′ R: 5′-ACATACTCTCTCTTGCCTTGACC-3′ Heat shock protein 90 alpha family class B member 1 HSP90AB1 F: 5′-CGCATGAAGGAGACACAGAA-3′ R: 5′-TCCCATCAAATTCCTTGAGC-3′ Platelet derived growth factor subunit A PDGFA F: 5′-TCCGTAGGGAGTGAGGATTCTTT-3′ R: 5′-GGCTTCTTCCTGACGTATTCCA-3′ Platelet derived growth factor subunit B PDGFB F: 5′-GATCCGCTCCTTTGATGATCTCC-3′ R: 5′-ATCTCGATCTTTCTCACCTGGAC-3′ Transforming growth factor alpha TGFA F: 5′-TGCCACTCAGAAACAGTGGTC-3′ R: 5′-AGTCCGTCTCTTTGCAGTTCTT-3′ F, forward primer; R, reverse primer. Int. J. Mol. Sci. 2018, 19, 2599 14 of 16 4.9. Statistical Analysis Quantitative data are shown as the mean ± standard deviation (SD). Data from MTT assays were analyzed using one-way ANOVA followed by Dunnett’s test; p < 0.05 was considered statistically significant. The qPCR data were analyzed using the Mann-Whitney test (two-tailed); p < 0.05 was considered statistically significant. 5. Conclusions To conclude, our work demonstrated that tumor cells with the c.1681C>T (p.R561C) mutation in PDGFRB show high levels of PDGFR-beta and ERK1/2 phosphorylation. Furthermore, our data support the use of specific tyrosine kinase inhibitors targeting PDGFR-beta phosphorylation as a treatment suitable for IM. This is the first study to show that sunitinib is able to reduce the proliferative activity of IM cells with a c.1681C>T (p.R561C) mutation in vitro. Author Contributions: J.N., R.V. and J.S. designed the study. J.S., P.M. (Peter Mudry) and K.P. provided tumor samples and relevant clinical data. H.N. and O.S. performed genetic analyses. M.S., P.M. (Petra Macigova) and J.N. designed and performed experiments with NSTS-47 cell line. M.S. and R.V. composed the manuscript. All authors reviewed and approved the final version of the manuscript. Funding: This study was supported by projects No. 16-34083A and No. 16-33209A from the Ministry of Healthcare of the Czech Republic and by project No. LQ1605 from the National Program of Sustainability II (MEYS CR). Acknowledgments: The authors thank Johana Maresova for her technical assistance and dr. Petr Chlapek for the derivation of the NSTS-47 cell line. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations DMEM Dulbecco’s modified Eagle’s medium DMSO dimethyl sulfoxide EGFR epidermal growth factor receptor ERK extracellular signal-regulated kinase FCS fetal calf serum IM infantile myofibromatosis MAPK mitogen-activated protein kinase MEK MAPK/ERK kinase MTT 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PDGFR platelet-derived growth factor receptor PKIs protein kinase inhibitors RTKs receptor tyrosine kinases TGFA transforming growth factor alpha WES whole exome sequencing References 1. Martignetti, J.A.; Tian, L.; Li, D.; Ramirez, M.C.; Camacho-Vanegas, O.; Camacho, S.C.; Guo, Y.; Zand, D.J.; Bernstein, A.M.; Masur, S.K.; et al. Mutations in PDGFRB cause autosomal-dominant infantile myofibromatosis. Am. J. Hum. Genet. 2013, 92, 1001–1007. [CrossRef] [PubMed] 2. Levine, E.; Fréneaux, P.; Schleiermacher, G.; Brisse, H.; Pannier, S.; Teissier, N.; Mesples, B.; Orbach, D. Risk-adapted therapy for infantile myofibromatosis in children. Pediatr. Blood Cancer 2012, 59, 115–120. [CrossRef] [PubMed] 3. Kim, E.J.; Wang, K.C.; Lee, J.Y.; Phi, J.H.; Park, S.H.; Cheon, J.E.; Jang, Y.E.; Kim, S.K. Congenital solitary infantile myofibromatosis involving the spinal cord. J. Neurosurg. Pediatr. 2013, 11, 82–86. [CrossRef] [PubMed] 4. Venkatesh, V.; Kumar, B.P.; Kumar, K.A.; Mohan, A.P. Myofibroma-a rare entity with unique clinical presentation. J. Maxillofac. Oral Surg. 2015, 14, 64–68. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 2599 15 of 16 5. Hausbrandt, P.A.; Leithner, A.; Beham, A.; Bodo, K.; Raith, J.; Windhager, R. A rare case of infantile myofibromatosis and review of literature. J. Pediatr. Orthop. B 2010, 19, 122–126. [CrossRef] [PubMed] 6. Weaver, M.S.; Navid, F.; Huppmann, A.; Meany, H.; Angiolillo, A. Vincristine and Dactinomycin in Infantile Myofibromatosis with a Review of Treatment Options. J. Pediatr. Hematol. Oncol. 2015, 37, 237–241. [CrossRef] [PubMed] 7. Cheung, Y.H.; Gayden, T.; Campeau, P.M.; LeDuc, C.A.; Russo, D.; Nguyen, V.H.; Guo, J.; Qi, M.; Guan, Y.; Albrecht, S.; et al. A recurrent PDGFRB mutation causes familial infantile myofibromatosis. Am. J. Hum. Genet. 2013, 92, 996–1000. [CrossRef] [PubMed] 8. Mudry, P.; Slaby, O.; Neradil, J.; Soukalova, J.; Melicharkova, K.; Rohleder, O.; Jezova, M.; Seehofnerova, A.; Michu, E.; Veselska, R.; et al. Case report: Rapid and durable response to PDGFR targeted therapy in a child with refractory multiple infantile myofibromatosis and a heterozygous germline mutation of the PDGFRB gene. BMC Cancer 2017, 17, 119. [CrossRef] [PubMed] 9. Gatibelza, M.E.; Vazquez, B.R.; Bereni, N.; Denis, D.; Bardot, J.; Degardin, N. Isolated infantile myofibromatosis of the upper eyelid: Uncommon localization and long-term results after surgical management. J. Pediatr. Surg. 2012, 47, 1457–1459. [CrossRef] [PubMed] 10. Murray, N.; Hanna, B.; Graf, N.; Fu, H.; Mylene, V.; Campeau, P.M.; Ronan, A. The spectrum of infantile myofibromatosis includes both non-penetrance and adult recurrence. Eur. J. Med. Genet. 2017, 60, 353–358. [CrossRef] [PubMed] 11. Lepelletier, C.; Al-Sarraj, Y.; Bodemer, C.; Shaath, H.; Fraitag, S.; Kambouris, M.; Hamel-Teillac, D.; Shanti, H.E.; Hadj-Rabia, S. Heterozygous PDGFRB Mutation in a Three-generation Family with Autosomal Dominant Infantile Myofibromatosis. Acta Derm. Venereol. 2017, 97, 858–859. [CrossRef] [PubMed] 12. Heldin, C.H. Targeting the PDGF signaling pathway in tumor treatment. Cell. Commun. Signal. 2013, 11, 97. [CrossRef] [PubMed] 13. Cao, Y. Multifarious functions of PDGFs and PDGFRs in tumor growth and metastasis. Trends Mol. Med. 2013, 19, 460–473. [CrossRef] [PubMed] 14. Andrae, J.; Gallini, R.; Betsholtz, C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 2008, 22, 1276–1312. [CrossRef] [PubMed] 15. Hoch, R.V.; Soriano, P. Roles of PDGF in animal development. Development 2003, 130, 4769–4784. [CrossRef] [PubMed] 16. Aster, J.C.; Pear, W.S.; Blacklow, S.C. The Varied Roles of Notch in Cancer. Annu. Rev. Pathol 2017, 12, 245–275. [CrossRef] [PubMed] 17. Jin, S.; Hansson, E.M.; Tikka, S.; Lanner, F.; Sahlgren, C.; Farnebo, F.; Baumann, M.; Kalimo, H.; Lendahl, U. Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells. Circ. 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PDGFRB mutants found in patients with familial infantile myofibromatosis or overgrowth syndrome are oncogenic and sensitive to imatinib. Oncogene 2016, 35, 3239–3248. [CrossRef] [PubMed] 22. Faivre, S.; Demetri, G.; Sargent, W.; Raymond, E. Molecular basis for sunitinib efficacy and future clinical development. Nat. Rev. Drug Discov. 2007, 6, 734–745. [CrossRef] [PubMed] 23. Sos, M.L.; Koker, M.; Weir, B.A.; Heynck, S.; Rabinovsky, R.; Zander, T.; Seeger, J.M.; Weiss, J.; Fischer, F.; Frommolt, P.; et al. PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res. 2009, 69, 3256–3261. [CrossRef] [PubMed] 24. Yap, J.L.; Worlikar, S.; MacKerell, A.D.; Shapiro, P.; Fletcher, S. Small-molecule inhibitors of the ERK signaling pathway: Towards novel anticancer therapeutics. ChemMedChem 2011, 6, 38–48. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 2599 16 of 16 25. Abouantoun, T.J.; Castellino, R.C.; MacDonald, T.J. Sunitinib induces PTEN expression and inhibits PDGFR signaling and migration of medulloblastoma cells. J. Neurooncol. 2011, 101, 215–226. [CrossRef] [PubMed] 26. Wetmore, C.; Daryani, V.M.; Billups, C.A.; Boyett, J.M.; Leary, S.; Tanos, R.; Goldsmith, K.C.; Stewart, C.F.; Blaney, S.M.; Gajjar, A. Phase II evaluation of sunitinib in the treatment of recurrent or refractory high-grade glioma or ependymoma in children: A children’s Oncology Group Study ACNS1021. Cancer Med. 2016, 5, 1416–1424. [CrossRef] [PubMed] 27. Jakacki, R.I.; Hamilton, M.; Gilbertson, R.J.; Blaney, S.M.; Tersak, J.; Krailo, M.D.; Ingle, A.M.; Voss, S.D.; Dancey, J.E.; Adamson, P.C. Pediatric phase I and pharmacokinetic study of erlotinib followed by the combination of erlotinib and temozolomide: A Children’s Oncology Group Phase I Consortium Study. J. Clin. Oncol. 2008, 26, 4921–4927. [CrossRef] [PubMed] 28. Altomare, D.A.; Testa, J.R. Perturbations of the AKT signaling pathway in human cancer. 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Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 2008, 3, 1101–1108. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Downloadedfromhttps://journals.lww.com/pidjbyBhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78=on12/18/2020 Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. The Pediatric Infectious Disease Journal  •  Volume 36, Number 1, January 2017 www.pidj.com | e1 ORIGINAL STUDIES Background: Data on safety and efficacy of voriconazole for invasive aspergillosis (IA) and invasive candidiasis/esophageal candidiasis (IC/EC) in pediatric patients are limited. Methods: Patients aged 2–<18 years with IA and IC/EC were enrolled in 2 prospective open-label, non-comparative studies of voriconazole. Patients followed dosing regimens based on age, weight and indication, with adjustments permitted. Treatment duration was 6–12 weeks for IA patients, ≥14 days after last positive Candida culture for IC patients and ≥7 days after signs/symptoms resolution for EC patients. Primary analysis for both the studies was safety and tolerability of voriconazole. Secondary end points included global response success at week 6 and end of treatment (EOT), all-causality mortality and time to death. Voriconazole exposure–response relationship was explored. Results: Of 53 voriconazole-treated pediatric patients (31 IA; 22 IC/EC), 14 had proven/probable IA, 7 had confirmed IC and 10 had confirmed EC. Treatment-related hepatic and visual adverse events, respectively, were reported in 22.6% and 16.1% of IA patients, and 22.7% and 27.3% of IC/EC patients.All-causality mortality in IA patients was 14.3% at week 6; no deaths were attributed to voriconazole. No deaths were reported for IC/EC patients. Global response success rate was 64.3% (week 6 and EOT) in IA patients and 76.5% (EOT) in IC/EC patients. There was no association between voriconazole exposure and efficacy; however, a slight positive association between voriconazole exposure and hepatic adverse events was established. Conclusions: Safety and efficacy outcomes in pediatric patients with IA and IC/EC were consistent with previous findings in adult patients. Key Words: voriconazole, aspergillosis, candidiasis, pediatric, exposure– response (Pediatr Infect Dis J 2017;36:e1–e13) Aspergillus and Candida species are the predominant causes of invasive fungal infection in pediatric patients.1 The incidence of invasive fungal infection has increased substantially in recent years, largely because of the increasing number of children at risk of acquiring these infections.1 Invasive aspergillosis (IA) is observed in children with compromised phagocytic function,2–4 as well as in patients with hematologic malignancies and specific immunosuppression, and recipients of allogeneic stem cell and solid organ transplants.4 Although the lungs are the most common infection site,4 the central nervous system, cardiovascular system and other tissues may be infected because of hematogenous dissemination in severely immunocompromised patients.5 Invasive candidiasis (IC) may present as catheter-associated candidemia, single-organ candidiasis or disseminated candidiasis, with or without candidemia.6 Risk factors for IC include intensive care unit admission, neutropenia, malignant diseases7 and congenital immunologic deficiencies.8 Voriconazole is a broad-spectrum triazole with activity against a wide range of yeasts and filamentous fungi. It is a substrate and inhibitor of the cytochrome P450 (CYP) isoenzymes CYP2C19, CYP2C9 and CYP3A4. Voriconazole exhibits nonlinear pharmacokinetics because of saturation of its metabolism; inter-individual variability in exposure is high.9–11 In healthy adults, it has been demonstrated that CYP2C19 genotyping status, gender and age are key factors, which contribute to this vari- ability.12 In healthy adults, poor metabolizers of CYP2C19 have, on average, approximately 2–4-fold higher voriconazole levels than their homozygous extensive metabolizers and heterozygous extensive metabolizers counterparts, respectively, independent of ethnicity.12,13 However, exposure of voriconazole varies widely within each genotype and overlaps considerably across genotypes.12 Therefore, no dose adjustment based on CYP2C19 genotyping status is warranted in the current product label for voriconazole. Although efficacy has been demonstrated in adults with IA, IC and esophageal candidiasis (EC),14–16 published data on voriconazole use in children are limited.10,11,17–20 Given the potentially life-threatening nature of invasive fungal infection, data on efficacy, safety and dosing of voriconazole in children will be of value to the medical community. Here, we evaluated safety, efficacy and exposure–response of voriconazole for the treatment of IA, IC and EC in pediatric patients using the recently revised dosing regimens in 2 prospective, open-label, noncomparative studies. MATERIALS AND METHODS Study A1501080 evaluated pediatric patients with IA (vori-IA study; NCT00836875), whereas Study A1501085 evaluated pediatric patients with IC/EC (vori-IC/EC study; NCT01092832). Both studies were conducted in compliance with the Declaration of Helsinki and International Conference on Harmonisation Good Clinical Practice Guidelines and were approved by the appropriate individual Institutional Review Boards for each study site. Investigators obtained written, informed consent from legally acceptable representatives and patient assent, where applicable. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0891-3668/17/3601-00e1 DOI: 10.1097/INF.0000000000001339 Safety, Efficacy, and Exposure–Response of Voriconazole  in Pediatric Patients With Invasive Aspergillosis, Invasive  Candidiasis or Esophageal Candidiasis Judith M. Martin, MD,* Mercedes Macias-Parra, MD,† Peter Mudry, MD,‡ Umberto Conte, PharmD,§ Jean L.Yan, MS,¶ Ping Liu, PhD,|| M. Rita Capparella, PharmD,** and Jalal A. Aram, MD†† Accepted for publication July 8, 2016. From the *Division of General Academic Pediatrics, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania; †Pediatric Infectious Diseases Department, Instituto Nacional de Pediatria, Mexico City, Mexico; ‡Department of Pediatric Oncology, University Children’s Hospital Brno, Brno, Czech Republic; §Antifungal Clinical Development, Pfizer Inc., NewYork, NewYork; ¶Global Established Pharma—Biostatistics, Pfizer Inc., New York, New York; ||Department of Clinical Pharmacology, Pfizer Inc., Groton, Connecticut; **Global Established Pharma, Pfizer PFE, Paris, France; and ††Global Medical Affairs, Pfizer Inc., Groton, Connecticut. J.M.M. has received research support from Genocea Biosciences. M.M.-P. has received travel support from Pfizer Inc. U.C., J.L.Y., P.L., M.R.C., and J.A.A. are employees of Pfizer Inc. Study A1501080 and Study A1501085 were sponsored by Pfizer Inc. Address for correspondence: Judith M. Martin, MD, Division of General Academic Pediatrics, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, 3414 Fifth Avenue, Floor 3, Pittsburgh, PA 15213. E-mail: judy. martin@chp.edu. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Martin et al The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 e2  |  www.pidj.com  © 2016 Wolters Kluwer Health, Inc. All rights reserved. Study Design and Treatment The vori-IA and vori-IC/EC studies were prospective, openlabel, noncomparative phase 3 studies. The vori-IA study was conducted at 16 centers (Asia, Europe and North America) from 2009 to 2013; the vori-IC/EC study was conducted at 11 centers (Asia, Europe, North America) from 2010 to 2013. In both studies, patients followed recently revised dosing regimens based on age, weight and indication (Table 1). These initial dosing regimens were based on an integrated population pharmacokinetic analysis of voriconazole data from children, adolescents and adults.21 Patients initiated treatment with intravenous (IV) voriconazole and continued IV therapy until clinical improvement was observed. Treatment for IA and IC started with loading doses of 9mg/kg every 12 hours (q12h) for the first 24 hours for children (aged 2–<12 years) and young adolescents (aged 12–14 years, weighing <50kg), followed by maintenance doses of 8mg/kg q12h. For all other adolescents (aged 12–<18 years, excluding 12–14-year olds weighing <50kg), the loading doses were 6mg/kg q12h for the first 24 hours followed by maintenance doses of 4mg/kg q12h. Children with EC did not receive loading doses of IV voriconazole. Dosage for children (aged 2–<12 years) and young adolescents (aged 12–14 years, weighing <50kg) began with 4mg/kg q12h. Dosage for all other adolescents (aged 12–<18 years, excluding 12–14 yearolds weighing <50kg) began with 3mg/kg q12h. Patients could switch to oral voriconazole after 1 week (voriIA) or 5 days (vori-IC/EC) of IV therapy. In the vori-IA study, patients received voriconazole for ≥6 weeks, up to a maximum of 12 weeks.A minimum treatment duration of 6 weeks was chosen based on recent clinical observations that this duration is sufficient to evaluate clinical efficacy in patients receiving therapy for IA.22 Duration of treatment was based on clinical improvement and improvement in radiologic findings. In the vori-IC/EC study, patients received voriconazole for ≥14 days after the last positive Candida culture from a normally sterile site (for IC) or ≥7 days after the resolution of clinical signs/symptoms (for EC), up to a maximum of 42 days. Patients had to return for the 1-month follow-up visit after end of treatment (EOT). Dose Adjustments Dose adjustments were made based on clinical response, tolerability or voriconazole plasma trough concentrations (Cmin ; collected before dosing on third day or later of IV therapy or after switching to oral therapy, as well as after each dose adjustment).Although no definitive relationship between voriconazole exposure and response has been established, provisional cut-off values of Cmin were used to inform dose adjustment. It is of note that children have less accumulation in response to a given dose of voriconazole than adults because of their faster metabolism of voriconazole; as detailed in an earlier analysis, to achieve the same total exposure [ie, area under the curve from 0 to 12h (AUC0–12 )], the corresponding Cmin in children is expected to be lower than that in adults.20 Therefore, the minimum of target voriconazole Cmin in children in this study was lower than that used in adults. For all treatments, the dose could be reduced by 1mg/kg steps (or 50mg steps if 350mg oral dose was used) if it exceeded 6 μg/mL. If Cmin was too high (eg, >10 μg/mL), the investigator could reduce the dose by >1mg/kg or 50mg, as needed and temporary discontinuation of dosing (eg, 24-hour washout) was allowed to avoid further accumulation of voriconazole in the body. For IA and IC treatment, the dose could be increased in 1mg/kg steps if Cmin was <0.5 μg/mL during IV therapy or increased in 1mg/kg or 50mg steps if Cmin was <0.2 μg/mL during step-down oral therapy. For EC treatment, the dose could be increased in 1mg/kg or 50mg steps if Cmin was <0.2 μg/mL during IV or oral therapy. Close monitoring of adverse events (AEs) was implemented when the dose was increased. To make voriconazole concentration data available to the investigators within 72 hours of receiving samples, trough plasma samples (approximately 1mL) were analyzed at designated reference laboratories or locally. CYP2C19 Genotyping Buccal swab samples were collected for CYP2C19 genotyping and analyzed at Pfizer Pharmacogenomics Laboratory (Groton, CT) using a published method.20 Patients Inclusion Criteria In the vori-IA study, eligible patients were aged 2–<18 years, immunocompromised with a clinically compatible illness and had proven, probable or possible IA based on European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions.23 Patients enrolled with possible IA were assessed again to determine whether they had proven or probable IA based on tests done within 7 days of the first dose of study drug. Patients with rare molds (eg, Scedosporium or Fusarium species) were also eligible. In the vori-IC/EC study, eligible patients were aged 2–<18 years with confirmed IC/EC. Invasive candidiasis diagnosis was based on growth of Candida species or mycologic evidence indicative TABLE 1. Initial Voriconazole Dosing Scheme by Age, Weight and Indication Loading Dose Maintenance Dose IV IV Switched to Oral Voriconazole Children (aged 2–<12 yr) and young adolescents (aged 12–14 yr weighing <50kg) IA/IC 9mg/kg q12h for first 24 h 8mg/kg q12 h 9mg/kg q12h (maximum dose 350mg) EC – 4mg/kg q12 h 9mg/kg q12h (maximum dose 350mg) Adolescents (aged 12–<18 yr) excluding those aged 12–14 yr weighing <50 kg IA/IC 6mg/kg q12h for first 24 h 4mg/kg q12 h 200mg q12 h* EC – 3mg/kg q12 h 200mg q12 h *At the investigator’s discretion, an oral dose of 300mg q12h may be used in adolescents with IA. EC indicates esophageal candidiasis; IA, invasive aspergillosis; IC, invasive candidiasis; IV, intravenous; q12h, every 12 hours. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 Voriconazole Use in Pediatric Patients © 2016 Wolters Kluwer Health, Inc. All rights reserved.  www.pidj.com  |  e3 of Candida species and later confirmed as Candida species from a specimen obtained from a sterile site within 7 days (primary therapy) or 14 days (salvage therapy) of first voriconazole dose. Patients with clinical and/or radiologic findings consistent with disseminated disease and a positive Candida culture from a normally sterile site within previous 2 weeks of diagnosis were also eligible. Esophageal candidiasis diagnosis was based on the presence of clinical symptoms/lesions consistent with EC, or positive microscopy and/or mycologic culture for Candida species from brush/tissue biopsy of esophageal lesions within 7 days of enrollment. Exclusion Criteria The vori-IA study excluded patients with sarcoidosis, aspergilloma, allergic bronchopulmonary aspergillosis or chronic IA with the duration of symptoms or radiologic findings for >4 weeks before entry. Patients who received previous treatment or prophylaxis with systemic agents against Aspergillus species or systemic antifungal treatment for the current episode of IA or rare molds for >96 hours were also excluded. Patients were excluded from the vori-IC/EC study (for primary therapy) if they required treatment with another systemic antifungal agent or had >48 hours of antifungal therapy before first voriconazole dose. Safety In both studies, the primary end point was safety and tolerability of voriconazole, as determined by the rate ofAEs and treatment discontinuations because of AEs. Adverse events were monitored by the study investigators from screening until the 1-month follow-up visit after EOT and were recorded and coded using the Medical Dictionary for RegulatoryActivities (MedDRA, v16.0). Investigators assessed the causality of allAEs.An investigator’s causality assessment was the determination of whether there was a reasonable possibility that the study drug caused or contributed to theAE.A serious adverse event (SAE) was defined as any untoward medical occurrence at any dose that resulted in death, was lifethreatening (immediate risk of death), required inpatient hospitalization or prolonged hospitalization, resulted in significant or permanent disability/incapacity (substantial disruption of the ability to perform normal life functions) or resulted in congenital abnormality/birth defect. Visual assessments were performed at baseline, and at weeks 1, 2, 4, 6 and 12 or EOT. In children aged ≥3 years, visual symptoms were assessedprimarilybyavisualquestionnaire,bytheHardy–Rand–Rittler colorvisiontestandbyacuityandfixationindicesoftheEarlyTreatment Diabetic Retinopathy Study chart, used at the investigator’s discretion. Patients with treatment-emergent visual AEs underwent ophthalmic fundoscopy, and all findings were recorded. Children aged <3 years had visual fixation assessed by the investigators. Liver function tests were monitored weekly up to week 6, at week 12 and at the 1-month followup visit. All clinically significant hepatic and other laboratory abnormalities were reported as AEs or SAEs, as appropriate. Potential Hy’s Law cases were reported as SAEs and were defined as patients who had aspartate aminotransferase (AST) or alanine aminotransferase (ALT) and total bilirubin baseline values within the normal range who, following treatment, presented withAST orALT 3 × the upper limit of normal (ULN) concurrent with a total bilirubin 2 × ULN with no evidence of hemolysis and alkaline phosphatase 2 × ULN. Alternatively, if patients with pre-existing ALT, AST, or total bilirubin values above the ULN, then presented with AST or ALT 2 × baseline values and 3 × ULN or 8 × ULN (whichever was smaller) or total bilirubin increased by 1 × ULN or ≥3 × ULN (whichever was smaller), this was also considered a serious hepaticAE. Efficacy Efficacy assessments were secondary end points and included global response (success rate) at week 6 (vori-IA) and EOT (vori-IA and vori-IC/EC), all-causality mortality, and time to death. In the vori-IA study, successful global response was defined as clinical resolution or improvement of signs/symptoms plus complete/partial resolution of radiologic findings. In the vori-IC/EC study, successful global response was defined as clinical cure/improvement plus confirmed/presumed microbiologic eradication. Exposure–Response Analyses Voriconazole concentration data from 48 patients (96 observations) were analyzed using a nonlinear mixed-effects model using NONMEM system (version 7.2). Individual exposure parameters [area under the curve from 0 to 12 hours (AUC0–12 ) and Cmin ] were estimated based on the final pharmacokinetic model. Relationships between voriconazole exposures and key safety (hepatic, visual, psychiatric, skin and subcutaneous tissue AEs) and efficacy (global response at EOT) end points were assessed by graphical examination or using a logistic regression model (NONMEM). The model selection was based on goodness-of-fit criteria, which included basic diagnostic plots, precision of parameter estimates and the objective function value. The graphic processing of the data and NONMEM output was performed with R (version 2.12.2). As there could be multiple AE observations per patient, both single-panel (without counting the frequency ofAE occurrence in each patient) and multiple-panel (includes all AE observations) analysis approaches were utilized for hepatic and visual AE analysis, including both all-causality and treatment-related events. As there were only a few psychiatric disorders and skin and soft tissue disorders reported, a simple descriptive check was performed for these 2 types of AEs. The following hepatic AE terms were included in the analysis: ALT increased or abnormal, AST increased or abnormal, γ-glutamyl transferase increased or abnormal, bilirubin increased, hyperbilirubinemia, transaminases increased, liver function tests abnormal, gallbladder disorder, hepatosplenomegaly, jaundice cholestatic, liver disorder and drug-induced liver injury. The following visual AE terms were included in the analysis: abnormal sensation in the eye, asthenopia, chromatopsia, diplopia, photophobia, visual impairment, vision blurred and visual acuity reduced. There were 5 all-causality psychiatric disorders: insomnia (n = 2), depression, affect lability, and intentional self-injury.There were 6 treatment-related skin and subcutaneous tissue disorders: dermatitis exfoliative, rash maculopapular, skin burning sensation, skin lesion and rash (n = 2). Statistical Analyses The safety population comprised all patients who received ≥1 voriconazole dose. The modified intent-to-treat population for efficacy evaluation comprised all patients with proven/probable IA, microbiologically confirmed IC, presumed EC (patients with neutropenia, thrombocytopenia or advanced HIV/AIDS concurrent with oral candidiasis) or microbiologically confirmed EC who received ≥1 voriconazole dose. Safety and efficacy data for both the studies were descriptive in nature; thus, no statistical testing was performed. RESULTS Patient Disposition and Baseline Demographics Vori-IA Study Thirty-one patients received voriconazole, of whom 16 completed the treatment and 25 completed the study (ie, returned for 1-month follow-up visit; patients who did not return for the 1-month follow-up visit were considered to have discontinued the study; Fig. 1). Patient demographics are presented in Table 2. Most patients (82.8%) had a recent history of neutropenia, and 17.2% were recipients of hematopoietic stem cell transplants (allogeneic: 13.8%, autologous: 3.4%). Median (range) duration of IV treatment Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Martin et al The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 e4  |  www.pidj.com  © 2016 Wolters Kluwer Health, Inc. All rights reserved. FIGURE 1. Patient disposition. a All patients who received ≥1 voriconazole dose. b All patients with proven/probable IA,  microbiologically confirmed IC, presumed EC or microbiologically confirmed EC who received ≥1 voriconazole dose.  c Patients who discontinued study treatment for any reason were not considered to have completed treatment. AE indicates  adverse event; EC, esophageal candidiasis; IA, invasive aspergillosis; IC, invasive candidiasis; MITT, modified intent-to-treat. TABLE 2. Patient Demographic Characteristics in the Vori-IA and Vori-IC/EC Studies (Safety Population) Vori-IA Study Vori-IC/EC Study 2–<12 Yr (n = 11) 12–<18 Yr (n = 20) Overall (n = 31) 2–<12 Yr (n = 14) 12–<18 Yr (n = 8) Overall (n = 22) Age in yr, mean (SD) 7.9 (2.3) 14.1 (1.7) 11.9 (3.5) 6.8 (2.9) 14.4 (1.7) 9.5 (4.5) Sex, n (%) Female 4 (36.4) 11 (55.0) 15 (48.4) 8 (57.1) 6 (75.0) 14 (63.6) Male 7 (63.6) 9 (45.0) 16 (51.6) 6 (42.9) 2 (25.0) 8 (36.4) Race, n (%) White 3 (27.3) 8 (40.0) 11 (35.5) 5 (35.7) 5 (62.5) 10 (45.5) Black - 1 (5.0) 1 (3.2) - - Asian 8 (72.7) 10 (50.0) 18 (58.1) 5 (35.7) 1 (12.5) 6 (27.3) Other - 1 (5.0) 1 (3.2) 4 (28.6) 2 (25.0) 6 (27.3) Weight in kg, mean (SD) 26.7 (9.7) 50.1 (15.3) 41.7 (17.6) 23.9 (11.1) 54.6 (19.7) 35.1 (20.8) Host factors for IA, n (%) Recent history of neutropenia 9 (81.8) 15 (83.3)* 24 (82.8)† - - Hematopoietic stem cell transplant 2 (18.2) 3 (16.7)* 5 (17.2)† - - Allogeneic 2 (18.2) 2 (11.1)* 4 (13.8)† - - Autologous - 1 (5.6)* 1 (3.4)† - - Risk factors for IC/EC, n (%) Broad-spectrum antibiotics - - - 12 (85.7) 7 (87.5) 19 (86.4) Chemotherapy - - - 12 (85.7) 7 (87.5) 19 (86.4) Neutropenia - - - 10 (71.4) 8 (100.0) 18 (81.8) Central venous catheter - - - 11 (78.6) 6 (75.0) 17 (77.3) Duration of IV treatment in days, median (range) 8.0 (3–33) 8.5 (5–22) 8.0 (3–33) 6.5 (2–24) 8.0 (5–17) 7.0 (2–24) Duration of oral treatment in days, median (range) 55.0 (2–78) 59.5 (8–81) 59.5 (2–81) 15.0 (3–37) 5.0 (2–8) 9.0 (2–37) Duration of total treatment in days, median (range) 37.0 (3–85) 43.5 (5–90) 41.0 (3–90) 16.5 (2–42) 14.0 (6–17) 14.0 (2–42) *n = 18; host factor case report form pages were not completed for 2 patients. †n = 29; host factor case report form pages were not completed for 2 patients. EC indicates esophageal candidiasis; IA, invasive aspergillosis; IC, invasive candidiasis; IV, intravenous; SD, standard deviation. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 Voriconazole Use in Pediatric Patients © 2016 Wolters Kluwer Health, Inc. All rights reserved.  www.pidj.com  |  e5 (n = 31), oral treatment (n = 22) and total treatment was 8.0 (3–33) days, 59.5 (2–81) days and 41.0 (3–90) days, respectively. Eleven patients (35.5%) required dose reduction, and 3 patients (9.7%) had a dose increase. Of 31 enrolled patients, 14 were diagnosed with proven/ probable IA and 17 were diagnosed with possible IA. Baseline characteristics for those patients with proven/probable IA are presented in Table 3. All patients had a blood and lymphatic system disorder. Metabolism and nutrition disorders, neoplasms, gastrointestinal disorders and infections were common.The lungs were a site of infection in all patients. All identified pathogens were Aspergillus species. Vori-IC/EC Study Twenty-two patients received voriconazole, of whom 13 completed the treatment and 21 completed the study. Patient demographics are presented in Table 2. Most patients had a recent history of broad-spectrum antibiotic therapy (86.4%), chemotherapy (86.4%), neutropenia (81.8%) and central venous catheter use (77.3%). Median (range) duration of IV treatment (n = 22), oral treatment (n = 13) and total treatment was 7.0 (2–24) days, 9.0 (2–37) days and 14.0 (2–42) days, respectively. Three patients (13.6%) required dose reduction, and 3 patients (13.6%) had a dose increase. TABLE 3. Patient Baseline Characteristics in the Vori-IA Study (MITT Population) Vori-IA Study 2–<12 Yr (n = 5) 12–<18 Yr (n = 9) Overall (n = 14) Most common (occurring in ≥5 patients) medical conditions by SOC, n (%) Blood and lymphatic system disorders 5 (100.0) 9 (100.0) 14 (100.0) Metabolism and nutrition disorders 3 (60.0) 7 (77.8) 10 (71.4) Neoplasms (benign, malignant, and unspecified) 3 (60.0) 7 (77.8) 10 (71.4) Gastrointestinal disorders 3 (60.0) 6 (66.7) 9 (64.3) Infections 4 (80.0) 5 (55.6) 9 (64.3) General disorders and administration site conditions 1 (20.0) 6 (66.7) 7 (50.0) Renal and urinary disorders 1 (20.0) 4 (44.4) 5 (35.7) EORTC criteria for IA, n (%) Proven 2 (40.0) 6 (66.7) 8 (57.1) Probable 3 (60.0) 3 (33.3) 6 (42.9) Host factors, n (%) Recent history of neutropenia 3 (60.0) 7 (87.5)* 10 (76.9)† Hematopoietic stem cell transplant 1 (20.0) 2 (25.0)* 3 (23.1)† Autologous 0 (0.0) 1 (12.5)* 1 (7.7)† Allogeneic 1 (20.0) 1 (12.5)* 2 (15.4)† Myeloablative 1 (20.0) 2 (25.0)* 3 (23.1)† Corticosteroid therapy 1 (20.0) 1 (12.5)* 2 (15.4)† Other T-cell immunosuppressants 0 2 (25.0)* 2 (15.4)† Site of infection, n (%)‡ Lung 5 (100.0) 9 (100.0) 14 (100.0) Sinus - 2 (22.2) 2 (14.3) Other 2 (40.0) - 2 (14.3) Baseline pathogen, n (%)§ Aspergillus species (unidentified) 3 (60.0) 7 (77.8) 10 (71.4) Aspergillus flavus - 1 (11.1) 1 (7.1) Aspergillus fumigates - 2 (22.2) 2 (14.3) *n = 8. In 1 patient, the host factor case report form was not completed as the patient’s medical condition (suspected congenital cystic adenomatoid malformation) was not prespecified; the patient was included in the efficacy (MITT) population based on recent lung lobectomy, lung tissue biopsy positive for Aspergillus species, positive serum galactomannan and pleural effusion. †n = 13. ‡Patients could have multiple sites at baseline. §Four patients did not have Aspergillus species isolated but were included in the efficacy (MITT) population based on the following: 3 patients had a positive serum galactomannan, 1 patient had sputum gram-stain sample positive for hyphae. EORTC indicates European Organisation for Research and Treatment of Cancer; IA, invasive aspergillosis; MITT, modified intent-to-treat; SOC, system organ class. TABLE 4. Patient Baseline Characteristics in the Vori-IC/EC Study (MITT Population) Vori-IC/EC Study 2–<12 Yr (n = 9) 12–<18 Yr (n = 8) Overall (n = 17) Most common (occurring in ≥5 patients) medical conditions by SOC, n (%) Neoplasms (benign, malignant and unspecified) 8 (88.9) 7 (87.5) 15 (88.2) Blood and lymphatic system disorders 6 (66.7) 8 (100.0) 14 (82.4) Infections 6 (66.7) 6 (75.0) 12 (70.6) Metabolism and nutrition disorders 5 (55.6) 5 (62.5) 10 (58.8) Gastrointestinal disorders 3 (33.3) 4 (20.0) 7 (41.2) General disorders and administration site conditions 3 (33.3) 3 (37.5) 6 (35.3) Nervous system disorders 1 (11.1) 5 (62.5) 6 (35.3) Psychiatric disorders - 5 (62.5) 5 (29.4) Respiratory, thoracic, and mediastinal disorders 1 (11.1) 4 (20.0) 5 (29.4) Fungal diagnosis, n (%) IC 7 (77.8) - 7 (41.2) Primary therapy 5 (55.6) - 5 (29.4) Salvage therapy 2 (22.2) - 2 (11.8) EC 2 (22.2) 8 (100.0) 10 (58.8) Primary therapy 2 (22.2) 6 (75.0) 8 (47.1) Salvage therapy - 2 (25.0) 2 (11.8) Candida risk factors, n (%) Chemotherapy 7 (77.8) 7 (87.5) 14 (82.4) Use of broad-spectrum antibiotics 7 (77.8) 7 (87.5) 14 (82.4) Neutropenia 5 (55.6) 8 (100.0) 13 (76.5) Use of central venous catheter 7 (77.8) 6 (75.0) 13 (76.5) Clinical sepsis 5 (55.6) 5 (62.5) 10 (58.8) Immunosuppressive therapy 5 (55.6) 5 (62.5) 10 (58.8) Mucosal colonization 5 (55.6) 5 (62.5) 10 (58.8) Use of systemic corticosteroids/ other immunosuppressive drugs 6 (66.7) 3 (37.5) 9 (52.9) Multifocal colonization 2 (22.2) 3 (37.5) 5 (29.4) Total parenteral nutrition 3 (33.3) 2 (25.0) 5 (2.4) Length of ICU stay >4 d, n (%) 2 (22.2) 2 (25.0) 4 (23.5) Surgery 3 (33.3) - 3 (17.6) Abdominal surgery 2 (22.2) - 2 (11.8) Other 1 (11.1) - 1 (5.9) Site of infection, n (%)* Esophagus 2 (22.2) 8 (100.0) 10 (58.8) Oropharynx 3 (33.3) 5 (62.5) 8 (47.1) Blood 7 (77.8) - 7 (41.2) Catheter 2 (22.2) - 2 (11.8) Left eye 1 (11.1) - 1 (5.9) Lung 1 (11.1) - 1 (5.9) Right eye 1 (11.1) - 1 (5.9) Skin (unspecified) 1 (11.1) - 1 (5.9) Baseline pathogen, n (%)† Candida albicans 4 (44.4) 8 (100.0) 12 (70.6) Candida tropicalis 3 (33.3) - 3 (17.6) Candida glabrata 1 (11.1) - 1 (5.9) Candida parapsilosis 1 (11.1) - 1 (5.9) *Patients could have multiple sites at baseline. †Patients could have multiple organisms at baseline. EC indicates esophageal candidiasis; IC, invasive candidiasis; ICU, intensive care unit; MITT, modified intent-to-treat; SOC, system organ class. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Martin et al The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 e6  |  www.pidj.com  © 2016 Wolters Kluwer Health, Inc. All rights reserved. Of 22 enrolled patients, 7 had confirmed IC and 10 had confirmed EC (the remaining 5 enrolled patients lacked microbiologic confirmation of Candida), with baseline characteristics presented in Table 4. The most common medical conditions were neoplasms, blood and lymphatic system disorders, infections and metabolism and nutrition disorders. The esophagus, oropharynx and blood were the most common sites of infection, and infection was related to central venous catheter use in 2 patients (data not shown). Most patients had infection caused by Candida albicans. Safety Vori-IA Study A safety summary is presented in Table 5. Sixteen of 31 patients experienced 35 treatment-related AEs, most commonly blurred vision (n = 3) and photophobia, increased ALT, abnormal liver function test and transaminases increased (n = 2 each). Most treatment-related AEs were mild or moderate in severity. Treatmentrelated hepatic AEs were experienced by 7 patients (22.6%), and except for 1 patient with severe drug-induced liver injury (discussed below), all were mild or moderate in severity. Treatment-related visual AEs were reported by 5 patients (16.1%) and were mild in severity. Four patients (12.9%) reported treatment-related skin AEs [exfoliative dermatitis (n = 1), maculopapular rash (n = 1), skin burning sensation (n = 1) and skin lesion (n = 1)], which were all mild in severity, and only 1 patient reported any psychiatric treatment-related AE (insomnia; data not shown). Serious adverse events were experienced by 15 of 31 patients. Two SAEs were considered treatment related. Specifically, an 11-year-old girl experienced acute renal failure on day 34. The patient also received concomitant treatment with other medications, including ganciclovir (days 7–36) and vancomycin (days 28–29), while receiving treatment with the study drug. On day 32, the patient switched from IV to oral voriconazole and continued treatment for an additional 5 days. The patient died on day 38 due to sepsis. A case of drug-induced liver injury leading to discontinuation was reported on day 40 in a 14-year-old boy; this patient’s underlying medical conditions at baseline included acute lymphocytic leukemia relapse, febrile neutropenia, herpes zoster oticus, hyperbilirubinemia, hypocalcemia, hypokalemia, hypomagnesemia, mucosal inflammation, pancytopenia, pneumonia, renal tubular disorder, rhinitis, sinusitis and thrombophlebitis. On day 40, the patient was hospitalized TABLE 5. Summary of Safety Data From the Vori-IA Study Vori-IA Study 2–<12 Yr (n = 11) 12–<18 Yr (n = 20) Overall* (n = 31) All- Causality Treatment- Related All- Causality Treatment- Related All- Causality Treatment- Related AEs, n 86 7 195 28 281 35 Patients with AEs, n (%) 11 (100.0) 5 (45.5) 19 (95.0) 11 (55.0) 30 (96.8) 16 (51.6) Hepatic AEs, n (%) - - 8 (40.0) 7 (63.6) 8 (25.8) 7 (22.6) ALT increased - - 2 (10.0) 2 (10.0) 2 (6.5) 2 (6.5) Liver function test abnormal - - 2 (10.0) 2 (10.0) 2 (6.5) 2 (6.5) Transaminases increased - - 2 (10.0) 2 (10.0) 2 (6.5) 2 (6.5) AST increased - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Blood bilirubin increased - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Drug-induced liver injury - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Jaundice cholestatic - - 1 (5.0) - 1 (3.2) Visual AEs, n (%) 3 (27.3) 1 (9.1) 6 (30.0) 4 (20.0) 9 (29.0) 5 (16.1) Vision blurred - - 3 (15.0) 3 (15.0) 3 (9.7) 3 (9.7) Visual impairment - - 2 (5.0) 1 (5.0) 2 (6.5) 1 (3.2) Photophobia 1 (9.1) 1 (9.1) 1 (5.0) 1 (5.0) 2 (6.5) 2 (6.5) Conjunctivitis - - 2 (10.0) - 2 (6.5) Abnormal sensation in the eye - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Asthenopia - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Chromatopsia - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Diplopia - - 1 (5.0) 1 (5.0) 1 (3.2) 1 (3.2) Cataract - - 1 (5.0) - 1 (3.2) Conjunctival hemorrhages 1 (9.1) - - - 1 (3.2) Dry eye 1 (9.1) - - - 1 (3.2) Eye discharge 1 (9.1) - - - 1 (3.2) Eye irritation - - 1 (5.0) - 1 (3.2) Eye pain - - 1 (5.0) - 1 (3.2) SAEs, n (%) 6 (54.5) 1 (9.1) 9 (45.0) 1 (5.0) 15 (48.4) 2 (6.5) Treatment discontinuation, n (%) 6 (54.5) 1 (9.1) 9 (45.0) - 15 (48.4) 1 (3.2) AEs 1 (9.1) - - - 1 (3.2) Insufficient clinical response 1 (9.1) 1 (9.1) - - 1 (3.2) 1 (3.2) No longer willing to participate - - 1 (5.0) - 1 (3.2) Patient died 3 (27.3) - 2 (10.0) - 5 (16.1) Other 1 (9.1)† - 6 (30.0)‡ - 7 (22.6) Study discontinuation, n (%) 3 (27.3) - 3 (15.0) - 6 (19.4) Patient died 3 (27.3) - 2 (10.0) - 5 (16.1) No longer willing to participate - - 1 (5.0) - 1 (3.2) *All patients received at least 1 dose of voriconazole. In the vori-IA study the median (range) duration of IV treatment (n = 31), oral treatment (n = 22) and total treatment was 8.0 (3–33) days, 59.5 (2–81) days and 41.0 (3–90) days, respectively. †Visual testing was not completed at screening and day 7. ‡Addition of another antifungal medication for additional coverage based on computed tomography findings and continued positive galactomannan with increasing titers (n = 1); IA not approved (n = 1); no proven or probable IA (n = 1); IA not identified, relapsing of lymphoma (n = 1); no longer possible IA (proven Candida tropicalis infection; n = 1); patient diagnosed with bacterial lung infection. AE indicates adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; IA, invasive aspergillosis; SAE, severe adverse event. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 Voriconazole Use in Pediatric Patients © 2016 Wolters Kluwer Health, Inc. All rights reserved.  www.pidj.com  |  e7 TABLE 6. Summary of Safety Data from the Vori-IC/EC Study (Safety Population) Vori-IC/EC Study 2–<12 Yr (n = 14) 12–<18 Yr (n = 8) Overall* (n = 22) All- Causality Treatment- Related All- Causality Treatment- Related All- Causality Treatment- Related AEs, n 78 13 35 5 113 18 Patients with AEs, n (%) 13 (92.9) 8 (57.1) 6 (75.0) 3 (37.5) 19 (86.4) 11 (50.0) Hepatic AEs, n (%) 6 (42.9) 5 (35.7) 1 (12.5) - 7 (31.8) 5 (22.7) ALT abnormal 3 (21.4) 1 (7.1) - - 2 (9.1) 1 (4.5) ALT increased 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) AST abnormal 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) AST increased 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) GGT abnormal 2 (14.3) 1 (7.1) - - 2 (9.1) 1 (4.5) Hepatic enzyme increased 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) Hyperbilirubinemia 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) Liver disorder 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) Blood ALP abnormal 1 (7.1) - - - 1 (4.5) Gallbladder disorder 1 (7.1) - - - 1 (4.5) GGT increased - - 1 (12.5) - 1 (4.5) Hepatosplenomegaly 1 (7.1) - - - 1 (4.5) Jaundice 1 (7.1) - - - 1 (4.5) Visual AEs, n (%) 6 (42.9) 3 (21.4) 3 (37.5) 3 (37.5) 9 (40.9) 6 (27.3) Photophobia 2 (14.3) 2 (14.3) 1 (12.5) 1 (12.5) 3 (13.6) 3 (13.6) Conjunctivitis 1 (7.1) 1 (7.1) - - 1 (4.5) 1 (4.5) Eye pruritus - - 1 (12.5) 1 (12.5) 1 (4.5) 1 (4.5) Retinal disorder - - 1 (12.5) 1 (12.5) 1 (4.5) 1 (4.5) Amaurosis 1 (7.1) - - - 1 (4.5) Corneal opacity 1 (7.1) - - - 1 (4.5) Eyelid disorder 1 (7.1) - - - 1 (4.5) Visual acuity reduced 1 (7.1) - - - 1 (4.5) SAEs, n (%) 2 (14.3) - 1 (12.5) 1 (12.5) 3 (13.6) 1 (4.5) Treatment discontinuation, n (%) 7 (50.0) 2 (14.3) 2 (25.0) 1 (12.5) 9 (40.9) 3 (13.6) AEs 2 (14.3) 2 (14.3) 2 (25.0) 1 (12.5) 4 (18.2) 3 (13.6) Medication error 1 (7.1) - - - 1 (4.5) Protocol violation 1 (7.1) - - - 1 (4.5) Other 3 (21.4)† - - - 3 (13.6) Study discontinuation, n (%) 1 (7.1) - - - 1 (4.5) Lack of confirmation of Candida infection 1 (7.1) - - - 1 (4.5) *All patients received at least one dose of voriconazole. In the vori-IC/EC study, the median (range) duration of IV treatment (n = 22), oral treatment (n = 13), and total treatment was 7.0 (2–24) days, 9.0 (2–37) days, and 14.0 (2–42) days, respectively. †Lack of confirmation of Candida infection. AE indicates adverse events; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EC, esophageal candidiasis; GGT, γ-glutamyl transferase; IC, invasive candidiasis; IV, intravenous; SAE, severe adverse event. FIGURE 2. Global response success rates at EOT  in patients with IA and IC/EC (MITT population).  EC indicates esophageal candidiasis; EOT, end of  treatment; IA, invasive aspergillosis; IC, invasive  candidiasis; MITT, modified intent-to-treat. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Martin et al The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 e8  |  www.pidj.com  © 2016 Wolters Kluwer Health, Inc. All rights reserved. for severe muscle weakness and fever. At that time, the patient’s blood bilirubin was 6.4mg/dL, AST 694 IU/L and ALT 684 IU/L. The patient was also diagnosed with steroid-related muscle weakness and parainfluenza type 1 bronchitis. The patient completed voriconazole therapy for the treatment of IA on day 40. Liver function tests returned to normal on day 64 (24 days after last voriconazole dose). Fifteen patients discontinued treatment. Only 1 patient (7-yearold male) discontinued treatment because of anAE; this patient discontinued on day 3 because of an SAE of sepsis (unrelated to voriconazole) and recovered by day 9. One treatment discontinuation was considered to be treatment related (insufficient clinical response); 6 patients were subsequently discontinued from the study for other reasons. Vori-IC/EC Study A safety summary is presented in Table 6. Eleven of 22 patients experienced 18 treatment-related AEs, most commonly photophobia (n = 3). Most treatment-relatedAEs were mild or moderate.Treatmentrelated hepatic AEs were reported in 5 patients (22.7%) and were mild or moderate in severity except for 1 case of severe liver disorder. Treatment-related visual AEs were reported by 6 patients (27.3%) and were mild or moderate in severity. Only 2 patients (9.1%) reported any treatment-related skin AEs [rash (=2); data not shown], which were both mild in severity; no psychiatric treatment-related AEs were observed. Serious adverse events were experienced by 3 of 22 patients; 1 SAE (EC patient), recorded as progression of suspected splenic candidiasis later confirmed by biopsy, was considered treatment related. Splenic candidiasis progressed to the kidneys and eye, despite systemic voriconazole treatment. Subsequent use of lipid amphotericin B and micafungin treatment did not lead to improvement; however, neutrophil reconstitution in addition to micafungin and posaconazole treatment led to remission on day 390 (373 days after last voriconazole dose). Nine patients discontinued the treatment. Four patients discontinued the treatment because of AEs and, of these, 3 discontinued because of treatment-related AEs. Specifically, a 9-year-old female with IC (salvage) and medical history of pancreatic tumor, hyperbilirubinemia and heart failure permanently discontinued treatment on A C B D FIGURE 3. Basic goodness-of-fit plots  for the final pharmacokinetic model,  showing: observed concentrations versus  population predicted concentrations  (A); observed concentrations versus  individually predicted concentrations (B);  conditional weighted residuals versus  individually predicted concentrations  (C); conditional weighted residuals  versus time (D). Open circles represent  observed data; the dashed line represents  the line of identity or unity; the solid line  represents the local regression smooth  line (loess smooth). The closer the  smooth line is to the line of identity or  unity, the more robust the model fit. TABLE 7. Summary of Estimated Voriconazole Exposures in Pediatric Patients Based on Final Pharmacokinetic Model Voriconazole AUC0–12 (μg·h/mL) Voriconazole Cmin (μg/mL) Children (n = 21) Regimen 8mg/kg IV q12 h 9mg/kg oral q12 h* 8mg/kg IV q12 h 9mg/kg oral q12 h* Geometric mean (CV%) 49.63 (57) 46.86 (60) 2.65 (77) 3.56 (64) Median (range) 51.54 (20.67–171.08) 45.66 (19.84–170.76) 2.95 (0.69–12.67) 3.48 (1.39–13.86) Young adolescents aged 12–14 yr weighing <50kg (n = 10) Regimen 8mg/kg IV q12 h 9mg/kg oral q12 h* 8mg/kg IV q12 h 9mg/kg oral q12 h* Geometric mean (CV%) 54.91 (40) 50.57 (43) 3.0 (52) 3.86 (46) Median (range) 68.24 (20.35–85.79) 62.02 (19.54–82.44) 4.19 (0.66–5.61) 4.84 (1.36–6.51) All other adolescents (n = 17) Regimen 4mg/kg IV q12 h 200mg oral q12 h 4mg/kg IV q12 h 200mg oral q12 h Geometric mean (CV%) 37.28 (59) 27.72 (65) 2.18 (75) 2.15 (67) Median (range) 33.78 (17.7–110.05) 25.07 (8.89–79.36) 1.97 (0.76–8.27) 1.94 (0.65–6.46) *Maximum oral dose was not to exceed 350mg q12h. AUC0–12 indicates area under the curve from 0 to12 hours; Cmin , minimum plasma concentration (trough); CV%, coefficient of variation in percentage. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 Voriconazole Use in Pediatric Patients © 2016 Wolters Kluwer Health, Inc. All rights reserved.  www.pidj.com  |  e9 day 12 after developing moderate hyperbilirubinemia, which resolved on day 17. A 5-year-old male with EC and medical history of acute lymphocytic leukemia, chemotherapy-related anemia, antithrombin III deficiency and hepatomegaly permanently discontinued the treatment on day 23 after developing severe liver disorder. On day 23, blood bilirubin was 2.1mg/dL, AST 661.2 IU/L and ALT 282 IU/L. The event resolved by day 27 (4 days after last voriconazole dose). Concomitant medications taken within 2 weeks of the event of severe liver disorder included cytarabine, cyclophosphamide, pegaspargase, methotrexate and tioguanine. A 12-year-old girl with EC permanently discontinued the treatment on day 17 after developing severe progression of suspected splenic candidiasis as described above. One patient lost to follow-up was discontinued from the study. Efficacy Vori-IA Study The week 6 global success rate in patients with proven/ probable IA (n = 14) was 64.3% [95% confidence interval (CI): 35.1–87.2] and was sustained at EOT. Success rates were numerically greater for adolescents aged 12–<18 years [77.8% (95% CI: 40.0–97.2)] versus children aged 2–<12 years [40.0% (95% CI: 5.3–85.3)]. EOT global response failures included an observed failure in 1 patient, indeterminate result in 1 patient and missing data in 3 patients. Four deaths due to septic shock (n = 3) and ruptured mycotic aneurysm (n = 1) were reported before week 6 (up to day 63), and 1 death due to acute lymphocytic leukemia was reported on day 75. There were 2 deaths in modified intent-to-treat patients aged <12 years; none were attributed to voriconazole. One patient died on day 30 and the other died on day 38. Vori-IC/EC Study EOT global success rate in patients with IC/EC (n = 17) was 76.5% (95% CI: 50.1–93.2). EOT global success rates were 88.9% (95% CI: 51.7–99.7) for patients aged 2–<12 years and 62.5% (24.5, 91.5) for those aged 12–<18 years. Global response for IC patients (n = 7) included success in 6 patients and indeterminate result in 1 patient. Global response for EC patients (n = 10) included success in 7 patients, failure in 1 patient and indeterminate results in 2 patients. Two EC patients with a successful EOT global response had recurrence of EC (14 and 16 days after last voriconazole dose). One EC patient with a successful EOT global response developed suspected splenic candidiasis during therapy. EOT global response success rates by therapy and baseline pathogen are presented in Figure 2. No patients died by the 1-month follow-up visit. Exposure–Response Analyses For all age groups, a 2-compartment pharmacokinetic model with first-order absorption and linear elimination reasonably described voriconazole data, with the caveat of some underestimation of high concentrations, as shown in the basic diagnostic plots (Fig. 3). These plots showed that the data points were generally distributed symmetrically across the line of identity or line of unity, although many data points appeared to be widely spread, and several higher concentration data points were skewed from the line of identity or unity. This is not unexpected given the sparse data from phase 3 studies. The equations for the final pharmacokinetic model are presented below, and interindividual variability was estimated for clearance only, given the limited concentration data. CL WT WT WT WT logi CL= ( ) = = = ( ) θ θ θ θ / / / / . . 70 70 70 70 0 75 2 2 3 3 0 75 V V Q V V Q tt Rate rate F k F a ka 1 1( ) = = = θ θ θ CL indicates linear clearance; V2 , central volume of distribution; V3 , peripheral volume of distribution; Q, intercompartmental clearance; F1, oral bioavailability; ka , first-order absorption rate constant; rate, infusion rate used to estimate voriconazole concentration from prior treatment; and θ, estimate of fixed effects in NONMEM. A B FIGURE 4. Observed and model-predicted probability of  treatment-related hepatic AEs versus voriconazole Cmin  (A)  and all-causality hepatic AEs versus voriconazole AUC0–12  (B)  using multiple-panel data. “|” symbols represent observed  individual data (AE present = 1, AE absent = 0); solid  circles represent the observed probability of an AE at each  concentration level (note: individual concentration values  were rounded up to the next integral value for summary  purposes). The line and the corresponding band represent  the population-predicted probability and its 95% CI  (computed with 1000 bootstrap). A wide 95% CI indicates  low precision on the probability prediction. AE indicates  adverse event; AUC0–12 , area under the curve from 0 to  12 hours; CI, confidence interval; Cmin , minimum plasma  concentration (trough). Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Martin et al The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 e10  |  www.pidj.com  © 2016 Wolters Kluwer Health, Inc. All rights reserved. At matching doses, voriconazole exposures in children and young adolescents with low body weight were comparable with those in heavier or older adolescents, given the large interindividual variability (Table 7). Although average voriconazole exposures tended to be greater in CYP2C19 poor metabolizers (n = 3) and heterozygous extensive metabolizers (n = 12) than homozygous extensive metabolizers (n = 17), substantial overlap in exposure distributions was seen across groups because of large interindividual variability (data on file; 16 patients did not have genotyping information available). An association between increased voriconazole exposures (AUC0–12 and Cmin ) and treatment-related hepatic AEs was established (Fig. 4A). For all-causality hepatic AEs, the association was only related to voriconazoleAUC0–12 but not Cmin (Fig. 4B).The wide 95% CIs for the population predictions of probability of hepatic AE occurrence as a function of voriconazole exposure reflect the large uncertainty of the prediction (Fig. 4A and B). Note that this positive association was identified only when multiple-panel data (all AE occurrences) were analyzed. When single-panel data (without counting AE frequency in each patient) were analyzed, this positive association diminished for both treatment-related and all-causality hepatic AEs. No associations between voriconazole exposures and visual AEs, psychiatric or skin and subcutaneous tissue disorders were identified. Given the limited sample size and high success rate, no association between voriconazole exposures and efficacy was established for IA and EC patients (Fig. 5A and B). All patients with IC for whom exposure data were available (n = 6) had global success at EOT; the exposure–response analysis was not performed because of the lack of failure cases. The average voriconazole AUC0–12 in IC patients ranged from 27.2 to 62 μg·h/mL, and average Cmin ranged from 1.09 to 4.32 μg/mL. DISCUSSION These data suggest that voriconazole is generally effective in pediatric patients with IA and IC/EC, with a favorable risk–benefit balance. Overall, the safety of voriconazole in this small number of pediatric patients was similar to the known safety profile in adults. Pediatric patients had a numerically greater frequency of hepatic-related AEs associated with liver enzyme elevations; however, the nature and severity of hepatic AEs in the pediatric population was similar to that seen in adults. Hepatic AEs (all-causality and treatment related) in the voriIA study only occurred in patients aged 12–<18 years, whereas most hepatic AEs in the vori-IC/EC study occurred in patients aged 2–<12 years. Because visual disturbances are known side effects of voriconazole use in adults, visual symptoms were A B FIGURE 5. EOT global response versus  voriconazole AUC0–12  and Cmin  in patients with IA  (A) and patients with EC (B). Horizontal center  line represents the median; box represents the  interquartile distance; whiskers represent ≤1.5  × interquartile range; solid circles represent  the estimated individual exposure parameters.  AUC0–12  indicates area under the curve from 0 to  12 hours; Cmin , minimum plasma concentration  (trough); EC, esophageal candidiasis; EOT, end  of treatment; IA, invasive aspergillosis. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 Voriconazole Use in Pediatric Patients © 2016 Wolters Kluwer Health, Inc. All rights reserved.  www.pidj.com  |  e11 closely monitored throughout these studies. However, whether the tests used accurately assess visual AEs in children is unclear. It may be the case that children are unable to accurately report visual symptoms using these tests, instead, any visual disturbances manifest themselves as atypical behaviors. Of note, we did attempt to assess behavioral change in patients by administering the visual questionnaire, but no clear pattern of change was observed. End of treatment global success rate in pediatric patients with IA was 64.3% (n = 9/14), similar to that seen in the adult therapeutic IA study (52.8%; n = 76/144) at 12 weeks.15 In addition, EOT global success rates in pediatric patients with IC and EC were 85.7% (n = 6/7) and 70.0% (n = 7/10; indeterminate: n = 2/10), respectively, and comparable with those reported in the adult therapeutic studies for IC (65.3%; n = 162/248) and EC (98.3%; n = 113/115).14,16 In the IA study, the success rate was numerically greater in patients aged 12–<18 years (77.8%) than in patients aged 2–<12 years (40.0%). In the IC/EC study, the reverse was true with greater success rate in patients aged 2–<12 years (88.9%) than in patients aged 12–<18 years (62.5%). However, any interpretation of these data is limited by the small subgroup sample sizes and by the open-label, non-comparative design of the presented studies. Compared with the previously developed pharmacokinetic model for immunocompromised pediatric patients,21 this model was simplified by removing the nonlinear component of clearance, without substantial degradation of model performance. The model fit voriconazole trough concentrations well although the absorption phase was poorly estimated, which was not unexpected as limited concentration data were available (particularly at absorption phase). On the basis of the totality of the model performance metrics, the simplified model was deemed acceptable to provide individual voriconazole exposure estimates. Typical voriconazole clearance in these pediatric patients was greater than that in adults with IA (7.79 versus 5.30L/h/70kg, respectively).24 Estimated oral bioavailability in pediatric patients was greater than that reported previously for immunocompromised pediatric patients and adults with IA (85% versus 64%, respec- tively).21,24 The oral bioavailability of voriconazole in healthy adults has been estimated to be greater than 90%.12,25 The large interindividual variability in oral bioavailability and voriconazole exposure seen in these patients may be because of them receiving many concomitant medications and having various serious underlying conditions, which could affect the oral absorption and disposition processes and could not be easily delineated. FIGURE 6. Comparison of estimated steady-state voriconazole AUC0–12  and Cmin  by age group at matching IV and oral  doses in patients with IA. Horizontal center line represents the median; box represents the interquartile distance; whiskers  represent ≤1.5 × interquartile range; outliers are represented by open circles beyond whiskers. Intravenous regimen: 8 mg/ kg q12 h for children (aged 2–<12 years) and young adolescents (aged 12–14 years weighing <50 kg); 4 mg/kg q12 h for all  other adolescents and adults with IA. Oral regimen: 9 mg/kg (max 350 mg) q12 h for children (aged 2–<12 years) and young  adolescents (aged 12–14 years weighing <50 kg); 200 mg q12 h for all other adolescents and adults with IA. Note that data  from adults with IA at 300 mg oral q12 h are also included here and denoted as “Adults with IA 300”. This figure was created  for easy comparison across different age groups. AUC0–12  indicates area under the curve from 0 to 12 h; Cmin , minimum  plasma concentration (trough); IA, invasive aspergillosis; IV, intravenous; q12 h, every 12 h. Copyright © 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Martin et al The Pediatric Infectious Disease Journal • Volume 36, Number 1, January 2017 e12  |  www.pidj.com  © 2016 Wolters Kluwer Health, Inc. All rights reserved. The current analysis is consistent with previous findings in adults with IA where CYP2C19 genotyping status did not have a clinically relevant effect on voriconazole exposure.24 A recently published article concluded that a CYP2C19 genotype-directed dosing algorithm (ie, 5, 6 or 7mg/kg q12h stratified by CYP2C19 status) allowed pediatric patients (n = 20) to reach target voriconazole concentration significantly sooner than pediatric patients with a standard dosing regimen (5mg/kg q12h, n = 25).26 Of note, the doses evaluated in that publication are lower than those investigated in our studies. It is possible that the use of lower doses in these pediatric patients might be an important factor in the delay of reaching target concentration, in addition to the CYP2C19 polymorphism. CYP2C19 is known to be the major pathway for voriconazole metabolism, but notably other pathways, such as CYP3A4 and CYP2C9, are also involved and consequently CYP2C19 genotype alone does not explain the variability in voriconazole exposure. The impact of genotype on voriconazole exposure can be influenced by a patient’s demographic characteristics, underlying disease and concomitant medications. Hence, voriconazole dose adjustment solely based on CYP2C19 genotype is not currently recommended. Approximately 42% of pediatric patients received omeprazole or esomeprazole (CYP2C19 inhibitors known to increase voriconazole exposure in healthy subjects27 ). Although no trend was identified in our assessment, the impact of these concomitant medications on voriconazole exposure cannot be ruled out. Similarly, approximately 30% of adults with IA for comparison also received concomitant omeprazole or esomeprazole.24 At matching IV doses, average exposure values and distributions were similar in these pediatric patients and adult patients with IA (Fig. 6). At matching oral doses, average exposures in pediatric patients were greater than that in adult patients with IA; however, substantial overlap in exposure distributions was observed between groups (Fig. 6). Considering that treatment is being provided for potentially lifethreatening infections, it is preferred to start with a dose with relatively high exposure to ensure sufficient coverage and then reduce to lower doses if needed. Although an association between increased voriconazole exposure and hepatic AEs was established (with multiple-panel data only), voriconazole concentrations could not be used to accurately predict hepatic AE occurrence given the large uncertainty of prediction (Fig. 4A and B). Note that the multiple-panel data analysis may have overestimated the AE occurrence probability, as a patient with multiple AEs would be counted several times. The lack of association of voriconazole exposure with efficacy and other safety end points may be because of an insufficient sample size. These findings are consistent with what has previously been reported in adult patients with IA.24 These patients typically had multiple comorbidities and received multiple medications. In addition, treatment effect is just one of the contributing factors leading to successful clinical outcomes for life-threatening fungal infections. Patients’ underlying conditions and ability to respond to the treatment are also important factors influencing the clinical outcomes. No consensus regarding correlations of voriconazole exposure with clinical outcomes and treatment-related toxicity has been established because of the complexity of fungal infections in the clinical setting, despite substantial efforts to do so.9,10,28–37 Therefore, the clinical response and tolerability of individual patients should continue to be the primary consideration for dose adjustment, and voriconazole Cmin (if available) should be considered as a secondary marker for the purpose of dose adjustment. In our studies, most pediatric patients (64%; n = 34) did not require dose adjustments; 26% (n = 14) had dose reductions and 11% (n = 6) had dose escalations. One patient had dose reduction and dose escalation during the treatment period. Among them, 9 patients had dose reductions because of high voriconazole concentrations, whereas 3 had dose escalations because of low concentrations based on predefined provisional instructions. Dose adjustment with 1mg/kg (50mg oral) was sufficient for all but 3 patients with IA, indicating that slight adjustment of the initial dose was generally adequate. Taken together, the proposed dosing regimens were deemed acceptable as the initial recommendation for pediatric patients. ACKNOWLEDGMENTS Editorial support was provided by Karen Irving of Complete Medical Communications and was funded by Pfizer Inc. REFERENCES 1. Brissaud O, Guichoux J, Harambat J, Tandonnet O, Zaoutis T. Invasive fungal disease in PICU: epidemiology and risk factors. Ann Intensive Care. 2012;2:6. 2. Gallin JI, Zarember K. Lessons about the pathogenesis and management of aspergillosis from studies in chronic granulomatous disease. Trans Am Clin Climatol Assoc. 2007;118:175–185. 3. Segal BH, DeCarlo ES, Kwon-Chung KJ, Malech HL, Gallin JI, Holland SM. Aspergillus nidulans infection in chronic granulomatous disease. Medicine (Baltimore). 1998;77:345–354. 4. Burgos A, Zaoutis TE, Dvorak CC, et al. 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The effect of therapeutic drug monitoring on safety and efficacy of voriconazole in invasive fungal infections: a randomized controlled trial. Clin Infect Dis. 2012;55:1080–1087. 33. Racil Z, Winterova J, Kouba M, et al. Monitoring trough voriconazole plasma concentrations in haematological patients: real life multicentre experience. Mycoses. 2012;55:483–492. 34. Tan K, Brayshaw N, Tomaszewski K, Troke P, Wood N. Investigation of the potential relationships between plasma voriconazole concentrations and visual adverse events or liver function test abnormalities. J Clin Pharmacol. 2006;46:235–243. 35. Trifilio S, Singhal S, Williams S, et al. Breakthrough fungal infections after allogeneic hematopoietic stem cell transplantation in patients on prophylactic voriconazole. Bone Marrow Transplant. 2007;40:451–456. 36. Troke PF, Hockey HP, Hope WW. Observational study of the clinical efficacy of voriconazole and its relationship to plasma concentrations in patients. Antimicrob Agents Chemother. 2011;55:4782–4788. 37. Ueda K, Nannya Y, Kumano K, et al. Monitoring trough concentration of voriconazole is important to ensure successful antifungal therapy and to avoid hepatic damage in patients with hematological disorders. Int J Hematol. 2009;89:592–599. Invasive aspergillosis in patients with hematological malignancies in the Czech and Slovak republics: Fungal InfectioN Database (FIND) analysis, 2005–2009 Zdenek Racil a,b, *, Barbora Weinbergerova a , Iva Kocmanova c , Jan Muzik d , Michal Kouba e , Lubos Drgona f , Lucia Masarova f , Tomas Guman g , Elena Tothova g , Kristina Forsterova h , Jan Haber h , Barbora Ziakova i , Eva Bojtarova i , Jan Vydra j , Peter Mudry k , Renata Foralova k , Daniela Sejnova l , Nada Mallatova m , Vit Kandrnal d , Petr Cetkovsky e , Jiri Mayer a,b a Department of Internal Medicine – Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic b CEITEC – Central European Institute of Technology, Masaryk University Brno, Brno, Czech Republic c Department of Microbiology, University Hospital Brno, Brno, Czech Republic d Institute of Biostatistics and Analyses at the Faculty of Medicine and the Faculty of Science of the Masaryk University, Brno, Czech Republic e Institute of Hematology and Blood Transfusion, Prague, Czech Republic f Second Department of Oncology, Comenius University and National Cancer Institute, Bratislava, Slovakia g Department of Hematology and Onco-hematology, Louis Pasteur University Hospital, Kosice, Slovakia h Department of Hemato-oncology, General Faculty Hospital, Prague, Czech Republic i Department of Hematology and Transfusiology, St. Cyril and Methodius Hospital – University Hospital Bratislava, Bratislava, Slovakia j Department of Hematology, University Hospital Kralovske Vinohrady, Prague, Czech Republic k Department of Pediatric Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic l Department of Pediatric Hematology and Oncology, Pediatric University Hospital Bratislava, Bratislava, Slovakia m Laboratory of Parasitology and Mycology, Ceske Budejovice Hospital, Ceske Budejovice, Czech Republic 1. Introduction Invasive fungal diseases (IFD) are an important cause of morbidity and mortality in patients with hematological diseases.1,2 The epidemiology of IFD in this group of severely immunocompromised patients has changed substantially during the last two decades, with invasive aspergillosis (IA) being a predominant infection.1 The incidence of this infection can vary and is mainly based on the underlying hematological malignancy; it can reach up to 10% among patients undergoing treatment for acute leukemia or allogeneic hematopoietic stem cell transplantation (HSCT).3 However, there have been several key advancements over the past decade that have significantly improved not only the diagnosis (widespread availability of high-resolution computed tomography (HRCT) and non-culture based diagnostic tools, such as the detection of galactomannan (GM)), but also treatment International Journal of Infectious Diseases 17 (2013) e101–e109 A R T I C L E I N F O Article history: Received 9 June 2012 Received in revised form 21 August 2012 Accepted 2 September 2012 Corresponding Editor: Meinolf Karthaus, Munich, Germany Keywords: Invasive aspergillosis Hematological malignancy Diagnosis Antifungal treatment S U M M A R Y Objectives: To evaluate risk factors, diagnostic procedures, and treatment outcomes of invasive aspergillosis (IA) in patients with hematological malignancies. Methods: A retrospective analysis of data from proven/probable IA cases that occurred from 2005 to 2009 at 10 hematology centers was performed. Results: We identified 176 IA cases that mainly occurred in patients with acute leukemias (58.5%), mostly those on induction/re-induction treatments (39.8%). Prolonged neutropenia was the most frequent risk factor for IA (61.4%). The lungs were the most frequently affected site (93.8%) and computed tomography detected abnormalities in all episodes; however, only 53.7% of patients had findings suggestive of IA. Galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (positive in 79.1% and 78.8% of episodes, respectively) played a crucial role in IA diagnosis. Neutrophil count and antifungal prophylaxis did not influence the GM positivity rate, but empirical therapy decreased this rate (in serum). Of the IA cases, 53.2% responded to initial antifungal therapy. The combination of voriconazole and echinocandin, even as initial or salvage therapy, did not perform better than voriconazole monotherapy (p = 0.924 for initial therapy and p = 0.205 for salvage therapy). Neutrophil recovery had a significant role in the response to initial (but not salvage) antifungal therapy. Conclusions: Our retrospective analysis identified key diagnostic and treatment characteristics, and this understanding could improve the management of hematological malignancy patients with IA. ß 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. * Corresponding author. Tel.: +420 602 564011. E-mail address: zracil@fnbrno.cz (Z. Racil). Contents lists available at SciVerse ScienceDirect International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid 1201-9712/$36.00 – see front matter ß 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. http://dx.doi.org/10.1016/j.ijid.2012.09.004 options (availability of new antifungal drugs, e.g., voriconazole and echinocandins) of IA. These events have led to the recently reported improvement in the prognosis of patients with this lifethreatening infection.2,4,5 Moreover, several observational registries in Europe, as well as worldwide, have been created with the goal of collecting real world data regarding incidence, risk factors, and treatment outcomes of patients with IA.1,2,4–6 In this multicenter study, we report data from IA episodes that occurred in patients with hematological malignancies. These data were retrospectively collected from the Fungal InfectioN Database (FIND), which holds data from almost all hematology centers in the Czech and Slovak republics. The aim of this study was to analyze the risk factors, diagnostic procedures, and treatment outcomes from the largest cohort of IA episodes in Central Europe published to date. 2. Methods 2.1. Design Thirteen hematology centers in the Czech and Slovak republics participate in the FIND project. The database consists of retrospectively collected data of proven and probable IA cases that occurred between 2001 and 2009, as well as a prospective collection of cases from 2010 onwards. This study was conducted by performing an analysis of proven and probable IA cases that occurred between January 1, 2005 and December 31, 2009, which had been retrospectively entered as electronic case report forms by 10 of 13 participating centers (seven adult and three pediatric centers). The distribution of episodes during this time period was not uniform and was mainly dependent on the extension of non-culture-based diagnostic techniques (e.g., GM detection) among centers. Therefore, the number of episodes in individual time intervals does not reflect the real incidence of infection. Forty-one percent of cases entered into the database and analyzed occurred between 2005 and 2007, 59% between 2008 and 2009. 2.2. Case identification Cases were identified in participating centers by reviewing the patient charts as well as laboratory, microbiology, and imaging results. Pathology reports from autopsies were also used. All identified episodes of IA during the observation period were included in the database. The variables collected in the electronic case report forms included the subject’s demographic characteristics, underlying hematological malignancy and treatment, clinical signs and symptoms, and the results of microbiological and histological investigations, as well as results of imaging studies, information regarding the use of mold-active antifungal prophylaxis and empirical antifungal treatment, targeted antifungal treatment and outcomes, neutrophil counts at the time of diagnosis as well as before and after each antifungal treatment, and finally patient survival. Due to the retrospective design of this study, a patient’s informed consent was not required. The Institutional Review Board of the University Hospital Brno approved this study. 2.3. Definitions Episodes of IA were defined according to the 2002 European Organisation for Research and Treatment of Cancer and Mycosis Study Group (EORTC/MSG) criteria.7 The day of diagnosis was defined as the day when criteria for proven or probable IA were fulfilled. Empirical antifungal therapy was defined as the administration of systemic antifungal treatment in patients with persistent fever only, or in patients who did not fulfill criteria for proven or probable IFD at the time of treatment initiation. Targeted antifungal therapy was started when patients fulfilled criteria for proven or probable IA. The overall outcome of therapy, as well as the outcome of each line of antifungal treatment, was classified according to published EORTC/MSG recommendations.8 The effect of therapy was evaluated only if the targeted antifungal therapy lasted at least 5 days. An independent, blinded evaluation of all the entered data was performed by a review board at the main study center, with special consideration to the fulfillment of EORTC/MSG criteria for the diagnosis of proven or probable IA, as well as treatment outcome. 2.4. Statistical analysis Frequency tables and standard descriptive statistics were used for summation of the patient characteristics. Proportions were compared with the maximum-likelihood Chi-square test or Fisher’s exact test. Continuous variables were compared with the Mann–Whitney or Kruskal–Wallis analysis of variance Table 1 Baseline characteristics Patients No. of patients 176 Age, years, median (range) 56 (3–77) Sex, male/female, n (%) 104 (59.1%)/ 72 (40.9%) Patient’s disease at baseline, n (%) AML + MDS 73 (41.5%) ALL 30 (17.0%) NHL + HL 27 (15.3%) CLL 20 (11.4%) MM 12 (6.8%) CML + CMPD 4 (2.3%) Other 10 (5.7%) Anticancer therapy during/before IA, n (%) Induction/reinduction therapy of acute leukemia 70 (39.8%) Allogeneic HSCT 30 (17.0%) Autologous HSCT 17 (9.7%) Other 52 (29.5%) None 7 (4.0%) Presence of risk factors for development of IA, n (%) Neutropenia <0.5  109 /l for >10 days 108 (61.4%) Administration of corticosteroids for >21 days 50 (28.4%) Pulmonary/respiratory tract disease in anamnesis (COPD, etc.) 22 (12.5%) GVHD 20 (11.4%) Other risk factors 41 (23.3%) Number of risk factors present at diagnosis, n (%) 0 29 (16.5%) 1 79 (44.9%) 2 44 (25.0%) !3 24 (13.6%) IA episodes No. of episodes 176 Certainty of diagnosis according to EORTC/MSG 2002 criteria, n (%) Proven IA 27 (15.3%) Probable IA 149 (84.7%) Site of infection, n (%) Lung 165 (93.8%) Sinuses 1 (0.6%) Disseminated 7 (4.0%) Other 3 (1.7%) ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; CMPD, chronic myeloproliferative disease; COPD, chronic obstructive pulmonary disease; EORTC/MSG, European Organisation for Research and Treatment of Cancer/Mycoses Study Group; GVHD, graft-versus-host disease; HL, Hodgkin lymphoma; HSCT, hematopoietic stem cell transplantation; IA, invasive aspergillosis; MDS, myelodysplastic syndrome; MM, multiple myeloma; NHL, non-Hodgkin lymphoma. Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109e102 (ANOVA) test, as appropriate. The probabilities of overall survival were estimated using the Kaplan–Meier method, and a comparison of survival in the groups of patients was performed using a logrank test. The point estimates were supplied with 95% confidence intervals (CI). A level of statistical significance a = 0.05 was used in all analyses. For the analysis of the role of neutrophil count at the defined time points in the efficacy of antifungal treatment, patients were divided into three groups: those with a neutrophil count 0.1, 0.1–1.0, and !1.0  109 /l. Analyses were performed using statistical software SPSS 12.0.2 for Windows (SPSS Inc., 2003) and STATISTICA 9.0.1 for Windows (StatSoft, Inc. 2010). 3. Results 3.1. Characteristics of patients and episodes of IA During the study period (2005–2009), 176 episodes of IA occurring in 176 patients were identified: 27 (15.3%) proven and 149 (84.7%) probable. Patient characteristics are shown in Table 1. Acute leukemias represented the majority of the underlying hematological diseases (58.5%), and induction or re-induction treatment for acute leukemia (but not allogeneic HSCT) represented the most frequent anticancer treatment (39.8%). Therefore, patients with active acute leukemia during the first induction or salvage therapy represented the typical population of hematological malignancy patients with the highest risk of IA. Based on these data, it is not surprising that the most common classical risk factor identified in 61.4% of IA episodes was profound and prolonged neutropenia (Table 1). The lung was the most commonly affected site (93.8%), with 21 (12.0%) proven and 144 (81.8%) probable episodes. In addition, disseminated and isolated extrapulmonary infections were rare (4.0% and 2.3%, respectively). 3.2. Signs of infection Out of the 176 patients with IA, 136 (77.3%) had fever at the time of diagnosis, with a median duration of 6 days before diagnosis (range 0–53 days before diagnosis; interquartile range (IQR) 3–11 days before diagnosis). Moreover, 54.0% of patients with IA fulfilled criteria for persistent fever despite the administration of broad-spectrum antibiotics for 5 days. Out of 165 patients with invasive pulmonary aspergillosis (IPA), 125 (75.8%) exhibited at least one sign that was suggestive of pulmonary disease, which developed within a median of 5 days before diagnosis (range 0–35 days; IQR 2–9 days). The spectrum of these signs is shown in Table 2. 3.3. Diagnostic procedures 3.3.1. Imaging studies A chest X-ray was performed at the time of diagnosis in 152/165 (92.1%) patients with IPA. However, abnormalities were only identified in 73.0% of those patients. Moreover, the most commonly observed abnormality was a non-specific infiltrate (44.7%) (Table 2). In contrast, chest HRCT, which was performed Table 2 Clinical manifestations and results of diagnostic tests at the time of diagnosis of invasive aspergillosis Clinical manifestations at the time of diagnosis, all patients (N = 176), n (%) Fever >38.0 8C 136 (77.3%) Fever not responding to 5 days of antibiotics 95 (54.0%) Presence of organ-specific clinical symptoms 134 (76.1%) Clinical signs in patients with IPA (n = 165), n (%) Any symptom 125 (75.8%) Cough 69 (41.8%) Dyspnea 37 (22.4%) Chest pain 11 (6.7%) Hemoptysis 2 (1.2%) Other 6 (3.6%) Chest X-ray abnormality in patients with IPA (n = 152),a n (%) Any abnormality 111 (73.0%) Non-specific infiltrate(s) 68 (44.7%) Nodule(s) 36 (23.7%) Interstitial process 1 (0.7%) Pleural effusion 1 (0.7%) Cavitation(s) 1 (0.7%) Other 4 (2.6%) Chest high-resolution CT abnormality in patients with IPA (n = 149),a n (%) Any abnormality 149 (100%) Predominant abnormality Non-specific infiltrate(s) 69 (46.3%) Halo sign 40 (26.8%) Macronodule(s) >1 cm 17 (11.4%) Cavitation 9 (6.0%) Micronodule(s) <1 cm 8 (5.4%) Pleural effusion 5 (3.4%) Air crescent sign 1 (0.7%) Laboratory test results at the time of diagnosis, all patients, n (%) Serum galactomannan positive (consecutive index of positivity >0.5) (n = 172)a 136 (79.1%) Serum (1!3)-b-D-glucan positive (single value >80 pg/ml) (n = 44)a 36 (81.8%) Mycological examination, microscopy positive (all materials) (n = 71)a 9 (12.7%) Mycological examination, culture positive (all materials) (n = 81)a 24 (29.6%) Histology positive (all materials) (n = 12)a 8 (66.7%) BAL fluid examination in patients with IPA, n (%) Mycological examination, microscopy positive (n = 49)a 5 (10.2%) Mycological examination, culture positive (n = 48)a 9 (18.8%) BAL fluid galactomannan positive (index of positivity >0.5) (n = 66)a 52 (78.8%) BAL, bronchoalveolar lavage; CT, computed tomography; IPA, invasive pulmonary aspergillosis. a Calculated only for patients for whom the test was performed. Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109 e103 in 149/165 (90.3%) patients with IPA at the time of diagnosis (2005–2007, 87.8% vs. 2008–2009, 90.2%, p = 0.620), detected an abnormality in all of these patients. Interestingly, the most frequently observed abnormality on these early HRCT scans was a non-specific infiltrate (46.3%). Signs that are more specific for IFD, such as a halo sign, nodules, or cavitations, were seen substantially less frequently (Table 2). There was no statistically significant difference in the frequency of individual abnormalities on HRCT scans between patients with neutropenia (neutrophils <1.0  109 /l) and those without (p = 0.378). 3.3.2. Non-culture diagnostic techniques—serum The GM test was performed at all centers for screening (2–3 times per week) in high-risk patients (e.g., patients receiving induction for acute leukemia or undergoing allogeneic HSCT) and on request in all other patients with abnormalities on imaging studies. GM assessment of at least two serum samples was performed in 172/176 (97.7%) patients with IA (2005–2007, 95.9% vs. 2008–2009, 99.0%, p = 0.176). Using the criterion of an index of positivity >0.5 from two consecutive serum samples as a positive test result, we found the test positive in 79.1% of tested episodes (Table 2), and a positive result of the GM assay (consecutive positivity) preceded the final diagnosis of IA by a median of 2 days (range 0–34 days; IQR 1–4 days). The rate of positive test results was not influenced by the neutrophil count at the time of diagnosis (p = 0.426) or by the administration of mold-active antifungal prophylaxis (p = 0.854). In contrast, empirical antifungal therapy using a mold-active antifungal drug at the time of diagnosis of IA significantly decreased the proportion of positive GM test results in serum compared to patients not receiving the treatment (67% vs. 88%, respectively; p = 0.001). The median serum GM index of positivity level at the time of IA diagnosis was 1.28 (range 0.11–11.46). The detection of 1!3-b-D-glucan (BG) was available at only one center, and therefore the test was performed in only 44/176 (25.0%) patients. A positive test result (BG concentration >80 pg/ml from a single serum sample as the cut-off) was recorded in 81.8% of these patients (Table 2). 3.3.3. Mycological examination Histological examination, microscopic evaluation, and cultures of any relevant clinical specimens were performed in 12/176 (6.8%), 71/176 (40.3%), and 81/176 (46.0%) patients with IA, respectively. However, with the exception of the histological examination, which was positive in 66.7% of a very limited number of samples obtained by biopsy, the rate of positive results of the other two conventional techniques was very low (12.7% and 29.6%, respectively) (Table 2). Aspergillus fumigatus represented 19/24 (79.2%) identified isolates, followed by Aspergillus flavus 1/24 (4.2%), Aspergillus niger 1/24 (4.2%), Aspergillus terreus 1/24 (4.2%), and other Aspergillus species 2/24 (8.3%). 3.3.4. Bronchoalveolar lavage (BAL) fluid analysis Since IPA predominated in our patient group, BAL fluid was the most frequent mycologically evaluated material (Table 2). However, conventional mycological techniques with a very low frequency of positive results (10.2% microscopy and 18.8% culture) did not contribute substantially to the diagnosis of IPA in this group of patients. In contrast, the GM assay was positive in 52 out of 66 (78.8%) obtained BAL fluids using a cut-off value of 0.5. The rate of GM assay positivity in BAL fluid was not influenced by neutrophil count (p = 0.580) or the administration of mold-active antifungal prophylaxis (p = 0.147), and in contrast to serum was not influenced by empirical antifungal therapy (76% vs. 81%; p = 0.607). 3.4. Prophylaxis and empirical treatment Of the 176 patients with IA, 44 (25.0%) had received mold-active antifungal prophylaxis, with a median treatment time of 24 days (range 4–227 days; IQR 16–42 days) (Table 3). More than half of these episodes developed under prophylaxis treatment with itraconazole (25/44, 56.8%); however, itraconazole was also the most frequently used anti-mold prophylaxis at the time our study was performed. Moreover, the azole plasma concentration before breakthrough infection was only available in two patients. At the time of diagnosis of IA, 76/176 (43.2%) patients had already received mold-active empirical antifungal treatment, and the most frequently used was conventional amphotericin B (30.3% of empirically treated patients) (Table 3). The length of empirical treatment before the definitive diagnosis of IA was short (median 6 days, range 2–44 days, IQR 4–11 days). Therefore, this relatively high number of empirically treated patients reflects the suspicions of the clinician to IFD and early administration of systemic antifungals, rather than a high number of breakthrough IFD cases during prolonged antifungal therapy. 3.5. Antifungal therapy Targeted antifungal therapy for proven and probable IA was administered in 156/176 (88.6%) patients. In addition, 71 (40.3%) patients received only one line of therapy, 61 (34.7%) patients received treatment with a second-line therapy for toxicity or failure of the previous therapy, and 24 (13.6%) patients received more than two lines of antifungal therapy. Neither the spectrum of antifungal drugs nor their combinations used for the treatment of IA differed between the two observed periods (p = 0.252, p = 0.229, and p = 0.622, for first line, second line, and further lines, respectively). A complete or partial response to treatment was achieved in 83/156 (53.2%) patients treated with first-line therapy (median length of first-line therapy 15 days, range 5–139 days, IQR 10– 25 days). There was no substantial difference in the response rate between the two most frequently used approaches: voriconazole monotherapy and a combination of voriconazole and echinocandin (61.9% vs. 61.0%, respectively; p = 0.924) (Table 4). Forty (25.6%) of the 156 patients treated with firstline therapy received salvage therapy for failure of this treatment (median duration 19 days, range 5–159 days, IQR 10–32 days). Although the number of these patients was limited, the combination of voriconazole and echinocandin did not provide a better therapeutic outcome in this setting compared to voriconazole monotherapy (p = 0.205) (Table 4). Table 3 Antifungal prophylaxis and empirical antifungal therapy Anti-mold prophylaxis at the time of IA diagnosis Present 44 (25.0%) Antifungal drug useda Itraconazole 25 (56.8%) Voriconazole 7 (15.9%) Posaconazole 6 (13.6%) Conventional amphotericin B 4 (9.1%) Echinocandin 2 (4.5%) Anti-mold empirical antifungal therapy at the time of IA diagnosis Present 76 (43.2%) Antifungal drug useda Conventional amphotericin B 23 (30.3%) Lipid formulation of amphotericin B 20 (26.3%) Voriconazole 13 (17.1%) Echinocandin 12 (15.8%) Other 9 (11.8%) IA, invasive aspergillosis. a Percentage calculated from patients receiving treatment. Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109e104 To shorten the period of neutropenia, 97/176 (55.1%) patients received granulocyte colony stimulating factors. Granulocyte transfusions were not used. Of the 176 patients, 10 (5.7%) underwent surgery in addition to chemotherapy. At the end of all targeted treatment approaches efficacy was evaluated. The median length of treatment was 32.5 days (range 5– 148 days, IQR 17–66 days), and 105 out of 156 (67.3%) patients responded; however, 50/156 (32.1%) patients failed and one patient was not evaluable. Secondary prophylaxis (mostly with voriconazole) was used in 71/176 (40.3%) patients with a median length of treatment of 48 days (range 10–512 days; IQR 21–78 days). 3.6. The role of neutrophils in the efficacy of antifungal treatment There was no statistically significant difference in the percentage of patients with a successful treatment outcome (complete and partial response) at the end of all antifungal therapies based on neutrophil count at the start of antifungal treatment (p = 0.423). This lack of difference was also found when the role of neutrophils at the start of the first treatment and salvage therapy and the treatment outcome at the end of these therapies was evaluated separately (Table 5). In contrast, there was a statistically significant increase in the percentage of patients who successfully responded (complete and partial response) at the end of all antifungal therapies with increasing neutrophil counts at the end of antifungal treatment (p < 0.001) (Table 5). A substantially higher response rate was identified in patients with neutrophil counts >1.0  109 /l at the end of the first-line treatment compared to patients with neutrophil counts of 0.1–1.0 (p = 0.007) and <0.1 (p < 0.001)  109 /l. However, we did not find a role of neutrophil counts at the end of salvage therapy in patients receiving this treatment (p = 0.432) (Table 5). Finally, the change in neutrophil count during IA therapy and treatment outcome was analyzed. During first-line treatment, patients with a successful treatment outcome (complete and partial response of IA) had a significant increase in neutrophil count (p < 0.001 and p = 0.003, respectively). Moreover, the median neutrophil count in patients with a complete or partial response increased during the treatment from neutropenic range (<1.0  109 /l) to non-neutropenic range (Figure 1A). In contrast, patients with treatment failure were persistently neutropenic (progression of IA) or did not reveal any significant increase in their neutrophil count during therapy (stable IA) (Figure 1A). A similar analysis was performed for patients receiving salvage therapy, and no significant increase in neutrophil count was observed in any treatment outcome group (Figure 1B); however, the number of patients was limited. 3.7. Survival The median survival in our patient group was 28.1 (95% CI 15.6– 40.7) weeks. The 3- and 12-month overall survival (OS) was 57.8% (95% CI 50.5–65.1%) and 43.0% (95% CI 35.4–50.5%), respectively. OS follows survival attributed to IA (OSIA), thus IA was the predominant cause of death during the first 3 months after diagnosis, while other causes (mainly underlying diseases) were Table 4 Targeted antifungal therapy—efficacy of first-line and salvage therapy Treatment response n Complete or partial response Stable disease Progression Not known First-line therapy 156 83 (53.2%) 20 (12.8%) 53 (34.0%) Voriconazole 63 39 (61.9%) 8 (12.7%) 16 (25.4%) Combination of echinocandin + voriconazole 41 25 (61.0%) 3 (7.3%) 13 (31.7%) Conventional AMB 13 4 (30.8%) 3 (23.1%) 6 (46.2%) Lipid formulation of AMB 13 7 (53.8%) 1 (7.7%) 5 (38.5%) Echinocandin 9 2 (22.2%) - 7 (77.8%) Other 17 6 (35.3%) 5 (29.4%) 6 (35.3%) Salvage therapy 40 15 (37.5%) 7 (17.5%) 17 (42.5%) 1 (2.5%) Voriconazole 9 2 (22.2%) 3 (33.3%) 3 (33.3%) 1 (11.1%) Combination of echinocandin + voriconazole 7 4 (57.1%) 2 (28.6%) 1 (14.3%) Lipid formulation of AMB 3 1 (33.3%) 1 (33.3%) 1 (33.3%) Other 21 8 (38.1%) 1 (4.8%) 12 (57.1%) AMB, amphotericin B. Table 5 The role of neutrophil count at the start and at the end of antifungal therapy in treatment outcome Patients with successful treatment outcome (complete or partial response) at the end of therapy (%)a Neutrophils <0.1  109 /l Neutrophils 0.1–1.0  109 /l Neutrophils >1.0  109 /l p-Valueb Neutrophil count at the start of: Any therapy (n = 143) 63.8% 76.7% 70.5% 0.423 First-line therapy (n = 144) 50.7% 53.3% 64.4% 0.341 Salvage therapyc (n = 30) 37.5% 66.7% 25.0% 0.195 Neutrophil count at the end of: All therapies (n = 128) 21.1% 50.0% 80.9% <0.001 First-line therapy (n = 129) 16.7% 35.0% 68.2% <0.001 Salvage therapyc (n = 32) 20.0% 57.1% 40.0% 0.432 a The treatment outcome was evaluated at the end of therapy given in the raw (i.e., the end of all received therapies, the end of first-line therapy, or the end of salvage therapy, respectively). b Maximum-likelihood Chi-square test, difference between all three groups according to neutrophil count. c Salvage was defined as treatment after failure of first-line therapy. Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109 e105 predominantly responsible for death in patients who survived longer than 3 months (Figure 2A). Patients with probable IA had significantly better OS as well as survival attributed to IA (OSIA) (Figure 2B). OS as well as OSIA did not differ between cases diagnosed during 2005–2007 compared to more recent episodes (2008–2009) (OS: p = 0.173, 63.4% (95% CI 52.4–74.4%) vs. 52.7% (95% CI 42.9–62.4%) at 3 months, respectively; OSIA: p = 0.366, 70.7% (95% CI 60.1–81.3%) vs. 60.8% (95% CI 51.0–70.6%) at 3 months, respectively). 4. Discussion This is the largest multicenter study published to date that has analyzed episodes of IA in hematological malignancy patients from Central Europe. FIND is a network of hematology centers that gather and share information to improve our understanding of epidemiology, diagnostics, therapy, and the outcome of IFD in hematological malignancy patients from the Czech and Slovak republics. Our analysis confirmed several published and generally accepted facts in the view of risk factors, diagnostics, and treatment of this infection among patients with hematological malignancies.2,4,6,9–16 The significance of our study clearly lies in several unique findings, which should be noted. First, although we have shown the importance of using early lung HRCT for the diagnosis of pulmonary abnormalities (all patients with IPA had some detectable abnormality), only 53.7% had findings that were described as ‘specific’ for invasive mold infection based on EORTC/MSG 2008 criteria.17 Therefore, half of our IPA patients had non-specific infiltrates on early HRCT scans, of which the performance was generally driven by persistent fever or GM results. Recent studies have shown that the neutrophil count plays a role in the pattern of findings on imaging studies.11,18 However, 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Start End Start End Start End Start End Neutrophilcountx109/l CR PR SD PROGRESSION N = 37* N = 32* N = 16* N = 42* Neutrophil count: Treatment response * Only patients with known neutrophil count at the start and at the end of therapy were evaluated Mean: 1.54 3.75 1.46 4.39 2.17 2.27 0.86 1.83 Median: 0.29 2.50 0.09 2.41 1.20 1.36 0.05 0.22 P < 0.001 P = 0.003 P = 0.910 P = 0.002 First line treatmentA 75% median 25% 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Start End Start End Start End Start End CR PR SD PROGRESSION N = 4* N = 7* N = 5* N = 12* Mean: 1.28 2.12 1.78 1.41 3.33 1.87 4.40 5.36 Median: 0.73 2.45 0.21 1.32 2.70 1.50 2.70 3.38 P = 0.465 P = 0.999 P = 0.080 P = 0.480 Salvage treatment Treatment response 75% median 25% B Neutrophil count: * Only patients with known neutrophil count at the start and at the end of therapy were evaluated Neutrophilcountx109/l Figure 1. Change in neutrophil count during (A) first-line and (B) salvage therapy of invasive aspergillosis (CR, complete response; PR, partial response; SD, stable disease). Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109e106 this does not explain our results. In the study by Nicolle et al.,4 the patient population and percentage of patients with prolonged neutropenia was similar to our observations in this analysis. However, the authors of that study found a ‘halo sign’ in 81% of the patients, whereas only 26.8% of patients had the sign in our study. On the other hand, a recent study by Lortholary et al.6 examining a mixed patient population with 77.6% of patients suffering from a hematological malignancy found nodules in the majority of patients (81.3%) with IPA. However, nodules were again rarely found in our study (16.8%). Moreover, we did not observe any significant difference in the frequency of individual abnormalities, including the frequency of non-specific infiltrates in patients with and without neutropenia. Therefore, despite the multicenter approach whereby CT evaluations were performed by local radiologists, one of the explanations for the significant proportion of non-specific findings could be the promptitude of HRCT usage in patients with persistent fever or GM positivity, which has been seen in the last few years due to better availability of this technique. The median time from an HRCT scan to diagnosis of IA in our study was 0 days. Thus, in daily clinical practice where early CT scans are commonly performed and non-specific infiltrates are more frequently seen, mycological examination of these nonspecific lesions for a differential diagnosis becomes very important. This finding was very recently supported by others.19,20 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 N = 176 Proportionofpatients Time from IA diagnosis (weeks) Survival – death of IA Overall survival Weeks from IA diagnosis Overall survival (%) Survival – death of IA (%) 2 82.4 (76.8; 88.0) 84.5 (79.2; 89.9) 6 64.2 (57.1; 71.3) 69.5 (62.5; 76.4) 12 57.8 (50.5; 65.1) 65.0 (57.7; 72.2) 30 48.9 (41.4; 56.4) 61.9 (54.4; 69.4) 52 43.0 (35.4; 50.5) 60.1 (52.4; 67.8) SurvivalA 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Probable IA (N = 149) Proven IA (N = 27) Survival according to probability of IA Survival – death of IA Overall survival Weeks from IA diagnosis Overall survival (%) p < 0.001 Survival – death of IA (%) p < 0.001 Probable IA Proven IA Probable IA Proven IA 2 87.9 (82.7; 93.2) 51.9 (33.0; 70.7) 90.5 (85.7; 95.2) 51.9 (33.0; 70.7) 6 69.1 (61.7; 76.5) 37.0 (18.8; 55.2) 75.4 (68.3; 82.5) 37.0 (18.8; 55.2) 12 62.9 (55.1; 70.7) 29.6 (12.4; 46.9) 71.6 (64.1; 79.1) 29.6 (12.4; 46.9) 30 53.8 (45.6; 62.0) 22.2 (6.5; 37.9) 69.7 (61.9; 77.4) 22.2 (6.5; 37.9) 52 47.5 (39.2; 55.8) 18.5 (3.9; 33.2) 67.5 (59.4; 75.5) 22.2 (6.5; 37.9) Proportionofpatients Time from IA diagnosis (weeks) B Figure 2. Overall survival and survival attributed to invasive aspergillosis (IA) for (A) all patients and (B) based on the probability of IA diagnosis. Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109 e107 Our study also demonstrated the essential role of GM testing for the diagnosis of IA.21 Since the vast majority of cases represented probable IA and the sensitivity of culture and/or cytology was very limited, the diagnosis of probable IA was typically made using a combination of pulmonary abnormalities on lung HRCT and positivity of a GM assay with serum and/or BAL fluid. In addition, the high rate of positive results of the GM assay in serum (79.1%) and BAL fluid (78.8%) was similar or higher than in a recently published series of hematological patients.4,6,22 This multicenter study also found that the routine use of regular and frequent (2–3/week) GM screening is widely used at all hematology centers in both countries and seems common in the countries of Europe,4,6 but is less frequent or limited in others countries,9 including the USA.5 Therefore, GM screening was often used in place of invasive procedures for the differential diagnosis of pulmonary infiltrates. In a study by Perkhofer et al.9 conducted in Austria, 34% of the patients with invasive mold infections had a biopsy performed, whereas only 9.6% of the patients in our study required a biopsy for final diagnosis. The authors of that study recommend performing biopsies in these patients due to the high frequency of invasive zygomycosis. However, the high rate of positive results of the GM assay (serum and/or BAL) in our study could limit biopsies to only GM-negative infiltrates that are very likely to be of IFD origin. Another reason for performing a biopsy given by Perkhofer et al. is the requirement for culture verification of the infection due to the high frequency of A. terreus cases; A. terreus is resistant to amphotericin B.9,23 However, in our study, A. fumigatus was still the predominant species, and non-fumigatus Aspergillus species were very rare, with A. terreus isolated in only one case from our large multicenter series. Finally, the importance of GM detection for the diagnosis of IA in daily clinical practice was demonstrated based on the investigator’s questionnaire, which is part of our database (data not shown). In 60.2% of IA episodes, investigators subjectively marked the GM assay result as the criterion on which the IA diagnosis was mainly based, followed by HRCT in 18.8% of episodes and histology in 10.2% of episodes. However, when discussing GM assay results, the possible limitation of the test (extensively reviewed in the last European Conference on Infections in Leukemia (ECIL-3) recommenda- tions24 ) given by the risk of lower sensitivity (e.g., caused by administration of mold-active antifungal drugs) or by falsepositive results must always be taken into account. Regardless of recently published and generally accepted guidelines,25 26.3% of patients with IA in our database received a combination antifungal treatment, which was mainly a combination of voriconazole and echinocandin, as an initial therapy of IA. This finding, which has also been reported in other registries,9,26 reflects the real-life situation, where the treating physician intends to maximize the efficacy of antifungal treatment in this group of highly immunocompromised and frequently critically ill patients, not only at the time when the initial treatment fails, but ideally at the start of therapy. However, regardless of promising results from in vitro27,28 and animal studies,29 there is limited evidence for such an approach in the salvage setting,30,31 and more in the initial treatment32,33 of IA in the literature. Although our study was retrospective and not randomized, we did not find any difference in the efficacy of voriconazole monotherapy compared to the combination of this azole with echinocandin when used as an initial or salvage therapy. The number of patients with neutropenia (<1.0  109 /l) and the length of therapy were not different between treatment groups. However, we did not collect information about performance status, and therefore we cannot exclude the possibility that patients with a severe clinical condition did not preferentially receive a combination therapy, at least during the initial treatment. Therefore, in order to finally resolve this issue, we should await the results of randomized studies comparing both of these approaches that are currently being conducted. Finally, even with the availability of new antifungal therapies, a large number of patients still fail. Therefore, the actual immunodeficiency status of each patient will play a crucial role in the treatment outcome. Although neutropenia was the most frequent risk factor found for the development of IA, the neutrophil level, in addition to the antifungal therapy used for treatment, would have an impact on patient prognosis.34 Cordonnier et al. found no impact of neutropenia on patient prognosis at the time of IA diagnosis.35 Similarly, in our analysis we did not find any significant role of neutrophil count at the start of antifungal therapy on the efficacy of antifungal treatment (primary as well as salvage). However, similar to data presented by Pagano et al.,2 which showed that acute myeloid leukemia patients with IA had a higher response rate when they had neutropenic recovery, we found a statistically significant increase in the response rate when the neutrophil count measured at the end of antifungal therapy had increased, regardless of the antifungal drug used for treatment. However, our sub-analysis found this crucial role of neutrophil count at the end of treatment was significant for primary therapy, but was not significant for salvage treatment, which was most likely due to the limited number of patients undergoing salvage therapy. An increase in neutrophil count greater than 1.0  109 /l during initial therapy was related to a complete and partial response, while patients with progression remained neutropenic. However, we found that the outcome of therapy in patients receiving a second-line treatment may be dependent on factors other than the development of neutrophil count during or at the end of therapy, such as the presence of graft-versus-host disease, persistent corticosteroid use, or hepatic insufficiency.34 In conclusion, IA is a life-threatening condition and the most frequent IFD in patients with hematological malignancies that requires rapid and specific diagnostics. Lung HRCT with high sensitivity allows for the detection of pulmonary abnormalities; however, these scans are often very non-specific. Therefore, the combination of HRCT with routine and regular screening of GM in serum and/or BAL fluid provides a better differential and rapid diagnosis of IA in this group of immunocompromised patients. While we do not have data that clearly support the benefit of combination antifungal treatment, we have clearly shown that the development of neutrophil count during IA treatment will be a key factor that will determine the treatment response regardless of the antifungal drug or strategy used. Acknowledgements We would like to thank all FIND database participating centers and colleagues: Department of Internal Medicine – Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic: Zdenek Racil, Barbora Weinbergerova, Jiri Mayer; Department of Microbiology, University Hospital Brno, Brno, Czech Republic: Iva Kocmanova; Institute of Hematology and Blood Transfusion, Prague, Czech Republic: Michal Kouba, Petr Cetkovsky; Second Department of Oncology, Comenius University and National Oncology Institute, Bratislava, Slovakia: Lubos Drgona, Lucia Masarova; Department of Hematology and Oncohematology, Louis Pasteur University Hospital, Kosice, Slovakia: Elena Tothova, Tomas Guman; Department of Hemato-oncology, General Faculty Hospital, Prague, Czech Republic: Jan Haber, Kristina Forsterova; Department of Hematology and Transfusiology, St. Cyril and Methodius Hospital – University Hospital Bratislava, Bratislava, Slovakia: Eva Bojtarova, Barbora Ziakova; Department of Hematology, University Hospital Kralovske Vinohrady, Prague, Czech Republic: Jan Vydra, Toma´sˇ Koza´k; Department of Pediatric Oncology, Masaryk University and University Z. Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109e108 Hospital Brno, Brno, Czech Republic: Peter Mudry, Renata Foralova, Jaroslav Sˇteˇrba; Department of Pediatric Hematology and Oncology, Pediatric University Hospital Bratislava, Bratislava, Slovakia: Daniela Sejnova; Laboratory of Parasitology and Mycology, Ceske Budejovice Hospital, Ceske Budejovice, Czech Republic: Nada Mallatova; Department of Pediatrics, Ceske Budejovice Regional Hospital, Ceske Budejovice, Czech Republic: Alena Smrckova. This work was supported by CELL – The CzEch Leukemia Study Group for Life – and by a research grant from the Ministry of Health (IGA NS10442-3/2009 and IGA NS10441-3/2009) of the Czech Republic. CELL received unrestricted grants for the FIND database from Astellas, Teva-Cephalon, Pfizer, and Merck Sharp and Dohme. Conflict of interest: ZR has served at the speakers’ bureau of Pfizer and Astellas Pharma, and has been a consultant to Astellas Pharma. LD has served at the speakers’ bureau of Pfizer and Merck Sharp and Dohme, and has been consultant to Pfizer, Astellas Pharma, Teva-Cephalon, and Merck Sharp and Dohme. JH has served at the speakers’ bureau of Pfizer, Astellas Pharma, Merck Sharp and Dohme, and Teva-Cephalon. JMa has served at the speakers’ bureau of Pfizer, Astellas Pharma, and Merck Sharp and Dohme, and has received scientific grants from Pfizer, Astellas Pharma, and Merck Sharp and Dohme. All other authors declare no competing financial interests. References 1. Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, et al. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001–2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) database. Clin Infect Dis 2010;50: 1091–100. 2. Pagano L, Caira M, Candoni A, Offidani M, Martino B, Specchia G, et al. 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Racil et al. / International Journal of Infectious Diseases 17 (2013) e101–e109 e109 Fusion Status in Patients With Lymph Node-Positive (N1) Alveolar Rhabdomyosarcoma Is a Powerful Predictor of Prognosis: Experience of the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) Soledad Gallego, MD, PhD 1 ; Ilaria Zanetti, MD2 ; Daniel Orbach, MD3 ; Dominique Ranche`re, MD4 ; Janet Shipley, FRCPath5 ; Angelica Zin, MD6 ; Christophe Bergeron, MD4 ; Gian Luca de Salvo, MD7 ; Julia Chisholm, MD8 ; Andrea Ferrari, MD9 ; Meriel Jenney, MD10 ; Henry C. Mandeville, MD8 ; Timothy Rogers, MD11 ; Johannes H.M. Merks, MD 12 ; Peter Mudry, MD13 ; Heidi Glosli, MD14 ; Giuseppe Maria Milano, MD15 ; Sima Ferman, MD16 ; and Gianni Bisogno, MD2 ; on behalf of the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) with lymph node involvement (N1 classification) accounts for up to 10% of all cases of RMS. The prognosis is poor, and is comparable to that of distant metastatic disease. In the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS2005 protocol, patients with a histologic diagnosis of aRMS/N1 received intensified chemotherapy with systematic locoregional treatment. METHODS: Patients with aRMS/N1 were enrolled prospectively after primary surgery/ biopsy and fusion status was assessed in tumor samples. All patients received 9 cycles of induction chemotherapy and 6 months of maintenance therapy. Local treatment included radiotherapy to the primary site and lymph nodes with or without secondary surgical resection. RESULTS: A total of 103 patients were enrolled. The clinical characteristics of the patients were predominantly unfavorable: 90% had macroscopic residual disease after initial surgery/biopsy, 63% had locally invasive tumors, 77% had a tumor measuring >5 cm, and 81% had disease at unfavorable sites. Fusion genes involving forkhead box protein O1 (FOXO1) were detected in 56 of 84 patients. Events occurred in 52 patients: 43 developed disease recurrence, 7 had disease that was refractory to treatment, and 2 patients developed second neoplasms. On univariate analysis, unfavorable disease site, tumor invasiveness, Intergroup Rhabdomyosarcoma Study group III, and fusion-positive status correlated with worse prognosis. The 5-year event-free survival rate of patients with fusion-positive tumors was 43% compared with 74% in patients with fusion-negative tumors (P 5.01). On multivariate analysis, fusion positivity and tumor invasiveness proved to be unfavorable prognostic markers. CONCLUSIONS: Fusion status and tumor invasiveness appear to have a strong impact on prognosis in patients with aRMS/N1. Fusion status will be used to stratify these patients in the next EpSSG RMS study, and treatment will be intensified in patients with fusion-positive tumors. Cancer 2018 American Cancer Society. KEYWORDS: alveolar rhabdomyosarcoma, lymph node involvement, paired box (PAX)-forkhead box protein O1 (FOXO1) fusion, prognostic factors, rhabdomyosarcoma. INTRODUCTION Rhabdomyosarcoma (RMS) is one of the most frequent extracranial solid tumors diagnosed in children and the most common form of soft-tissue sarcoma diagnosed in children and young adults.1 The prognosis of patients with localized RMS has improved considerably over time thanks to numerous clinical trials conducted by collaborative groups working in North America (Children’s Oncology Group [COG]) and Europe (International Society of Pediatric Oncology [SIOP] Malignant Mesenchymal Tumor Group [MMT], Italian Soft Tissue Sarcoma Committee [STSC], and German Cooperative Soft Tissue Sarcoma Study Group [CWS]). The presence of disseminated disease at the time of diagnosis is Corresponding author: Soledad Gallego, MD, PhD, Pediatric Oncology and Hematology, Children’s Hospital Vall d’Hebron, P Vall d’Hebron 119-129, 08035 Barcelona, Spain; sgallego@vhebron.net 1 Pediatric Oncology and Hematology, Children’s Hospital Vall d’Hebron, Barcelona, Spain; 2 Padova University Hospital, Padova, Italy; 3 Pediatric Oncology, SIREDO Oncology Center, Institute Curie, Paris Sciences and Letters University, Paris, France; 4 IHOPE/Center Leon Berard, Lyon, France; 5 Institute of Cancer Research, London, United Kingdom; 6 Pediatric Research Institute Citta della Speranza, Padova, Italy; 7 Clinical Trials and Biostatistics Unit, Veneto Oncologic Institute IOV-IRCCS, Padova, Italy; 8 The Royal Marsden National Health Service Foundation Trust, Sutton, United Kingdom; 9 National Tumor Institute, Milan, Italy; 10 Pediatric Oncology, Children Hospital for Wales Cardiff and Vale University Health Board, Cardiff, United Kingdom; 11 University Hospitals Bristol National Health Service Foundation Trust, Bristol, United Kingdom; 12 Pediatric Oncology, Emma Children’s Hospital-Academic Medical Center, Amsterdam, The Netherlands; 13 Pediatric Oncology, University Children’s Hospital Brno, Brno, Czech Republic; 14 Pediatric Oncology, Oslo University Hospital, Oslo, Norway; 15 Pediatric Oncology, Children’s Hospital Bambino Gesu, IRCCS, Rome, Italy; 16 Pediatric Oncology, National Cancer Institute, Rio de Janeiro, RJ, Brazil We thank Ms. Christine O’Hara for English language correction. Additional supporting information may be found in the online version of this article. DOI: 10.1002/cncr.31553, Received: March 27, 2018; Revised: April 19, 2018; Accepted: April 23, 2018, Published online May 24, 2018 in Wiley Online Library (wileyonlinelibrary.com) Cancer Original Article 3201 2018;124:3201-9. VC August 1, 2018 the most powerful prognostic factor in RMS. Although the probability of cure in pediatric patients with localized disease is >70%, the prognosis of those with distant metastatic disease remains poor.2-8 In patients with localized disease, clinical and tumor characteristics have been used to classify RMS into different risk categories and to determine treatment intensity. Unfavorable characteristics include alveolar histology, invasive tumor (T2 classification), tumor location, lymph node involvement, tumor size >5 cm, and patient age 10 years9-11 and constitute the basis for the risk stratification system used in the recent European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS2005 study. Previous experience has suggested that patients with alveolar RMS (aRMS) and regional lymph node involvement represent a group with a particularly poor prognosis.11 Approximately 70% of patients with aRMS present with the fusion genes paired box 3 (PAX3)-forkhead box protein O1 (FOXO1) or paired box 7 (PAX7)-FOXO1 as a consequence of the reciprocal chromosomal translocations t(2;13)(q35;q14) or t(1;13)(p36;q14).12 Recent data have suggested that the PAX3/7-FOXO1 fusion genes have prognostic significance.13,14 This observational study reports on the results obtained in this very high-risk population, and focuses on the prognostic role of fusion gene status. MATERIALS AND METHODS Patients The RMS2005 protocol was initiated in October 2005 and opened in 14 countries. Eligibility criteria for inclusion in the RMS2005 protocol were age >6 months to <21 years, a pathologically proven diagnosis of RMS, no evidence of distant metastatic lesions, tumor previously untreated except for primary surgery, no preexisting illness preventing treatment, no previous malignant tumors, and an interval between diagnostic surgery and treatment of 8 weeks. Patients with localized aRMS and regional lymph node involvement (N1 classification) were assigned to the very high-risk group according to the EpSSG stratification system. This group is the focus of the current analysis, with particular attention to the group of patients who underwent molecular analysis of PAX3/7FOXO1 fusions. Only patients enrolled before December 31, 2013 were included in this analysis to ensure an adequate follow-up. The cutoff date for the analysis was April 4, 2017. Staging Disease was staged according to the TNM classification and the Intergroup Rhabdomyosarcoma Study Group (IRS) postsurgical grouping system.15 Regional lymph node involvement was indicated as N0 or N1 and distant metastases at the time of onset as M0 or M1 based on histologic or clinical/radiologic assessments. Tumor location was considered favorable if arising from the orbit, genitourinary region other than the bladder or prostate (ie, paratesticular and vagina/uterus), and nonparameningeal head and neck, and was considered unfavorable when arising from any other site. Regional lymph nodes were defined as those appropriate to the site of the primary tumor. Any evidence of distant lymph node involvement other than these was considered metastasis and patients were treated according to the protocol for those with metastatic disease at the time of diagnosis. Surgical exploration of regional lymph nodes was mandatory in cases of RMS arising in the limbs. In tumors originating in other locations, regional lymph node involvement was determined clinically and by imaging, including magnetic resonance imaging and/or positron emission tomography (PET)-computed tomography scan. In doubtful cases, a lymph node biopsy was recommended. Systematic sentinel lymph node examination was suggested but implemented only at a small number of centers. Treatment Patients received intensified initial chemotherapy and additional maintenance chemotherapy with systematic local treatment to the primary and lymph node sites. Induction chemotherapy comprised 4 cycles of 21 days each of ifosfamide at a dose of 3 g/m2 on days 1 to 2 with mesna; vincristine at a dose of 1.5 mg/m2 (maximum, 2 mg) on days 1, 8, and 15 in the first 2 cycles and day 1 in cycles 3 and 4; actinomycin D at a dose of 1.5 mg/m2 (maximum, 2 mg) on day 1; and doxorubicin at a dose of 30 mg/m2 on days 1 to 2 (IVADo) followed by 5 cycles of 21 days each of ifosfamide at a dose of 3 g/m2 on days 1 to 2 with mesna, vincristine at a dose of 1.5 mg/m2 on day 1, and actinomycin D at a dose of 1.5 mg/m2 on day 1 (IVA) and 6 cycles of 28 days each of maintenance chemotherapy comprising continuous daily oral cyclophosphamide at a dose of 25 mg/m2 and intravenous vinorelbine at a dose of 25 mg/m2 on days 1, 8, and 15 of each cycle.16 Local treatment after the initial 4 cycles of IVADo (week 13) included delayed (secondary) surgery to remove macroscopic residual tumor and radiotherapy (RT). External beam RT was scheduled to be given to the primary tumor area and the affected lymph node region. Doses varied according to chemotherapy response and surgical results and were administered in 1.8-gray (Gy) Cancer3202 August 1, 2018 Original Article daily fractions. The total dose to the primary tumor in postsurgical IRS group II and group III patients with complete remission after secondary surgery was 41.4 Gy. For patients in IRS group III with incomplete secondary resection or when secondary surgery was not feasible, the total dose was 50.4 Gy with an optional additional boost of 5.4 Gy in 3 fractions for large tumors with poor responses to chemotherapy. RT to the involved lymph nodes was recommended at a dose of 41.4 Gy regardless of surgical resection. Treatment was delivered with megavoltage photons at 1 fraction per day for 5 days per week. Response was evaluated after initial chemotherapy (week 9) and at the end of treatment by 3-dimensional volumetric assessment using the formula: tumor volume (cm3 ) 5 0.52 3 length (cm) 3 width (cm) 3 thickness (cm). Responses were defined as complete response (clinically or histologically confirmed complete disappearance of disease), partial response (at least a two-thirds reduction in tumor volume), minor response (a reduction in tumor volume greater than one-third but less than two-thirds), stable disease (a modification in tumor volume of less than one-third), and progressive disease (an increase in tumor size >30% or the detection of new lesions). The site of first disease recurrence was defined as local if the tumor recurred at the site of primary disease, lymph node if regional lymph nodes were involved, locoregional in cases of local and lymph node disease recurrence, distant in cases with the appearance of metastatic disease, and combined when locoregional plus metastatic disease recurrence were evident. Pathology and Biology Histologic analysis was performed locally at participating EpSSG centers using routine hematoxylin and eosin staining. Following protocol guidelines, a panel of appointed pathologists reviewed 2 to 12 tumor slides from each patient and confirmed the diagnosis of aRMS. The molecular characterization of aRMS was part of several translational studies to be implemented in the RMS2005 protocol. The analysis was strongly recommended and should be conducted at a single laboratory for each participating national group. However, fusion status data were not available for the entire population because of a shortage of suitable or fresh biologic material. Molecular analysis of the PAX3/7-FOXO1 fusion was performed by fluorescent in situ hybridization (FISH) in paraffin blocks and/or by reverse transcriptase-polymerase chain reaction (RT-PCR) in frozen tissue. Interphase and metaphase FISH studies for RMS translocations were performed using chromosome 13 cosmids flanking the FOXO1 gene using a commercial break-apart probe as described.12 RNA from snap-frozen tumor was assayed by single-round RT-PCR using the primer pairs and conditions as described.17 Only samples with a sufficient number of tumor cells (>50%) were considered for the analysis. Alternative PAX3 fusions with partners other than FOXO1 were not analyzed. Samples with FOXO1 gene disruption (ie, positive PAX3-FOXO1, PAX7FOXO1, or FOXO1 with an unknown gene partner) were considered fusion status positive. Statistical Analysis Data were collected via a Web-based system and analyzed at Veneto Oncologic Institute (Padova, Italy). Continuous variables were summarized with the median, minimum, and maximum, whereas categorical variables were reported as counts and percentages. Survival was calculated from the date of diagnosis to the time of the event or last follow-up. Tumor progression, disease recurrence, occurrence of a second malignancy, or death due to any cause were considered for event-free survival (EFS). Overall survival (OS) was measured from the date of diagnosis to the date of death from any cause. Patients who still were alive at the end of the study were censored at the date of the last observation. Survival probability was computed using the Kaplan-Meier method and heterogeneity in survival among strata of selected variables was assessed with the log-rank test. The 5-year EFS and OS rates of the patient subgroup with available molecular data were reported along with their 95% confidence intervals (95% CIs), computed using the Greenwood formula. The Cox proportional hazards regression method was used to ascertain whether fusion-positive status may have prognostic significance in this cohort of patients. A stepwise variable selection procedure was applied to the covariates with a P value  .25 in the univariate analysis. Hazard ratios (HRs) with 95% CIs according to the Wald method were reported for independent selected variables. All data analyses were performed using the SAS statistical package (release 9.4; SAS Institute Inc, Cary, North Carolina). Ethics The EpSSG RMS2005 treatment protocol was submitted to the institutional and national review boards of each participating country for review and approval before the enrollment of patients. Written informed consent for participation was obtained from patients, parents, or legal guardians in all cases. The study was conducted in Cancer 3203August 1, 2018 Fusion Status Predicts Prognosis in aRMS N1/Gallego et al accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines (European Union Drug Regulating Authorities Clinical Trials EUDRACT No. 2005-000217-35). RESULTS Patient and Tumor Characteristics From December 2005 to December 2013, a total of 103 patients with aRMS/N1 were included, accounting for 8.1% of all patients (1272 patients) enrolled in the EpSSG RMS2005 protocol. PAX3/7-FOXO1 fusion was analyzed in 85 patients (82.5%). FOXO1 gene disruption was detected by FISH or RT-PCR in 56 patients, 31 of whom had PAX3-FOXO1, 8 of whom had PAX7FOXO1, and 17 of whom were FOXO1 positive with an unknown gene partner. Twenty-eight patients had fusionnegative tumors and 1 sample was inadequate for analysis. Molecular study was not performed in 18 patients. The clinical characteristics of the entire cohort (Table 1) demonstrated a predominance of unfavorable prognostic factors: 90% of patients had IRS group III tumors, 81% of tumors were located at unfavorable sites, 77% of tumors measured> 5cm, invasive (classified as T2) tumors represented approximately 63% of all cases, and approximately 50% of patients were aged >10 years. No significant differences were found with regard to the prognostic factors considered in the current study between patients with or without biologic data, with the exception of the predominance of age 10 years in the group without molecular study (P5 .0339). For this reason, inferential statistical analyses were performed in patients with available biological data. Treatment Chemotherapy Of the 103 enrolled patients, 73 received chemotherapy as per protocol and 30 received treatment with modifications. Of these 30 patients, 19 had interrupted chemotherapy before completing treatment (18 because of progressive disease or disease recurrence and the parents of 1 patient refused to continue treatment) and in 11 patients chemotherapy was modified because of (CTCAE v4.03) toxicity in 2 patients (septic shock and hemorrhagic cystitis, respectively), a lack of tumor response in 2 patients, and by the attending physician’s decision in 7 patients. All patients presented with at least 1 episode of TABLE 1. Clinical Characteristics of Patients With aRMS and Lymph Node Involvement (N1 Classification) Molecular Biology Not Performed N518 Molecular Biology Performed N585 Total N5103 Characteristic No. of Patients % No. of Patients % No. of Patients % P Age at diagnosis <10 y 5 27.8 47 55.2 52 50.5 .0339 10 y 13 72.2 38 44.8 51 49.5 Sex Female 6 33.3 35 41.2 41 39.8 .5369 Male 12 66.7 50 58.8 62 60.2 Postsurgical IRS group II 1 5.6 9 10.6 10 9.7 .5124 III 17 94.4 76 89.4 93 90.3 Tumor invasiveness (T classification) T1 5 27.8 33 38.8 38 36.9 .3776 T2 13 72.2 52 61.2 65 63.1 Tumor size a: 5 cm 3 16.7 20 23.5 23 22.3 0.7224 b: >5 cm 15 83.3 64 75.3 79 76.7 x: Not evaluable - - 1 1.2 1 1.0 Site of origin of primary tumor Favorable site 1 5.6 19 22.4 20 19.4 0.1017 Unfavorable site 17 94.4 66 77.6 83 80.6 Fusion status PAX3-FOXO1 positive - - 31 36.5 31 30.1 PAX7-FOXO1 positive - - 8 9.4 8 7.8 FOXO1 positive - - 17 20.0 17 16.5 FOXO1 negative - - 28 32.9 28 27.2 Sample inadequate - - 1 1.2 1 0.9 Not analyzed 18 100.0 - - 18 17.5 Abbreviations: aRMS, alveolar rhabdomyosarcoma; FOXO1, forkhead box protein O1; IRS, Intergroup Rhabdomyosarcoma Study; PAX3, paired box 3; PAX7, paired box 7. Cancer3204 August 1, 2018 Original Article grade 3 to 4 hematologic toxicity. The most frequent nonhematologic toxicity was gastrointestinal (mucositis) and neurologic (peripheral neuropathy and ileus) (see Supporting Table 1). Surgery Ten patients (10%) underwent primary surgery: 6 in IRS group IIb (primary complete resection without microscopic residual disease and lymph node involvement) and 4 in IRS group IIc (primary complete resection with microscopic residual disease and lymph node involvement). A total of 48 patients underwent secondary surgery (resection of the primary tumor in 29 patients, combined resection of the tumor and lymph nodes in 15 patients [1 bilateral lymphadenectomy, 7 unilateral lymphadenectomies, and 7 biopsies], and surgery to the lymph nodes alone in 4 patients [2 biopsies and 2 unilateral lymphadenectomies]). Among the 44 patients who underwent delayed surgical resection of the primary tumor, complete local resection (R0) was performed in 29 patients, with microscopic residual disease (R1) noted in 8 patients, macroscopic residual disease (R2) noted in 4 patients, and no residual tumor noted in 3 patients. Radiotherapy Overall, 92 of 103 patients (89.3%) were irradiated. RT was not administered because of progressive disease in 4 patients, amputation in 2 patients, parental refusal in 2 patients, and physician decision in 3 patients. Eight patients received irradiation to the primary tumor area alone, 81 to the primary tumor and lymph nodes, and 3 to lymph nodes alone (2 patients after limb amputation and 1 with a completely resected primary tumor at the time of diagnosis). The median dose to the primary tumor for the overall population was 50.4 Gy (range, 36.0-59.4 Gy) and that to the lymph nodes was 41.4 Gy (range, 24.0-54.4 Gy). Fifteen of 103 patients were aged 3 years: 11 received RT and 4 did not receive RT because of parent refusal in 1 patient, physician decision in 1 patient, and tumor progression before the initiation of RT in 2 patients. Outcome With a median follow-up of 64.9 months (range, 19.8- 116.3 months), 52 patients developed an event and 47 died of disease. Seven patients had refractory disease (no response or disease progression at week 9) and presented with early disease progression (median time to disease progression of 6.2 months [range, 2.1-9.7 months]), 2 patients developed secondary neoplasms, and 43 patients developed disease recurrence. The site of the first recurrence was local in 10 patients, lymph node in 6 patients, and locoregional in 2 patients. Seventeen patients had distant disease recurrence and 8 patients had combined disease recurrence. Local and locoregional events (18 of 43 patients) accounted for approximately 42% of the cases of disease recurrence and lymph node recurrences were present in 13 patients as the first event (33%). The median TABLE 2. Association Between Potential Prognostic Factors and Outcome in Patients With Fusion Status Analyzed EFS OS No. of Patients Failed 5-Year (95% CI) P Failed 5-Year (95% CI) P Age <10 y 47 18 60.4 (44.7-73.0) .0596 16 60.6 (43.4-74.0) .0797 10 y 37 22 44.0 (27.3-59.5) 19 47.9 (30.2-63.6) Tumor size 5 cm 20 7 64.3 (39.3-81.2) .3475 7 59.8 (32.9-78.8) .6395 >5 cm 63 33 49.9 (36.8-61.6) 28 52.8 (38.6-65.1) Tumor invasiveness (T classification) T1 33 10 67.3 (47.3-81.1) .0137 7 71.5 (48.6-85.5) .0040 T2 51 30 44.8 (30.8-57.8) 28 45.2 (30.8-58.5) Fusion status Positive 56 33 43.0 (29.5-55.7) .0101 28 45.5 (30.8-59.2) .0548 Negative 28 7 74.4 (53.6-87.0) 7 73.7 (52.4-86.6) IRS group II 9 1 88.9 (43.3-98.4) .0367 1 87.5 (38.7-98.1) .0533 III 75 39 49.0 (37.0-60.0) 34 51.0 (38.1-62.6) Site of primary tumor Favorable site 19 4 75.7 (46.9-90.3) .0177 3 81.2 (51.9-93.6) .0293 Unfavorable site 65 36 46.9 (34.2-58.5) 32 48.2 (34.7-60.4) Abbreviations: 95% CI, 95% confidence interval; EFS, event-free survival; IRS, Intergroup Rhabdomyosarcoma Study; OS, overall survival. Cancer 3205August 1, 2018 Fusion Status Predicts Prognosis in aRMS N1/Gallego et al time from diagnosis to disease recurrence was 16.4 months (range, 2.1-63.5 months). At the time of last follow-up, among the 5 patients surviving tumor recurrence, 3 were alive with disease and 2 were in complete response after second-line chemotherapy and RT. The 5year EFS rate for the entire population was 50.1% (95% CI, 39.8%-59.5%) and the 5-year OS rate was 50.6% (95% CI, 39.7%-60.5%). The median time from first event to death was 8.8 months (range, 0-41.0 months). In the univariate analysis performed in the group of patients for whom fusion status data were available (Table 2), the following factors were found to be associated with an increased risk of disease recurrence or death: unfavorable primary site, invasive tumor (T2 classification), the presence of the FOXO1 translocation, and classification into IRS group III. Significant variables (P < .25) emerged from univariate analysis (patient age at the time of diagnosis, primary tumor site, tumor invasiveness, fusion status, and IRS group) and were included in the Cox model. Only fusion gene status and tumor invasiveness remained as independent prognostic factors for the risk of disease recurrence. Fusion-positive aRMS was associated with EFS with an HR of 2.6 (95% CI, 1.1-5.9; P 5 .0226) and tumor invasiveness (T2 classification) was associated with an HR of 2.2 (95% CI, 1.1-4.6; P 5 .0296). Fusion gene status and tumor invasiveness also remained as independent prognostic factors for the risk of death with an HR associated with fusion-positive tumors of 2.5 (95% CI, 1.1-5.6; P 5 .0300) and an HR related to tumor invasiveness (T2 classification) of 2.2 (95% CI, 1.1-4.6; P 5 .0298). The 5-year EFS rate in patients with fusionpositive tumors was 43.0% (29.6%-55.7%) compared with 74.4% (53.6%-86.9%) in those with fusion-negative tumors (P 5 .0101) (Fig. 1). The 5-year OS rate for patients with fusion-positive tumors was 45.5% (95% CI, 30.8%-59.2%) compared with 74.7% (95% CI, 52.4- 86.6) for patients with fusion-negative tumors (P 5 .0548) (Fig. 2). DISCUSSION The results of the current study provide evidence of the prognostic impact of fusion status and tumor invasiveness in patients with aRMS and lymph node involvement. Results from previous European and North American cooperative studies have demonstrated very poor survival in patients with aRMS and lymph node involvement, who account for up to 10% of all patients with RMS. In the CWS/RMS86 study, the 3-year EFS rate was 28% and the OS rate was 29%.18 Results in the SIOP experience were only slightly better, with a 5-year EFS rate of 39% in the SIOP MMT84 study,19 which is comparable to that of stage IV disease. The impact of lymph node involvement on prognosis in patients with RMS remains a matter of controversy. Rodary et al20 evaluated a cohort of 951 international patients with nonmetastatic RMS and identified tumor invasiveness, tumor size, primary tumor site, and N1 disease as prognostic factors. Similarly, in their analysis of patients with nonmetastatic RMS enrolled in American IRS protocols, Meza et al10 demonstrated that only stage of disease and IRS group were significantly associated with EFS for the majority of patients with aRMS. However, for patients in group III with aRMS, N1 disease was Figure 1. Kaplan-Meier curves representing 5-year event-free survival (EFS) by fusion status. The EFS rate for patients with fusion-positive tumors was 43% compared with 74.4% for those with fusion-negative tumors (P 5.01). Figure 2. Kaplan-Meier curves representing 5-year overall survival (OS) by fusion status. The OS rate for patients with fusion-positive tumors was 45.5% compared with 74.7% for those with fusion-negative tumors (P 5.05). Cancer3206 August 1, 2018 Original Article associated with poorer EFS and OS. These observations influenced the development of the current EpSSG treatment protocol, which assigned patients with aRMS of N1, but not embryonal N1 RMS, to the very high-risk group, for whom a more intensive treatment was recommended.21 Rodeberg et al22 investigated the contribution of regional lymph node disease to the prognosis of patients enrolled in the IRS-IV study. They included 125 patients with localized RMS and lymph node involvement. Patients with alveolar histology and positive lymph nodes were found to have significantly worse 5-year failure-free survival compared with those with alveolar histology without lymph node involvement (43% and 73%, respectively). Moreover, in patients with alveolar histology and N1 disease, outcomes were more similar to those of patients with solitary metastatic disease compared with patients with N0 disease. These results are consistent with the results of the current study. The main difference between the aforementioned study and the current report is that the former included both alveolar and embryonal tumors with lymph node involvement; however, as in the current study, patients with tumors located at unfavorable sites, those with disease at advanced stages, and those with large and invasive tumors were predominant. All these characteristics have been associated with an increased risk of distant metastatic disease.3,5,23,24 Conversely, involvement of regional lymph nodes in patients with embryonal tumors did not prove to have any negative effect on outcome in the study by Rodeberg et al22 or in the more recent report by Rogers et al.25 This could be due at least in part to the intensified treatment with RT and chemotherapy administered, suggesting that patients with lymph node-positive embryonal tumors can attain equivalent outcomes when given intensified treatment. To the best of our knowledge, the overall outcome of the current study cohort was better than the historical series reported to date. The reasons for the apparent improvement in outcome among these patients could be due in part to better risk stratification, more adequate treatment with intensified chemotherapy, systematic local treatment, and improvements in supportive care. In the current study, a significant number of patients had tumors that did not respond to initial chemotherapy and these individuals presented with progressive disease shortly after diagnosis, thereby representing 14% of those patients who developed disease recurrence. A recent report from Vaarwerk et al26 demonstrated the lack of correlation between early radiologic response and outcome in patients enrolled in the MMT95 protocol, even though patients with progressive disease were excluded from the analysis. It must be emphasized that the patients with progressive disease in the current study failed to respond to further treatment and the chance of cure after disease recurrence was very low (5% of the entire cohort), which suggests that patients with refractory disease or disease recurrence could be offered experimental therapy immediately after tumor events. Nevertheless, even with the implementation of combined local therapy with delayed surgery and systematic RT to the primary tumor site and lymph nodes in the current study protocol, locoregional disease recurrences were frequent and accounted for approximately 42% of the initial events. Furthermore, lymph node failures occurred in approximately 33% of the disease recurrences. Some authors have recommend that the in-transit lymphatics be imaged at the time of diagnosis.27 The involvement of in-transit lymph nodes could be better assessed by performing systematic [18 F]fludeoxyglucose (FDG) positron emission tomography-computed tomography at the time of diagnosis, a procedure that was not performed routinely in the cohort of patients in the current study. Moreover, the question of whether in-transit lymph nodes should be irradiated routinely remains unsolved, given the risk of significant toxicity associated with extensive irradiation in pediatric patients.28,29 In the current series, we identified some variables found to have prognostic significance on univariate analysis (unfavorable site of tumor origin, tumor invasiveness, FOXO1 fusion, and IRS group III). However, on multivariate analysis, only tumor invasiveness and the presence of a characteristic fusion gene associated with aRMS resulted in independent predictors of disease recurrence or death. This is consistent with several studies that correlated the presence of a fusion gene with poorer outcome; however, to the best of our knowledge, the real contribution of the presence of PAX3/7-FOXO1 fusions to the outcome of aRMS remains to be elucidated.30-32 In the current series, approximately 66% of tumors were fusion positive. These figures are lower than the rate of 70% to 75% reported in the literature, which could be due in part to the fact that fusions involving PAX3 with partners other than FOXO1 were missed in the current analysis.33 We will attempt to avoid these false-negative results in the future EpSSG protocol: in an alveolar tumor that is negative for PAX3/7-FOXO1 by RT-PCR and for FOXO1 rearrangement by FISH, additional FISH assessments for the disruption of PAX3 will be made. In the current study, fusion status appeared to identify the “real” very high-risk population, thereby Cancer 3207August 1, 2018 Fusion Status Predicts Prognosis in aRMS N1/Gallego et al highlighting the importance of performing biologic studies in all patients. We did not attempt to analyze outcome according to the type of fusion because of the limited number of patients with the PAX7-FOXO1 fusion. Survival in this newly defined very high-risk group is comparable to results observed in patients with metastatic aRMS treated in the high-risk COG studies D9802 and ARST0431.34 In those studies, fusion status was not found to be an independent prognostic factor, despite better EFS noted in patients with fusion-negative aRMS. Poorer outcomes for patients with metastatic disease in the COG report were most closely related to other clinical risk factors, including age, primary tumor site, and number of metastatic sites. The clinical implications of the current study will include a new stratification for patients with aRMS/N1 disease according to fusion status in the future EpSSG RMS study. Patients with fusion-negative N1 tumors will be treated with a strategy similar to that for those with eRMS/N1 disease, with no reduction in treatment intensity. Patients with fusion-positive N1 disease will be treated in the same group as patients with metastatic tumors. For patients with refractory disease or disease recurrence, the EpSSG is working to establish an effective, innovative strategy for the study of new agents and the inclusion of patients in phase 1 and 2 clinical trials. FUNDING SUPPORT Fondazione Citta della Speranza, Italy, has supported the overall organization of this study. CONFLICT OF INTEREST DISCLOSURES Soledad Gallego has acted in a paid advisory role for Loxo Oncology (one conference) and Clinigen Group (one conference) for work performed outside of the current study. Christophe Bergeron is supported by the Association Leon Berard Enfant Cancereux (ALBEC). Julia Chisholm has acted as a paid consultant for F. Hoffman La Roche for work performed outside of the current study and she and Henry Mandeville have received funding from the National Institute for Health Research (NIHR) Biomedical Research Centre at the Royal Marsden National Health Service Foundation Trust and the Institute of Cancer Research for work performed as part of the current study. Gianni Bisogno has acted in a paid advisory role for Loxo Oncology (one conference) and Clinigen Group (one conference) and as a paid member of the Speakers’ Bureau for Merck and Company and received a grant from INDENA S.p.A. for work performed outside of the current study. AUTHOR CONTRIBUTIONS Soledad Gallego: Conception and design, collection and assembly of data, and data analysis and interpretation. Ilaria Zanetti: Data analysis and interpretation. Gian Luca de Salvo: Conception and design and data analysis and interpretation. Gianni Bisogno: Conception and design, collection and assembly of data, and data analysis and interpretation. 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Histology, fusion status, and outcome in metastatic rhabdomyosarcoma: a report from the Children’s Oncology Group. Pediatr Blood Cancer. 2017;64(12). Cancer 3209August 1, 2018 Fusion Status Predicts Prognosis in aRMS N1/Gallego et al Original research Conservative strategy in infantile fibrosarcoma is possible: The European paediatric Soft tissue sarcoma Study Group experience Daniel Orbach a, *, Bernadette Brennan b , Angela De Paoli c , Soledad Gallego d , Peter Mudry e , Nadine Francotte f , Max van Noesel g , Anna Kelsey h , Rita Alaggio i , Dominique Ranche`re j , Gian Luca De Salvo c , Michela Casanova k , Christophe Bergeron l , Johannes H.M. Merks m , Meriel Jenney n , Michael C.G. Stevens o , Gianni Bisogno p , Andrea Ferrari k a Department of Pediatric, Adolescent and Young Adult Oncology, Institut Curie, Paris, France b Department of Pediatric Oncology, Royal Manchester Children’s Hospital, Manchester, United Kingdom c Clinical Trials and Biostatistics Unit, IRCCS Istituto Oncologico Veneto, Padova, Italy d Paediatric Oncology, Hospital Universitario Vall d’Hebron, Barcelona, Spain e Department of Pediatric Oncology, University Children’s Hospital, Brno, Czech Republic f Department of Pediatrics, CHC-Clinique Esperance, Montegne´e, Belgium g Princess Ma´xima Center for Pediatric Oncology, Utrecht, Netherlands h Department of Diagnostic Paediatric Histopathology, Royal Manchester Children’s Hospital, Manchester, United Kingdom i Pathology Department, Padova University, Padova, Italy j Pathology Department, Institut d’Hematologie et d’Oncologie Pediatrique, Centre Le´on Be´rard, Lyon, France k Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy l Department of Pediatric Oncology, Institut d’Hematologie et d’Oncologie Pe´diatrique, Centre Le´on Be´rard, Lyon, France m Department of Pediatric Oncology, Emma Children’s Hospital-Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands n Department of Pediatric Oncology, Children’s Hospital for Wales, Heath Park, Cardiff, United Kingdom o Department of Pediatric Oncology, Royal Hospital for Children, University of Bristol, United Kingdom p Pediatric Hematology and Oncology Division, Padova University, Padova, Italy Received 14 October 2015; received in revised form 29 December 2015; accepted 31 December 2015 Available online 2 February 2016 * Corresponding author: Pediatric Adolescent Young Adult Department, Institut Curie, 26, rue d’Ulm, 75005 Paris, France. Tel.: þ33 0 144324550; fax: þ33 0 153104005. E-mail address: daniel.orbach@curie.fr (D. Orbach). http://dx.doi.org/10.1016/j.ejca.2015.12.028 0959-8049/ª 2016 Elsevier Ltd. All rights reserved. Available online at www.sciencedirect.com ScienceDirect journal homepage: www.ejcancer.com European Journal of Cancer 57 (2016) 1e9 KEYWORDS Infantile fibrosarcoma; Newborn; Infant; Cancer; Chemotherapy; ETV6-NTRK3 transcript Abstract Background: Infantile fibrosarcoma (IFS) is a very rare disease occurring in young infants characterised by a high local aggressiveness but overall with a favourable survival. To try to reduce the total burden of therapy, the European pediatric Soft tissue sarcoma Study Group has developed conservative therapeutic recommendations according to initial resect- ability. Material and methods: Between 2005 and 2012, children with localised IFS were prospectively registered. Initial surgery was suggested only if possible without mutilation. Patients with initial complete (IRS-group I/R0) or microscopic incomplete (group II/R1) resection had no further therapy. Patients with initial inoperable tumour (group III/R2) received first-line vincristine-actinomycin-D chemotherapy (VA). Delayed conservative surgery was planned after tumour reduction. Aggressive local therapy (mutilating surgery or external radiotherapy) was discouraged. Results: A total of 50 infants (median age 1.4 months), were included in the study. ETV6NTRK3 transcript was present in 87.2% of patients where investigation was performed. According to initial surgery, 11 patients were classified as group I, 8 as group II and 31 as group III. VA chemotherapy was first delivered to 25 children with IRS-III/R2 and one with IRS-II/ R1 disease. Response rate to VA was 68.0%. Mutilating surgery was only performed in three cases. After a median follow-up of 4.7 years (range 1.9e9.0), 3-year event-free survival and overall survival were respectively 84.0% (95% confidence interval [CI] 70.5e91.7) and 94.0% (95% CI 82.5e98.0). Conclusions: Conservative therapy is possible in IFS as only three children required mutilating surgery, and alkylating or anthracycline based chemotherapy was avoided in 71.0% of patients needing chemotherapy. VA regimen should be first line therapy in order to reduce long term effects. ª 2016 Elsevier Ltd. All rights reserved. 1. Introduction Although infantile fibrosarcoma (IFS) is a rare tumour, it is the commonest soft tissue sarcoma in children less than 1 year of age. IFS is currently classified as a soft tissue tumour of intermediate malignancy characterised by a quite specific t(12;15)(p13;q25) translocation coding for a ETV6-NTRK3 gene fusion [1e3]. It arises below the age of 2e5 years with survival rates between 80 and 100% [1,4,5]. It often presents with initial rapid growth, sometimes with indolent evolution and metastatic spread is uncommon (1e13%). Local recurrence may occur after initial conservative surgery (17e43%), the latter being the mainstay of treatment, aiming for a conservative resection. However, IFS may present with locally advanced disease and surgery maybe mutilating or cause functional damage [4,5]. Since IFS is a chemosensitive tumour, chemotherapy may play a major role in the treatment strategy [1,6,7]. Recently, the VA regimen (vincristine-actinomycin-D), has confirmed its efficacy and allows important tumour reduction [1]. The International Society of Pediatric OncologyeMalignant Mesenchymal Tumour Committee and the Associazione Italiana Ematologia Oncologia PediatricaeSoft Tissue Sarcoma Committee (previously called the Italian Cooperative Group) founded the European-paediatricSoft-tissue-Sarcoma-Study Group (EpSSG) in 2005. The group developed treatment guidelines for IFS, with the major goal to make uniform the treatment of IFS patients across Europe, according to a conservative approach based on non-mutilating surgery and alkylating-anthracycline-free chemotherapy (EpSSG non-rhabdomyosarcoma soft tissue sarcomas [NRSTS] 2005 study e European Union Drug Regulating Authorities Clinical Trial No. 2005-001139-31) This present paper reports the results of a prospective cohort of IFS patients treated between 2005 and 2012 aiming to propose a conservative strategy in this disease. 2. Patients and methods 2.1. Study population All infants aged from birth to 24 months with localised IFS were prospectively registered in the EpSSG database using a web-based system, from October 2005 to 30th June 2012. Patients were classified according specific tumour sites [8]. Clinical staging was defined according to the tumour node metastases system: T1 or T2 according to the invasion of contiguous organs; N0/N1, and M0/M1 according to the presence of lymph node or distant metastases [8]. Lymph node involvement was evaluated clinically or by imaging and confirmed when necessary by cytological or histological biopsy. The status of resection margins was classified according to the UICC-R classification and the Intergroup D. Orbach et al. / European Journal of Cancer 57 (2016) 1e92 Rhabdomyosarcoma Staging (IRS) system which is generally used for primary surgery in paediatric rhabdomyosarcomas [9]. UICC-R R0 or IRS group I correspond to complete tumour resection with histologically free margins, UICC-R R1 or IRS II correspond to macroscopic resection, but invaded margins on histology, UICC-R R2 or IRS III correspond to macroscopic residual tumour after surgery (III b) or biopsy (III a). Patients with metastatic tumours were excluded from the analysis. Cytogenetic and molecular evaluation to identify the presence of ETV6-NTRK3 transcript derived by the specific translocation by FISH and RT-PCR were recommended to confirm the diagnosis [10]. Where there was doubt, tumours were prospectively reviewed at the time of diagnosis by national and/or international panel of pathologists [11,12]. Exclusion criteria were: histological review did not confirm IFS diagnosis (n Z 3); tumours negative for the ETV6-NTRK3 transcript or not tested in the absence of pathologic panel review (n Z 4) (Fig. 1). Institutional ethics board approval was obtained for all participating centres according to the rules established in Europe. Written consent for treatment and the use of data were obtained from parents or guardians according to local research ethics requirements. 2.2. Treatment Primary surgery after initial biopsy was recommended only when en bloc resection, removing the tumour through normal tissue with clear margins, might be achieved without significant long-term functional or cosmetic impairment. In the other cases, a biopsy was required followed by chemotherapy and, if necessary, delayed surgery. No adjuvant chemotherapy was recommended if resection was complete or microscopicallyincomplete (IRS group-I/R0 or II/R1). The VA regimen was the treatment of choice in patients with unresectable disease (IRS group III/R2), with the exception of patients with congenital tumours (age <3 months at diagnosis), for which an optional ‘wait and see’ strategy was considered to evaluate the possibility of spontaneous regression or time to facilitate subsequent surgery. VA chemotherapy was continued, in a responsive tumour, until tumour resectability was possible. If the tumour shrinkage was not sufficient to permit conservative surgery, ifosfamide (IVA regimen) or cyclophosphamide (VAC regimen) was added (Fig. 2). Where there was no response to VA, or tumour progression, ifosfamide-doxorubicin (ID) chemotherapy was recommended. Mutilating surgery and external radiotherapy was strongly discouraged. Additional dose reductions were applied for infants <8 kg and <6 months (30% reduction), and for newborns <5 kg and <3 months (50%). Moreover, the initial doses were delivered at 50%, progressively increasing to 75% and 100% to verify overall tolerance in infants, with specific attention to neurologic and hepatic toxicity, particularly constipation and venoocclusive disease (VOD). No alkylating agent was administered before 1 month and no anthracycline before 3 months of age. 2.3. Response assessment In patients with measurable disease, response to chemotherapy was assessed after three cycles of chemotherapy by assessment of radiologically-identified tumour volume reduction: i.e. complete response (CR) Fig. 1. Patient’s charts. Abbreviations: IFS, infantile fibrosarcoma; IRS-I/R0, complete exeresis; IRS-II/R1, microscopic residue; IRS-III/R2, macroscopic residue; pts, patients. D. Orbach et al. / European Journal of Cancer 57 (2016) 1e9 3 Z complete disappearance of visible tumour with no residual disease; major partial response (PR !2/3) Z volume response 66e99%; minor PR (<2/3) Z volume response 34e65%; stable disease (SD) 33% reduction in tumour volume; progressive disease (PD) Z more than 40% increase in the sum of the volumes of all measurable lesions, or the appearance of new lesions [13]. Response rate to specific regimen of chemotherapy was considered as follows: (CRþ PR !2/3þ PR <2/3). 2.4. Statistical methods Data were analysed by the International Data Center (Istituto Oncologico Veneto I.R.C.C.S., Padua; Italy), considering information within the Remote Data Entry system as at May 2015. Outcome was defined as overall survival (OS) and event-free survival (EFS). The definition of OS was measured from the date of diagnosis to death from any cause. Events were defined for EFS as progression during chemotherapy, relapse after CR or death from any cause. Local control was defined as disappearance of all radiological signs of disease at the site of the primary or stable residual radiographic images for at least 6 months after completion of treatment. Survival curves were calculated by the Kaplan-Meier method [14]. The 3-year EFS and OS were reported along with their 95% confidence intervals (CI). Fig. 2. Chemotherapy schedules. Chemotherapy dosages: Vincristine and actinomycin-D: 50 mg/kg/injection; if age >12 months and weight >10 kg: 1.5 mg/m2 . Endoxanâ -cyclophosphamide: 50 mg/kg; if age >12 months and weight >10 kg: 1.5 g/m2 . Holoxanâ -ifosfamide: 100 mg/kg  2 d for patients >3 months and 5e10 kg; of age >12 months and weight >10 kg: 3 g/m2  2 d. See text for further reductions in young children. D. Orbach et al. / European Journal of Cancer 57 (2016) 1e94 3. Results A total of 50 cases with a diagnosis of IFS and age <2 years were considered during the study period. They represent 6.5% of all registered patients with NRSTS and 30.1% of those aged less than 2 years included in the NRSTS EpSSG database. Four older patients (>2 years) were registered during the same time in this database but their tumours did not manifest the specific transcript and were not included in the analysis. Overall clinical characteristics of the population are indicated in Table 1. Most tumours were not associated with specific congenital abnormalities (95.9%). Median age at diagnosis was 1.43 months (range: 0.03e18.73). The diagnosis was made before birth or during the first month of life for 40.0% of the patients and in 68.0% of the cases before the age of 3 months (so called ‘congenital forms’). Tumours occurred mainly in the limbs (54.0%), with more than half !5 cm at diagnosis and none had lymph node spread. Histological local diagnosis was confirmed by national and/or international histology review in 39 and 15 cases respectively. The identification of ETV6-NTRK3 transcript was tested in 39/50 patients: FISH showed the presence of the fusion gene in 9/11 samples, RT-PCR was positive in 19/21 samples, and both tests were positive in 6/7 additional patients. All cases without histology review harboured the ETV6-NTRK3 translocation. In summary, the characteristic biological translocation was identified in 87.2% of tumours. 3.1. Treatment according to IRS group According to initial surgery, eleven patients were classified as IRS-group I/R0, eight as IRS-group II/R1, thirty one as IRS-group III/R2, four after resection with macroscopic residual tumour and twenty seven after biopsy (Fig. 1). IRS I-II-group (n [ 19): three out of 19 patients underwent primary re-excision of the tumour. No adjuvant chemotherapy was given according to the guideline recommendations in all but one case (a ruptured atypical hypercellular mesoblastic nephroma primary, IRSgroup II/R1) that received 6 months of VA (treating physician’s decision) with additional vincristinecyclophosphamide for 2 months due to mild hepatic toxicity. Surgery comprised of wide tumour excision (18 cases), associated with a partial colectomy (three cases) or unilateral nephrectomy (two cases) and a limited chest wall excision (one case). One local relapse occurred in this group: a 17-d-old baby with a right wrist IFS who suffered a local relapse 2.5 months after microscopic incomplete surgery and then underwent radical surgery. He remains in 2nd CR 4 years after diagnosis. All 19 patients were alive in remission at the time of the analysis. IRS III-group (n [ 31): Chemotherapy was administered to 27 patients for a median duration of 4.14 months (range: 0.46e12.06). The remaining four had a ‘wait and see’ strategy. Overall 25 patients started chemotherapy according to the protocol with VA regimen for 14 d to 12 months, median 4.14 months. Six patients then switched chemotherapy to IVA (three cases), VAC (two cases), or ID regimen (one case) either due to SD or PD (three cases), to facilitate surgery (two cases) or for a life threatening scenario (one case). Finally, one patient received the IVA regimen due to an initially incorrect diagnosis and another one received VAC by physician preference due to rapid growth of the tumour after diagnosis. A wait and see approach was used for four IRS group-III/R2 patients. Among them, three patients needed delayed VA chemotherapy from 2e4 months Table 1 Clinical characteristics of the population. Number of patients n Z 50 % Age at diagnosis (months) <1 20 40.0 1e3 14 28.0 4e12 12 24.0 >12 4 8.0 Congenital abnormalities associated Yes 2 4.0 Ductus arteriosus persistens 1 50.0 Occipital haemangioma 1 50.0 No 47 94.0 Missing data 1 2.0 Gender Female 18 36.0 Male 32 64.0 Post-surgical tumour staging (IRS) Group I (R0) 11 22.0 Group II (R1) 8 16.0 Group III a (biopsy) (R2) 27 54.0 Group III b (incomplete surgery) (R2) 4 8.0 Primary tumour invasiveness (T) T1 e Localised to the organ or tissue of origin 33 66.0 T2 e Extending beyond the tissue or organ of origin 17 34.0 Tumour size a: 5 cm 23 46.0 b:>5 cm 27 54.0 Regional lymph node involvement N0-No evidence of lymph node involvement 50 100 Site of origin of primary tumour Extremities 27 54.0 Axial sites 14 28.0 Abdomen 7 Paraspinal 2 Retroperitoneal 2 Thorax 2 Trunk 1 Non-parameningeal head and neck 4 8.0 Parameningeal 3 6.0 Genito-urinary non Bladder Prostate 2 4.0 Kidney 2 D. Orbach et al. / European Journal of Cancer 57 (2016) 1e9 5 after diagnosis, all with response (1 CR, 2 PR>2/3) and are alive in remission at the end of follow up. One is alive with a residual mass after spontaneous tumour reduction (Table 2). The overall response rate to chemotherapy was 62.9% (17/27 evaluable patients) and 68.0% specifically to VA regimen (17/25 cases). Tolerance of chemotherapy was manageable overall but seven cases had specific grade IIIeIV toxicity: three reversible VOD, one peripheral neurotoxicity with ptosis, one haematological grade IV neutropenia with grade III anaemia, one haemorrhage in the tumour during progression. A 1-month old patient received by error an overdose (100 fold) of actinomycin-D and died despite supportive care. Delayed tumour surgery was performed after a median of 4.9 months (range: 1.2e20.5) from diagnosis in 19 cases, with a wide excision in 13 patients including a conservative parotidectomy (one case), a limited perineal excision (one case), and nephrectomy with adrenalectomy (one case). Limb amputation was performed for two children and an exenteration in one patient. Overall, resection was complete in 14 cases, with microscopic residual in four cases and a macroscopically incomplete resection in one patient. No further surgery was done for 11 IRS-III/R2 patients. In seven cases, this was due to physician’s decision (despite a residual images following chemotherapy in four cases; after an initial wait and see strategy in three cases), after histological remission of a residual mass confirmed by biopsy (two cases), and a clinical complete remission after chemotherapy (one case). 3.2. Total burden of therapy Among the 50 cases, 40 (80.0%) had tumour surgery: resection alone in 19 cases and associated with chemotherapy in 21 cases. Surgery was mostly conservative (37 cases) whereas three needed mutilating surgery. Only one patient with a progressive orbital parameningeal IFS despite VA than VAC regimens received proton radiotherapy at 54 Gy after an orbital exenteration. Two other patients had limb amputation (finger, hand). Overall, among the 47 survivors, chemotherapy was delivered in 29 cases (61.7%) and comprised a VA regimen alone (22 cases), with additional alkylating agents (six cases) and/or anthracycline drug (one case). 3.3. Congenital cases Among the 34 infants with congenital IFS, 59.0% were discovered antenatally or before 1 month of age. The site was the limbs (47.1%), ‘other’ sites (35.3%), headand-neck (11.8%) and genito-urinary (5.9%). 3.4. Outcome At the time of analysis, 35 patients are in first complete remission (CR), one is alive after 1st line chemotherapy; one is alive with a residual mass after therapy, seven are in 2nd or greater CR off therapy, three have died and three are lost to follow-up in CR (Fig. 1). Ten patients had a tumour event, nine initially classified as IRS group III/R2 and one as IRS group II/R1: seven tumours progressed, one patient experienced a metastatic relapse, one had a local relapse and one patient died due to toxicity. Among the 10 cases with tumour events, eight had tumours with ETV6-NTRK3 transcript, one without and in one case the analysis had not been performed. Tumour progression occurred in two cases after a wait and see strategy (Table 2), after VAC-IVA/ID regimens (two cases with refractory disease responsible for patients’ death), and after the VA regimen (three cases were treated with VAC and surgery, surgery alone and ID regimen, and are in subsequent CR). One patient developed lung metastases 2.5 years after a head and neck tumour initially unresponsive to VA but Table 2 IRS III/R2 patients with a ‘wait and see’ strategy. Patient no 1 Patient no 2 Patient no 3 Patient no 4 Age at diagnosis 10 d 41 d 68 d 11 months ETV6-NTRK3 transcript Presence Presence Presence Presence Site of primary tumour Foot Shoulder Retroperitoneal Tight Invasiveness T1 T2 T1 T1 Tumour size 5 cm >5 cm >5 cm 5 cm Time from diagnosis to start of CT 4 months e 3 months 2 months Reason for treatment Progressive disease e Progressive disease Absence of regression Therapy CT (VA regimen for 5 months) e CT (VA regimen for 3 months) CT (VA regimen for 5 months) þ HCR after delayed surgery Status Alive in CR off therapy Alive with tumour decreased from diagnosis Alive in CR off therapy Alive in 1st CR off therapy Time from diagnosis to last FUP 5 years and 7 months 2 years and 11 months 3 years 7 years and 10 months Abbreviations: CT chemotherapy, HCR histologic complete response; CR complete remission; VA vincristine-actinomycin-D, FUP follow-up. D. Orbach et al. / European Journal of Cancer 57 (2016) 1e96 responding to subsequent ID chemotherapy, (allowing a R1 resection). At the time of the report, this patient was alive in secondary remission after second-line chemotherapy and pulmonary metastasectomy. One patient died due to an overdosage of chemotherapy. Two patients died from disease. The three patients that received mutilating surgery are alive and in continuing complete remission off therapy. After a median follow-up of 4.7 years (range 1.9e9.0), 3-year EFS and OS were respectively 84.0% (95% CI 70.5e91.7) and 94.0% (95% CI 82.5e98.0) (Fig. 3). 4. Discussion This study demonstrates that a conservative treatment approach is feasible in young children with IFS without jeopardising survival. Despite many having large tumours at diagnosis, mutilating surgery was only required in three cases and alkylating-anthracycline-free chemotherapy sufficient to achieve cure in 74.2% of patients requiring chemotherapy. Our experience also confirms that prospective multi-institutional trials are possible even in very rare tumours in children at an European level [15]. The very good compliance with treatment guidelines within the different European countries involved, e.g. 94.7% of IRS I-II group patients were treated with surgery alone, and 93.3% of IRS group III patients received the VA regimen as first line therapy as recommended, shows that the goal to standardise the IFS treatment was achieved. This series confirms some of the general clinical characteristics of IFS as a rare disease occurring in very young patients (median age 1.43 months), predominantly in males (64.0%), and mainly in limbs (54.0%) [16,17]. According to some authors, IFS can be either a histological or a biological defined entity [4,11,18]. This series reported that the ETV6-NTRK3 fusion gene documented by FISH or RT-PCR was present in 87.2% of the patients with IFS where the investigation was performed. This is a helpful tool where there is pathological difficulty in confirming the diagnosis of IFS [10]. Nevertheless, it is important to note that the ETV6NTRK3 fusion gene is not totally specific to IFS. It has been described in congenital hypercellular mesoblastic nephroma, mammary analogue secretory carcinoma of salivary glands and of the breast, and in some leukaemias [19]. The definition of the ‘infantile’ nature of fibrosarcoma is not precise in the literature and an age limit up to 2 years is used by most authors [16,17,20,21]. Even if some rare series consider patients with IFS up to 3 years, we focused on the population of very young children ( 2 years of age at diagnosis) for whom the consequences of treatment (chemotherapy, radical surgery and radiotherapy) are a major factor guiding treatment decisions, and also to be consistent with other analyses [1,6,22]. Other studies previously reported the very good OS of children with IFS, and emphasised the challenge of tumour resectability without anatomical or functional damage. Even if surgery should still be seen as the cornerstone of therapy in this tumour, our experience highlighted that the use of chemotherapy may also play a critical role in large diffuse inoperable tumours. Initial grossly tumour resection (IRS-I/IIeR0/1) was only possible in 38.0% of patients but tumour shrinkage achieved with chemotherapy in the majority of initially unresected tumours allowed a secondary conservative surgical approach in the majority. It is clear, however, that postoperative chemotherapy is not necessary after a delayed complete macroscopic tumour resection (IRSI/ II-R0/1) or total necrosis. Similarly, adjuvant chemotherapy was unnecessary for IRS group I/R0 patients but also for IRS group II/R1. In this cohort, only one local recurrence occurred out of 19 patients, and was successfully treated with further surgery. Nevertheless, the overall consensus should be to try to avoid incomplete surgery with macroscopic residue. The VA regimen, a combination that does not contain alkylating agents or anthracyclines, appears to be very active in IFS. Acute toxicity was not negligible but despite three mild episodes of VOD and one toxic death (associated with a dose error) we believe that VA is more advantageous compared with VAC chemotherapy (cyclophosphamide) or anthracycline containing regimens, as it reduces the gonadal and mutagenic toxicity of cyclophosphamide/ifosfamide and the cardiac toxicity of anthracyclines in very young children, previously used in up to 53e87% of all patients [6,20,23,24]. The optimal duration of preoperative chemotherapy was not defined in our protocol and still needs to be clarified. Previously it was unclear whether it is possible to avoid delayed surgery in IRS-group III/R2 patients, after successful use of neoadjuvant chemotherapy with Fig. 3. Event-free survival and overall of the entire population. Abbreviations: OS, overall survival; EFS, event-free survival. D. Orbach et al. / European Journal of Cancer 57 (2016) 1e9 7 radiological complete remission. In our experience, 35.4% (11/31) of IRS-III/R2 patients did not need delayed resection due to a radiologic CR or VGPR after neoadjuvant chemotherapy, and we therefore recommend this approach. The possibility of spontaneous regression in IFS has already been reported [25e27]. The observation that one patient in our series showed a spontaneous tumour shrinkage supports a ‘wait and see’ strategy especially in very young patients, i.e. patients <3 months with a nonresectable primary in a non-threatening situation, in whom tolerance to chemotherapy may be poor. This approach may be extended to older infants, if strict follow-up could be ensured. If progression does occur, then neoadjuvant chemotherapy with VA should be started. The small number of relapses in our cohort does not allow further analysis of prognostic factors and subsequent risk-stratification. A recent epidemiological retrospective study among a large cohort of 224 children 2 years with IFS did not show any significant survival difference according to various risk factors such as margin status, nodal involvement, tumour size or treatment modalities [17]. In conclusion, this study highlights the importance of paediatric international cooperation in developing prospective studies for very rare childhood tumours. Due to the rarity of this tumour all medical decisions should be shared through multidisciplinary discussions at a regional, national or international level [15]. This should allow a conservative treatment approach where feasible in young children with IFS without jeopardising survival. Funding The EpSSG is supported by la Fondazione “la Citta` della Speranza”. This work is partially financially supported by “La ligue pour la vie” (Grant number MMR 7825). Conflict of interest statement All authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organisations within that could inappropriately influence (bias) their work. Acknowledgement Authors want to thank Dr O Oberlin, Villejuif, France, Pr M Carli, Padova, Italy for their help and Ilaria Zanetti, Padova, Italy for extensive data management. References [1] Orbach D, Rey A, Cecchetto G, Oberlin O, Casanova M, Thebaud E, et al. Infantile fibrosarcoma: management based on the European experience. J Clin Oncol 2009;16:16. [2] Orbach D, Rey A, Oberlin O, Sanchez de Toledo J, TerrierLacombe MJ, van Unnik A, et al. 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[27] Kihara S, Nehlsen-Cannarella N, Kirsch WM, Chase D, Garvin AJ. A comparative study of apoptosis and cell proliferation in infantile and adult fibrosarcomas. Am J Clin Pathol 1996; 106(4):493e7. D. Orbach et al. / European Journal of Cancer 57 (2016) 1e9 9 Clinical Trial Outcome of extracranial malignant rhabdoid tumours in children registered in the European Paediatric Soft Tissue Sarcoma Study Group Non-Rhabdomyosarcoma Soft Tissue Sarcoma 2005 StudydEpSSG NRSTS 2005 Bernadette Brennan a, *, Gian Luca De Salvo b , Daniel Orbach c , Angela De Paoli b , Anna Kelsey d , Peter Mudry e , Nadine Francotte f , Max Van Noesel g , Gianni Bisogno h , Michela Casanova i , Andrea Ferrari i a Paediatric Oncology, Royal Manchester Children’s Hospital, Manchester, UK b Clinical Trials and Biostatistics Unit, IRCCS Istituto Oncologico Veneto, Padova, Italy c Pediatric, Adolescent, Young Adult Department, Institut Curie, Paris, France d Department of Diagnostic Paediatric Histopathology, Royal Manchester Children’s Hospital, Manchester, UK e Department of Pediatric Oncology, University Children’s Hospital, Brno, Czech Republic f Department of Pediatrics, CHC-Clinique Esperance, Montegne´e, Belgium g Princess Ma´xima Center for Pediatric Oncology, Utrecht, The Netherlands h Pediatric Hematology and Oncology Division, Padova University, Padova, Italy i Fondazione IRCCS Istituto Nazionale Tumori Milano, Milan, Italy Received 23 October 2015; received in revised form 22 December 2015; accepted 23 February 2016 Available online 13 April 2016 KEYWORDS Malignant rhabdoid tumour; Paediatric; Prospective registry; Survival; Prognostic factors Abstract Background: Extracranial malignant rhabdoid tumours (MRT) are rare lethal childhood cancers that often occur in infants and have a characteristic genetic mutation in the SMARCB1 gene. The European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) conducted a multinational prospective study of registered cases of extracranial MRT to test an intensive multimodal approach of treatment for children with newly diagnosed extracranial MRT. Methods: Between December 2005 and June 2014, we prospectively registered 100 patients from 12 countries with a diagnosis of MRT tumour at an extracranial site on the EpSSG Non-Rhabdomyosarcoma Soft Tissue Sarcoma 2005 Study (NRSTS 2005). They were all treated on a standard multimodal protocol of surgery, radiotherapy, and chemotherapy over 30 weeks as follows: vincristine, cyclophosphamide, and doxorubicin (VDCy) at weeks 1, 10, 13, 22, and 28; vincristine was also given alone on weeks 2, 3, 11, 12, 14, 15, 23, 24, 29, and 30. * Corresponding author: Royal Manchester Children’s Hospital, Oxford Road, Manchester, M13 9WL, UK. Tel.: þ44 161 701 8430; fax: þ44 161 70 18410. E-mail address: Bernadette.brennan@cmft.nhs.uk (B. Brennan). http://dx.doi.org/10.1016/j.ejca.2016.02.027 0959-8049/ª 2016 Elsevier Ltd. All rights reserved. Available online at www.sciencedirect.com ScienceDirect journal homepage: www.ejcancer.com European Journal of Cancer 60 (2016) 69e82 Cyclophosphamide, carboplatin, and etoposide (Cy*CE) was given at weeks 4, 7, 16, 19, and 25. Radiotherapy was recommended for all primary tumour sites and all sites of metastatic disease. Results: Forty-three patients completed the protocol treatment. Median follow-up for alive patients of the complete cohort was 44.6 months (range 11.5e84.6). For the whole cohort, the 3-year event-free survival (EFS) was 32.3% (95% confidence interval [CI] 23.2e41.6%) with a 3-year overall survival (OS) of 38.4% (95% CI 28.8e47.9%). For localised disease, the 4-year EFS was 39.3% (95% CI 28.2e50.1%) with a 4-year OS of 40.1% (95% CI 28.4e51.5%). For metastatic disease, the 2-year EFS was 8.7% (95% CI 1.5e24.2%) with a 2-year OS of 13.0% (95% CI 3.3e29.7%). Multivariable analysis disclosed that all patients 1 year of age were associated with at higher risk of death (hazard ratio [HR]: 2.6; 95% CI 1.0e6.8; p-value Z 0.0094). Risk of death was also related with gender in metastatic patients (HR for males: 2.9, 95% CI 1.0e8.0; p-value Z 0.0077). Conclusions: The EpSSG NRSTS 2005 protocol of intensive therapy can be delivered to extracranial MRT patients, with a possible improvement in outcome. The outcome, however, remains poor for patients who progress or with metastatic disease. ª 2016 Elsevier Ltd. All rights reserved. 1. Introduction Extracranial MRT tumours are rare and often occur in infants with an age standardised incidence ratio of 0.6 per million children in the United Kingdom (UK), 61% of cases in the first of year of life [1]. The vast majority contain a somatic bi-allelic inactivating mutation in the SMARCB1 gene, which is part of the chromatin remodelling complex SW1/SWF, important in cell cycle control, and functions as a classic tumour suppressor gene [2]. MRT are often described as lethal, with little evidence of improved survival in recent years. In the UK population-based National Registry of Childhood Tumours during 1993 to 2010, the 1-year overall survival (OS) was only 31% [1]. This poor survival is also reflected in the National Wilms’ Tumour Study (NWTS) series, and in the United States, Surveillance Epidemiology and End Results (SEER) programme, OS, at 4 years was 23.3% and 33.0%, respectively [3,4]. Given the rarity of extracranial MRT, there is no standard therapeutic pathway, and there has been no randomised or prospective trials examining the role of chemotherapy combinations or, indeed, the addition of new agents. Instead, there have been small retrospective series published either from single institutions or larger series of MRT at single anatomical sites from other site-specific studies, such as NWTS [3,5]. Despite the challenging nature of this tumour and its treatment, two case reports including two and one patients, respectively, with metastatic renal MRT are often cited in view of their successful outcome [6,7]. Based on these reports, the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) conducted a multinational prospective study of registered cases of extracranial MRT to test an intensive multimodal approach of treatment for children with newly diagnosed extracranial MRT. 2. Methods 2.1. Patients and study design One hundred patients with a diagnosis of MRT at an extracranial site were registered on the EpSSG NonRhabdomyosarcoma Soft Tissue Sarcoma 2005 Study (NRSTS 2005). This was a prospective observational study for all NRSTS patients, with recommended treatment for MRT. The study was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines. Informed written consent was obtained for all patients/parents. The study was managed via a Web-based system provided by CINECA, an Inter-University Computing Consortium (Casalecchio, Italy). 2.2. Pathological analysis National and international review by the pathology panel of the histological diagnosis was advised but not considered mandatory. Patients were included if their local histological diagnosis of MRT was supported by immunohistochemistry demonstrating loss of nuclear expression of INI-1 (BAF47 antibody) and/or molecular testing demonstrated deletion of the SMARCB1 gene [2]. Additional national and/or an international review by the EpSSG panel of pathologists were performed in 64 of the cases. 2.3. Staging and surgery Following staging investigations, including either computed tomography (CT) or magnetic resonance imaging (MRI) of the primary site, CT scan chest, MRI/ CT scan of brain, and for some bone scan and bone marrow assessment, it was recommended for all patients B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8270 to undergo surgical resection of primary tumour but if deemed unresectable, biopsy only. The Intergroup Rhabdomyosarcoma Study (IRS) and TNM postsurgical staging was used [8]. Complete resection with no microscopic disease was R0, with microscopic disease was R1, and macroscopic disease was R2. 2.4. Chemotherapy Following initial surgery or biopsy, the recommended chemotherapy was given over 30 weeks as follows: vincristine, cyclophosphamide, and doxorubicin (VDCy) at weeks 1, 10, 13, 22, and 28; vincristine was also given alone on weeks 2, 3, 11, 12, 14, 15, 23, 24, 29, and 30; cyclophosphamide, carboplatin, and etoposide (Cy*CE) given at weeks 4, 7, 16, 19, and 25 (see Appendix 1 for full dose and schedule plan). Dosages were adapted to infant weight and progressively increased. No details about doses of chemotherapy were collected, but data were available on whether treatment was received and if completed. 2.5. Radiotherapy Radiotherapy was recommended for all primary tumour sites and all sites of metastatic disease, either following up-front surgery at week 2 or following delayed surgery at week 14. The chemotherapy schedule allowed concomitant radiotherapy. The dose up to a maximum of 50.4 Gy, treatment volume, and fractionation depended on the site of the primary tumour, degree of resection, site, and type of metastases (Appendix 2 for full details). 2.6. Toxicity and disease evaluation Severe toxicity and serious adverse events were recorded on the end of treatment form but as a registry this was not graded according to the National Cancer Institute Common Toxicity Criteria. If no signs of tumour progression were present, a formal tumour revaluation was advised at the end of treatment in patients without measurable disease and after 12 weeks of chemotherapy in patients with measurable disease, including those patients with metastases. 2.7. Statistical analyses Data were collected via a web-based system and analysed at Istituto Oncologico Veneto (Padua, Italy) considering information reported up to 27th May 2015. Continuous variables were summarised with median, minimum and maximum, and categorical variables were reported as counts and percentages. Survival time was calculated from the date of diagnosis to the time of event or last follow-up. Tumour progression, relapse or death due to any causes were considered for event-free survival (EFS). OS was measured from the date of diagnosis to death for any reason. Patients still alive at the end of the study were censored at the date of last observation. The survival probability was computed by means of the KaplaneMeier method and heterogeneity in survival among strata of selected variables was assessed through the log-rank test. The 3-year EFS and OS (4-year EFS for localized tumours) were reported along with their 95% confidence intervals (CIs). To investigate the impact of the variables gender, age category ( 1 year; >1 year), tumour size ( 5 cm; >5 cm), primary site (favourable: orbit, head and neck non-parameningeal, genitourinary non-bladdereprostate; unfavourable: parameningeal, bladder-prostate, extremities, “other”; according to rhabdomyosarcoma classification, IRS group and initial surgery (performed; not performed) on EFS for localized patients and OS for localized and metastatic patients, survival multivariable analysis were conducted using the Cox proportional hazard regression method [8]. A stepwise variable selection procedure was applied to the covariates with a p-value of at least 0.05 at univariate analysis. Hazard ratios (HRs) with their 95% CI calculated according to the Wald method was reported for significant variables. To check the proportional hazards assumption, a score process (which is a transformed partial sum process of the martingale residuals) was compared with the simulated processes under the null hypothesis that the proportional hazards assumption holds [14]. All data analyses were performed using the SAS statistical package (SAS, release 9.4; SAS Institute Inc, Cary, NC). 3. Results 3.1. Patients Between December 2005 and June 2014, 110 patients were enrolled on the study but 10 were excluded due to adherence to other protocols (3), immunohistochemistry and molecular data missing (1), histological diagnosis after pathology review was not MRT (2) or immunohistochemistry did not demonstrate loss of nuclear expression of INI-1, and/or molecular testing did not demonstrate deletion of the SMARCB gene (4), leaving in total 100 eligible patients. There was an even distribution between the sexes, 49 female and 51 male. The median age at diagnosis was 1.4 years (range 3 de10.9 years) with 41 patients 1 year of age. The majority (56 patients) were between 2 and 9 years (39 patients between the ages 1 and 3 years) and only 3 were older than 10 years. Patient staging data and site and size of primary tumour are listed in Table 1. The majority in the series had localised disease (77 patients) and of those 19 (25%) had surgical resection up front. The primary site of the tumour was across multiple B. Brennan et al. / European Journal of Cancer 60 (2016) 69e82 71 anatomical sites, the commonest site in this series was “other” sites (44 patients) followed by genitourinary non-bladdereprostate (18 cases). Twenty-three patients had distant metastases. The majority (17 patients) had metastases to the lung: four patients lung alone and 13 with other metastases. Two cases had brain tumour metastases. Thirteen patients had congenital MRT as defined by diagnosis within the first 4 weeks of birth. Five of them (39%) had metastatic disease, with the majority having tumours greater than 5 centimetres (62%). Primary sites were multiple but the largest group was “other”dparaspinal, thorax, retroperitoneal or liver. One case had brain tumour metastases. 3.2. Treatment and toxicity Forty-three patients completed the protocol treatment in a median period of 8.4 months (minimum 6.5emaximum 13.0) of chemotherapy. Fifty-five patients discontinued chemotherapy due to toxicity (3), early progressive disease (49) between 3 d and 10.9 months or physician’s choice (3). One patient did not receive any treatment due to death before starting treatment and for one patient no treatment data are available. There were dose adjustments due to delays in starting the next course of chemotherapy or mucositis in 10 patients. The most frequent reported toxicities included bone marrow suppression, febrile neutropenia, infection, mucositis, anorexia, and electrolyte disturbances. In those who completed all courses of chemotherapy, there were no permanent toxicities, such as renal impairment, and there were no toxic deaths. All those younger than 12 months were able to receive chemotherapy except one patient who died before the start of treatment. They were no more likely to have toxicities than older patients but had doses of chemotherapy adjusted for their age and weight. Fifty-four patients from the whole cohort did not receive radiotherapy, 39 had progressive disease during first-line treatment prior to the planned radiotherapy, whereas in 15 patients, no radiotherapy was delivered by physicians choice probably due to the very young age of the patient. One patient developed radiation colitis but there were no other radiation-recorded toxicities. For the localised patients, 25 progressed before planned radiotherapy with only 37 of the remaining 52 patients receiving radiotherapy. Up-front complete surgical resection of the primary tumour was performed in 8 (R0 resection), including 1 metastatic patient, and in 12 patients R1 resection. In 73, only a surgical/trucut biopsy or lymph node exploration was performed at diagnosis (53 localised and 20 metastatic patients). For the remaining seven patients, macroscopic tumour was present after surgical resection of primary tumour (five localised and two metastatic patients). Thirty-nine patients had second surgery, for Table 1 Clinical characteristics. Localised patients, N Z 77 Metastatic patients, N Z 23 Total Total % Age (years) at diagnosis Median (minemax) 1.51 (0.01e 10.93) 0.60 (0.01e 0.60) 1.38 (0.01e 10.93) 1 29 12 41 >1 48 11 59 Gender Female 35 14 49 Male 42 9 51 Post-surgical tumour staging (IRS) Group I 7 e 7 Group II 12 e 12 Group III 58 e 58 Group IV e 23 23 Primary tumour Invasiveness (T) T0dno detectable e 1 1 T1dlocalized to the organ or tissue of origin 34 6 40 T2dextending beyond the tissue or organ of origin 42 14 56 Txdinsufficient information about the primary tumour 1 2 3 Tumour size a: 5 cm 19 3 22 b: >5 cm 56 19 75 X: not evaluable 2 1 3 Regional lymph node involvement N0dNo evidence of lymph node involvement 67 10 77 N1dEvidence of regional lymph node involvement 9 11 20 NxdNo information on lymph node involvement 1 2 3 Site of origin of primary tumour Orbit 1 e 1 Head neck 12 e 12 Parameningeal 7 e 7 Bladder-prostate 4 e 4 Genitourinary non- Bladdereprostate 11 7 18 Kidney 10 7 94 Uterus 1 e 5 Extremities 8 6 14 Other sites 34 10 44 Abdomen 2 e 2 Liver 10 5 15 Paraspinal 13 1 14 Pelvis 1 e 1 Perineum 1 e 1 Retroperitoneal e 2 2 Thorax 6 2 8 Trunk 1 e 1 Number of metastatic sitesa 1 e 9 39 2 e 8 35 3 e 3 13 4 e 3 13 IRS, Intergroup Rhabdomyosarcoma Study; max, maximum; min, minimum. a Percentage computed considering metastatic patients only. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8272 26 after 3e4 cycles of chemotherapy, for 8 after 5e8 cycles and for 3 at another time. Additional surgeries were necessary for two patients. This resulted in a 17 with a R0 resection including 1 with a liver transplant, R1 resection in 13, and macroscopic residual tumour in 8. In one case, no tumour was found. 3.3. Outcome data Median follow-up for alive patients of the complete cohort was 44.6 months (range 11.5e84.6), for localized patients was 49.8 months (range 11.5e84.6), whereas for metastatic patients was 32.1 months (range 14.9e38.8). Sixty-seven patients developed an event (46 in localized and 21 metastatic patients) and subsequently 65 died (45 in localized and 20 metastatic patients). Median time to progression was 5.0 months (minimum 3 d, maximum 31.5 months), for localised patients 7.5 months (1.4e31.5) and for metastatic patients 2.7 months (3 de14.9 months). In the total cohort, 35 were alive at the time of this analysis. For the whole cohort, the 3-year EFS was 32.3% (95% CI 23.2e41.6%) with a 3-year OS of 38.4% (95% CI 28.8e47.9%; Fig. 1A and B). For localized disease, the 4-year EFS was 39.3% (95% CI 28.2e50.1%) with a 4-year OS of 40.1% (95% CI 28.4e51.5%; Fig. 1C and D). For metastatic disease, the 2-year EFS was 8.7% (95% CI 1.5e24.2%) with a 2-year OS of 13.0% (95% CI 3.3e29.7%; Fig. 1C and D). For IRS III disease, achieving a complete response (CR) at any time point occurred in 30 patients leading to a statistically significant (p<0.0001) survival advantage with a 4-year EFS of 66.3% (95% CI 46.5e80.3%) and 4-year OS of 66.8% (95% CI 44.6e81.7%) compared with no CR in 28 patients with a 4-year EFS of 4.8% (95% CI 0.4e18.9%) and 4-year OS of 4.8% (95% CI 0.4e18.9%). 3.4. Prognostic factors Table 2 lists the estimated EFS and OS for the patient’s clinical characteristics in those with localized tumours. On univariate analysis, patient age only significantly influenced the EFS and OS, with those 1 year of age having a significantly worse outcome, with a 4-year EFS of 17.2% (95% CI 6.3e32.7%) and an HR of 2.9 (95% CI 1.6e5.3) and a 4-year OS of 20.1% (95% CI 7.9e36.3%) with an HR of 2.7 (95% CI 1.5e5.0). Table 3 lists the estimated 1-year OS by main characteristics of Fig. 1. Survival analysis of whole cohort, localised and metastatic extracranial MRT. (A) Event-free survival and (B) overall survival of 100 patients with extracranial MRT registered on EpSSG NRSTS 2005. (C) Event-free survival and (B) overall survival of localised and metastatic patients separately. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e82 73 metastatic patients. Patients 1 year of age had the worst prognosis, as well as male patients. Multivariable analysis disclosed that all patients 1 year were associated with at higher risk of death (HR: 2.6; 95% CI 1.0e6.8; p-value Z 0.0094). Risk of death was also related with gender in metastatic patients (HR for males: 2.9, 95% CI 1.0e8.0; p-value Z 0.0077) 4. Discussion Our results demonstrate that in this first large prospective study of extracranial MRT treated in multiple European countries for what is a very rare soft tissue sarcoma, intensive therapy can be delivered to a very young paediatric population of patients, with possibly an improvement in outcome, be it in comparison with historical series. Furthermore, a substantial proportion of the patients in this EpSSG protocol had an extrarenal tumour site, which confers a poorer prognosis [1]. The outcome remains poor for the majority of patients in this series, in particular patients with metastatic disease and those who progressed, who universally had a fatal outcome. In the NWTS series of renal MRT, over a much longer historical period between 1969 and 2002, OS at 4-year was 23.2% [3]. This compares to, perhaps, our superior results with an OS of 38.4%, and perhaps, it is significant in terms of a better outcome, as the NWTS series only contained patients with a renal primary, thought to have a better outcome, maybe in part because a larger proportion can have up-front resection of the primary tumour. In our series, it is noteworthy that only 24% had up-front surgery with no survival advantage, and with surgery following chemotherapy 73% were in CR. CR, by either surgery or chemotherapy in IRS III patients, had a survival advantage but also reflects those patients who had not progressed before delayed local control and, therefore, must be read with caution. The role of a CR maybe important for Table 2 Estimated EFS and OS for localised patients (univariate analysis). Characteristics N No. events EFS 1-Year EFS (95% CI) 4-Year EFS (95% CI) p-Value No. events OS 1-Year OS (95% CI) 4-Year OS (95% CI) p-Value IRS group 0.2961 0.3234 I 7 2 85.7 (33.4e97.8) 68.6 (21.3e91.2) 2 85.7 (33.4e97.8) 68.6 (21.3e91.2) II 12 8 41.7 (15.2e66.5) 33.3 (10.3e58.8) 8 75.0 (40.8e92.2) 41.7 (15.2e66.5) III 58 36 44.8 (31.8e57.0) 36.9 (24.4e49.4) 35 53.4 (39.9e65.2) 35.9 (22.7e49.3) Age at diagnosis (years) 0.0002 0.0005 1 year 29 24 24.1 (10.7e40.5) 17.2 (6.3e32.7) 23 34.5 (18.2e51.4) 20.1 (7.9e36.3) >1 year 48 22 62.4 (47.2e74.4) 52.8 (37.5e66.1) 22 75.0 (60.2e85.0) 52.1 (35.7e66.2) Gender 0.6600 0.6288 Male 42 23 45.0 (29.6e59.2) 45.0 (29.6e59.2) 23 61.8 (45.4e74.6) 45.6 (29.5e60.4) Female 35 23 51.4 (34.0e66.4) 33.3 (18.3e49.1) 22 57.1 (39.3e71.5) 33.4 (17.4e50.3) Ta 0.2193 0.1196 T0eT1 34 17 55.7 (37.6e70.5) 49.3 (31.6e64.8) 16 64.6 (46.1e78.1) 52.4 (32.7e68.9) T2 42 28 42.8 (27.8e57.0) 32.6 (19.0e47.0) 28 57.1 (40.9e70.4) 31.9 (18.2e46.5) Sizea (cm) 0.6555 0.6671 5 19 10 52.6 (28.7e71.9) 47.4 (24.4e67.3) 10 63.2 (37.9e80.4) 47.4 (24.4e67.3) >5 56 34 48.1 (34.5e60.4) 37.4 (24.4e50.3) 33 60.6 (46.6e72.0) 38.0 (24.0e51.9) Site 0.2765 0.3525 Favourable 24 12 57.8 (35.7e74.7) 48.9 (27.8e67.0) 12 75.0 (52.6e87.9) 52.3 (30.4e70.2) Unfavourable 53 34 43.3 (29.9e56.1) 35.5 (22.8e48.3) 33 52.8 (38.6e65.1) 35.5 (22.3e48.9) Initial surgery 0.7451 0.8096 No 49 29 44.9 (30.7e58.1) 40.2 (26.4e53.7) 28 55.1 (40.2e67.7) 39.7 (24.9e54.1) Yes 28 17 53.3 (33.5e69.7) 37.2 (19.4e55.1) 17 67.7 (47.0e81.7) 40.0 (21.5e57.9) CI, confidence interval; EFS, event-free survival; IRS, Intergroup Rhabdomyosarcoma Study; OS, overall survival. a The sum does not add up to the total because of missing values. Table 3 Estimated OS for metastatic patients (univariate analysis). Characteristic N No. events 1-year OS (95% CI) p-Value Age at diagnosis (years) 0.0094 1 year 12 12 0 >1 year 11 8 27.3 (6.5e53.9) Gender 0.0077 Male 9 9 0 Female 14 11 21.4 (5.2e44.8) Ta 0.3709 T0eT1 7 7 0 T2 14 11 21.4 (5.2e44.8) Sizea 0.1913 5 cm 3 2 33.3 (9.0e77.4) >5 cm 19 17 10.5 (1.8e28.4) Site 0.6406 Favourable 7 7 0 Unfavourable 16 13 18.8 (4.6e40.2) Initial surgery 0.4330 No 19 17 10.5 (1.8e28.4) Yes 4 3 25.0 (8.9e66.5) CI, confidence interval; OS, overall survival. a The sum does not add up to the total because of missing values. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8274 long-term survival as suggested in previous small series [11]. The small numbers with a concomitant CNS primary compared to the NWTS series reflect selection of these patients into CNS protocols rather than our study [3]. The small numbers also makes it hard to comment on therapy, but at present most will receive similar intensive chemotherapy, surgical resection if possible plus or minus radiation. We showed that age continues to be an important prognostic factor and remains the only factor in multivariable analysis for OS in localised patients and univariate analysis for metastatic patients. The importance of age, in particular the negative effect of younger age on outcome, confirms the findings in the NWTS series [3], the SEER database series [4], and the UK population-based registry [1]. Uniquely, we analysed the congenital cases separately (13 cases) with 12 events (all died) and a median time to event of 3.1 months (3e11.7 d). It might be expected that these cases had a germline mutation of the SMARCB1 gene, thought to confer a poorer prognosis, but our data are incomplete [12]. Their outcome may also question the role of intensive therapy in congenital cases or, indeed, in the very young cohort. For parents, however, offering palliative therapy as the first line of treatment may not be acceptable. Progression on treatment remains an important finding, 49.5% progressed on treatment, which was an important factor for those subjects not receiving the recommended protocol radiotherapy. Of course, age of the patient may also be a further factor for no radiotherapy as in the 15 patients with physicians choice for no radiotherapy, 14 were younger than 2 years. The role of radiotherapy as an important factor affecting outcome could not be shown in our series, confounded by the number who progressed prior to delivering radiotherapy and the reluctance to give radiotherapy to very young children especially in infants or with a planned delay. This echoes the findings of the NWTS series, as the possible benefit of radiotherapy again was difficult to define, and also confounded by the patient’s age [3]. Radiotherapy tended to be given to those with a higher clinical stage and in an older age group, who received a higher dose. This is in contrast, however, to the SEER database series [4]. In particular where the use of radiotherapy remained a significant predictor of survival (p Z 0.0006). Radiotherapy was only used in 35% of patients in total, but there was no significant difference in its use at the different primary tumour sites (p Z 0.90). Less was used, however, in those younger than 3 years. For localised disease, stage was not an important predictor of outcome but a statistically significant difference in EFS and OS is evident comparing localised with metastatic patients (p-value for log-rank test <0.0001 in EFS and OS). In both the NWTS series and the SEER database series, stage also determined outcome, with a 41.8% 4-year OS for stage I to II tumours compared with 15.9% in those with stage III, IV, or V disease in NWTS, and in for the SEER database series in a multivariable model applied only to children and adolescents with extracranial MRT, tumour stage remains a significant predictor of survival (p Z 0.00014) [3,4]. Like any discussion comparing historical series, the staging systems used, the patient selection and the numbers at each anatomical site are not directly comparable and, hence, cannot replace a randomised study. The lack of prospective historical series in MRT at all sites hampers this further. Extracranial MRT continue to be aggressive tumours with poor survival. The young age at presentation often limits the ability to deliver multimodal therapy, in particular radiotherapy, which seems to be important. Further research needs to allow better understanding of MRT biology and the role of the SMARCB1 gene in MRT development. The later information could also determine better and more targets for therapy. A recent eloquent study in the molecular subgroups of primary brain atypical teratoid rhabdoid tumours, biologically the same tumour, allowed further stratification of these tumours for future biologically based trials [10]. Our results may allow us to use this protocol as a standard chemotherapy backbone in order to add small molecule inhibitors against what we currently know are targets. Recent data on EHZ2 inhibitors is promising as an epigenetic regulator and should be in phase I studies shortly in children [13]. We may need to take a leap of faith based on cell line data and pre-clinical mouse models to put these agents into phase III clinical trials while not having data from phase II trials in MRT, as it is so rare, but at least toxicity data from phase I studies in paediatric tumours. We will not improve the outcome with this protocol which is already at maximal tolerance but we may alter how we deliver conventional chemotherapy as successfully demonstrated in the Ewings sarcoma study of interval compressed chemotherapydAEWS0031 [9], with new targeted agents, to be given in combination, in a multiple arm randomised study using an innovative statistical plan for rare cancers. Conflict of interest statement None declared. Funding This study was supported by Roche, Chugai, and Citta` della Sperenza. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e82 75 Appendix 1. Chemotherapy schedule and drug doses for rhabdoid tumours registered on the EpSSG NRSTS 2005 study Chemotherapy schedule Week number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 V V V V V V V V V V V V V V V D Cy* Cy* D D Cy* Cy* D Cy* D Cy C C Cy Cy C C Cy C Cy E E E E E V Vincristine 0.025 mg/kg/d i.v.  1 as bolus for infants <12 months 0.05 mg/kg/d i.v.  1 as bolus for children 12 months to 3 years 1.5 mg/m2 /d  1 as bolus for children !3-year old D Doxorubicin 1.25 mg/kg/d i.v.  2 d over 15 min for infants <12 months 37.5 mg/m2 /d i.v.  2 d over 15 min for children !12 months Cy Cyclophosphamide 40 mg/kg/d i.v.  1 d over 1 h for infants <12 months 1200 mg/m2 /d i.v.  1 d over 1 h for children !12 months Cy* Cyclophosphamide 14.7 mg/kg/d i.v. over 1 h  5 d for infants <12 months 440 mg/m2 /d i.v. over 1 h  5 d for children !12 months C Carboplatin See nomogram in protocol E Etoposide 3.3 mg/kg/d i.v. over 1 h  5 d for infants <12 months 100 mg/m2 /d i.v. over 1 h  5 d for children !12 months Administration schedule for cycles VDCy weeks 1, 10, 13, 22 and 28 Drug Route Dose Week (s) Day(s) Vincristine i.v. over 1 min 0.025 mg/kg/d for infants <12 months 0.05 mg/kg/d for children 12 mo. to 3 years 1.5 mg/m2 /d for children !3-year old 1, 2, 3, 10, 11, 12, 13, 14, 15, 22, 23, 24, 28, 29, 30 1 Doxorubicin i.v. over 15 min 1.25 mg/kg/d for infants <12 months 37.5 mg/m2 /d for children !12 months Consideration for the use of a cardioprotective agent. Individual groups may prefer to infuse over 1 h. 1, 10, 13, 22, 28 1e2 Cyclophosphamide with MESNA hydrationa i.v. over 1 h 40 mg/kg/d for infants <12 months 1200 mg/m2 /d for children !12 months 1, 10, 13, 22, 28 1 a MESNA and hydration guidelines: MESNA 1440/m2 /dose (48 mg/kg/dose for infants <12 months old) should be added to the hydration (2000 ml/m2 /16 h) of 0.45% saline/2.5% dextrose and run for 3 h pre- and with cyclophosphamide and at least 12 h post-cyclophosphamidedtotal 16 h. Urine output at least 3 ml/kg/h. Administration schedule for cycles Cy*CE weeks 4, 7, 16, 19, and 25 Drug Route Dose Week Day(s) Cyclophosphamide with MESNA hydration i.v. over 1 h 14.7 mg/kg/d for infants <12 months 440 mg/m2 /d for children !12 months 4, 7, 16, 19, 25 1e5 Carboplatin i.v. over 1 h GFR Dose 4, 7, 16, 19, 25 1 >150 ml/min/1.73 m2 560 mg/m2 (18 mg/kg for infants) 100e150 ml/min/1.73 m2 500 mg/m2 (16.6 mg/kg for infants) 75e99 ml/min/1.73 m2 370 mg/m2 (12.3 mg/kg for infants) 50e74 ml/min/1.73 m2 290 mg/m2 (9.7 mg/kg for infants) 49 ml/min/1.73 m2 Discuss with study coordinators Etoposide i.v. over 1 h 3.3 mg/kg/d for infants <12 months 100 mg/m2 /d for children !12 months 4, 7, 16, 19, 25 1e5 Hydration: prehydrate with 0.45% saline/2.5% dextrose at 125 ml/m2 /h for 2 h. Then continue at 125 ml/m2 /h for 2 h following completion chemotherapydtotal fluids 500 ml/ m2 with 530 mg/m2 of MESNA added. i.v., intravenous; MESNA, 2-mercaptoethane sulfonate sodium. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8276 Appendix 2. Radiotherapy guidance for the rhabdoid tumours registered on the EpSSG NRSTS 2005 Renal rhabdoid tumours Indications for post-operative flank radiotherapy  Stage IeIII renal rhabdoid tumour (19.8 Gy in 11 fractions of 1.8 Gy over 15 d for patients ! 12 months; 10.5 Gy in 7 fractions of 1.5 Gy over 9 d for patients < 12 months) Indications for whole-abdominal and pelvic radiotherapy  Stage III with cytology positive ascites  Pre-operative intraperitoneal rupture  Diffuse operative spill and peritoneal seeding (19.5 Gy in 13 fractions of 1.5 Gy over 17 d for patients ! 12 months; 10.5 Gy in 7 fractions of 1.5 Gy over 9 d in the case of infants) Indications for pulmonary radiotherapy  Lung metastases (15 Gy with lung correction in 10 fractions of 1.5 Gy over 12e14 d for patients ! 12 months; 10.5 Gy in 7 fractions of 1.5 Gy over 9 d for patients < 12 months) Indications for liver radiotherapy  Liver metastases (19.8 Gy in 11 fractions of 1.8 Gy for patients ! 12 months; 15 Gy in 10 fractions of 1.5 Gy for patients < 12 months.) Indications for whole-brain radiotherapy  Brain metastases (21.6 Gy in 12 fractions of 1.8 Gy) þ boost of 10.6 Gy Indications for bone metastases radiotherapy  None metastases (25.2 Gy in 14 fractions of 1.8 Gy) Timing of radiation therapy All radiation therapy should begin as soon as it is logistically possible concurrent with the initiation of chemotherapy after surgery which is either up front or after 12 weeks of chemotherapy. Equipment All patients will be treated with megavoltage equipment (4e20 MV linear accelerator. The use of colbalt-60 equipment is not acceptable for radical therapy.) Treatment planning All patients should have a planning CT scan to enable three-dimensional conformal planning, generation of dose volume histograms for organs at risk, and lung correction where necessary. The dose is prescribed according to international commission on radiation units and measurements (ICRU) 50. Fractionation Treatment is given with conventional fractionation, treating all fields each day, with one treatment daily, 5 d a week. The fraction size should be 1.8 Gy except with large fields (whole-abdominal and pelvic radiotherapy, and whole-lung irradiation) and in infants. Once treatment is started, there will be no interruptions in treatment unless absolutely necessary. It is not necessary to suspend treatment because of uncomplicated myelosuppression, supportive care should be given for neutropenia and thrombocytopaenia according to local protocols. Haemoglobin levels should be maintained at 12 g/dl or above during the time of radiotherapy. <<7-d split 1.8 8 14.4 Patients prescribed 19.8 Gy Timing Fx size # Fx Total dose (Gy) Normal and/or up to 3-d split 1.8 11 19.8 4- to 7-d split 1.8 12 21.6 >7-d split 1.8 13 23.4 B. Brennan et al. / European Journal of Cancer 60 (2016) 69e82 77 associated tumour. The PTV should not extend more than 2 cm beyond the defined GTV, except where necessary to allow the superior and inferior field borders to lie within an intervertebral space, and the medial border to fully encompass the entire vertebral width without significantly overlapping the contralateral kidney. In patients where the tumour prior to resection bulged into the contra lateral flank without tumour invasion into the contra lateral kidney, it is not necessary for the CTV to encompass the medial extent of the GTV, and so the PTV can lie so that the full vertebral width is covered without overlap of the contralateral kidney. In most patients, the superior border of the radiation therapy field will be well below the diaphragmatic dome. The radiation therapy field should not be extended to the dome of the diaphragm unless there is tumour extension to that height. When there are positive lymph nodes that have been surgically removed, the entire length of the para-aortic chain of lymph nodes should be included in the radiotherapy field. An anteroposterior parallel-opposed (AP-PA) technique is recommended for flank irradiation. The borders of the radiation fields should be placed so that the PTV is encompassed by the 95% isodose. The flank irradiation dose is 19.5 Gy in 13 fractions of 1.5 Gy over 17 d for those 12 months or older, and 10.5 Gy in 7 fractions of 1.5 Gy over 9 d in the case of infants. Dose volume histograms should be performed for liver and the remaining kidney to ensure that the doses to these organs at risk are kept within tolerance levels. At least two thirds of the remaining kidney should not receive a dose greater than 14.4 Gy, and at least half the liver should not receive a dose greater than 19.8 Gy. Whole-abdominal and pelvic irradiation For whole-abdominal and pelvic radiotherapy, the CTV will be the entire peritoneal cavity that extends from the dome of the diaphragm superiorly to the pelvic diaphragm inferiorly and laterally from the right to the left lateral abdominal wall. The superior border of the whole-abdominal and pelvic field will be placed approximately 1 cm above the dome of the diaphragm. The inferior border of the field will be placed at the bottom of the obturator foramen. The lateral borders of the field will be placed approximately 1 cm beyond the lateral abdominal wall. The femoral heads should be shielded during radiotherapy. An AP-PA is recommended for whole-abdominal and pelvic irradiation. The dose/fractionation schedule for whole-abdominal and pelvic radiotherapy is 19.5 Gy in 13 fractions of 1.5 Gy over 17 d for those 12 months or older. For these patients, the remaining kidney should be shielded to limit the dose to 14.4 Gy. In the case of infants, the wholeabdominal and pelvic dose is 10.5 Gy in 7 fractions of 1.5 Gy over 9 d. This treatment should be CT planned to allow dose volume histograms to be generated for organs at risk. This is especially important if a second phase of treatment to boost the dose to macroscopic residual disease is being contemplated (Section 9.1.8). Boost for gross residual disease Patients with gross residual disease after surgery may receive a second phase of treatment after flank or wholeabdominal and pelvic radiotherapy. This requires individualised consideration. Depending on factors such as the volume which would require treatment, and the age of the patient, a lower dose may be deemed safer, or the boost may be omitted. The GTV will be defined on the post-operative planning CT scan used for planning the first phase of treatment. The CTV will usually be the same as the GTV, but may be extended to ensure uniform irradiation of vertebral bodies. The PTV will be the CTV þ 1 cm unless departmental quality control data indicate that a different margin is appropriate. The organs at risk will already have been delineated on the planning CT scan. Fields will be shaped with multileaf collimator (MLC) or customised blocks to conform to the PTV. The most appropriate field arrangement will be selected by the clinician taking into account the composite dose volume histograms for phase I and phase II combined, with respect to coverage of the PTV and the dose constraints to organs at risk as stated in Section 9.1.6. The dose will usually be 10.8 Gy in six fractions of 1.8 Gy over 8 d, but 10.5 Gy in seven fractions over 9 d may be more appropriate in infants or if the volume is large. Whole-lung irradiation Both lungs are irradiated regardless of the number and location of the metastases. Treatment should be CT planned with patient lying supine with the arms to the side, slightly away from the body. The CTV includes the entire lungs, mediastinum and the pleural recesses. The CTV to PTV margin should take account of respiratory movement and is likely to be about 1 cm superiorly and laterally and 2 cm inferiorly. AP-PA and posterior parallel-opposed field will be used such that the PTV is encompassed with the 95% isodose. CT planning will take into account and correct the increased transmission through lung tissue. The inferior border of the field should lie in an intervertebral space, often below L1. The shoulder joints should be protected by MLC or cerrobend shielding. The whole-lung irradiation (WLI) dose/ fractionation schedule for those aged 12 months or over is 15 Gy with lung correction in 10 fractions of 1.5 Gy over 12e14 d. For infants, it is 10.5 Gy in seven fractions of 1.5 Gy over 9 d. If patients require both whole-lung and infra-diaphragmatic irradiation, then both fields should be treated simultaneously whenever possible. As the volumes for WLI often abut or overlap with the volumes for flank or whole-abdominal and pelvic radiotherapy, the contiguous areas should be treated in B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8278 the first instance as a single volume with a single pair of appropriately shaped AP-PA and posterior parallelopposed fields. For such a large volume, a fraction size of 1.5 Gy will be used. The fields will be reduced in size (off the lungs) after 10 fractions (15 Gy) to cover only the infra diaphragmatic volume. If the WLI volume and the flank volume appear well separated, they may be treated simultaneously as two separate areas, but great care must be taken when planning to ensure an adequate gap so that there is no overlap. Similarly, if WLI and infradiaphragmatic radiotherapy are given at different times, care must be taken to ensure that there is no overlap. Localized foci of lung disease persisting 2 weeks after the delivery of WLI may either be excised or given an additional 7.5 Gy in five fractions. The volume of the lungs included in this boost irradiation field should be <30% in order to limit the acute and long-term pulmonary complications that could result from higher doses of irradiation. Liver irradiation The entire liver is included in the irradiation portal only if the liver is diffusely involved (19.8 Gy in 11 fractions of 1.8 Gy.) In infants the dose/fractionation schedule should be 15 Gy in 10 fractions of 1.5 Gy. If the entire liver volume is not involved, then only the metastases with a margin of 2 cm is irradiated. Additional boost irradiation doses of 5.4 to 10.8 Gy may be administered to limited volumes (<75% of the entire liver) at the discretion of the clinical oncologist. While irradiating the liver, the dose to the upper pole of the remaining kidney should be monitored. A posterior kidney block may be inserted in order to limit the remaining kidney to 14.4 Gy. An AP-PA technique is recommended for liver irradiation. Brain irradiation In patients with brain metastases, the whole brain is included in the irradiation field to a dose of 21.6 Gy in 12 fractions of 1.8 Gy. A boost of at least 10.8 Gy is required to site of metastases. In patients with 3 circumscribed lesions especially in patients younger than 3 years, a limited volume (tumour or tumour bed only with 0e1 cm margin) boost dose of 10.8 Gy in 6 fractions using intensity-modulated radiation therapy (IMRT) or sterotactic radiotherapy may be administered after whole-brain irradiation to 21.6 Gy. A lateral parallel-opposed technique (right and left lateral) is recommended for whole-brain irradiation. Bone irradiation In patients with bone metastases, the GTV is the lesion as shown on appropriate imaging, which may include skeletal scintiography, plain radiographs MRI and CT. The clinical target volume will usually include a margin of apparently healthy bone up to 2 cm. A narrower margin may be appropriate where the metastasis is close to the edge of the bone. Irradiation of the epiphyses should be avoided where possible to diminish late effects. An appropriate margin should be added for the PTV, taking into account the technique of immobilisation used. The entire bone need not be irradiated. An AP-PA technique is usually recommended for bone irradiation, depending on the anatomical site. Thebone irradiation dose is 25.2 Gy in 14 fractions of 1.8 Gy, but may be modified if appropriate. Lymph node irradiation Lymph nodes with metastatic tumour that have not been surgically removed should receive radiation therapy. Groups of lymph nodes which were involved at presentation should be irradiated in their entirety. The GTV will be the nodal area including any residual mass after chemotherapy as defined on the planning CT scan. The CTV will usually be a 1 cm margin around the GTV. The margin for PTV definition will depend on immobilisation and individual departmental data. If vertebrae are to be irradiated, the whole vertebral body shall be included in the fields. For mediastinal and abdominal nodes, a parallel-opposed field arrangement usually gives best coverage of the PTV. Where possible, nodal areas will be treated in continuity with the primary tumour site or other metastatic sites requiring irradiation. The dose will usually be 19.8 Gy in 11 fractions of 1.8 Gy. Target dose The daily dose to ICRU prescription points shall be 1.8 Gy, except in younger children (e.g. <3 years) or when large volumes (e.g. whole lung or whole abdomen and pelvis) are to be treated. Extrarenal non-CNS rhabdoid tumour All patients should have a consultation by a radiation oncologist at the time of study entry so that the radiation oncologist can assist in providing appropriate staging/grouping of the patient and review the adequacy of the initial diagnostic imaging studies for subsequent local control treatment with RT. Extrarenal non-CNS rhabdoid tumours Gross total resection with no residual disease (microscopic negative margins) (group I) 36 Gy in 20 fractions Gross total resection with microscopic residual disease (microscopic positive margins) (group II) 45 Gy in 25 fractions Biopsy only or gross residual disease (group III) 50.4 Gy in 28 fractions These total doses and fractionation schedules may need to be modified taking into account factors including the age of the child, the volume requiring irradiation, critical normal structures and co-morbidity. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e82 79 Equipment Treatment will usually be with x-ray photons of 4e20 MV from a linear accelerator. The use of cobalt teletherapy is not acceptable. In some circumstances, the use of electrons may result in a more favourable dose distribution. Similarly, interstitial or intacavitary brachytherapy may be preferable in certain circumstances, such as with tumours at gynaecological, extremity and some non-parameningeal head and neck primary sites. Brachytherapy should not be used without careful discussion and is only appropriate in specialised treatment centres. Proton therapy is permitted in this study in specialised treatment centres. Protocol target volumes Three-dimensional treatment planning is strongly encouraged for patients treated on this study. All treatment planning, regardless of whether it is standard or three-dimensional conformal/IMRT, will be based upon the following target definitions. Treatment will be prescribed to the PTV, which will be derived from the GTV and CTV as follows: GTV The GTV is defined as the pre-treatment visible and/or palpable disease defined by physical examination, operative surgical findings, computer tomography, or magnetic resonance imaging. The T1 MR image with contrast is usually optimal imaging study. In special circumstances, changes can be made in this definition based upon the post-operative geometry of the target volume. In patients who have undergone primary surgical tumour resection, the entire surgical scar should be included in the GTV. However, in general, the GTV does not change based on any surgical resection or chemotherapy response. CTV For all Clinical Groups, the CTV is defined as the GTV þ 1.5 cm (but not extending outside of the patient). For some sites, the definition of the CTV is modified to account for specific anatomic barriers to tumour spread. The CTV will always include the entire draining lymph nodes chain if the regional nodes are clinically or pathologically involved with tumour. Patients with gross residual disease and primary sites in the head and neck and vulva/uterus who do not undergo second look operations may have second CTV and PTV defined for a cone down boost. The patients will receive a total dose of 50.4 Gy given to the PTV. PTV For all Clinical Groups, the PTV is defined as the CTV plus an institution specific margin to account for day-to-day setup variation related to the ability to immobilise the patient and physiological motion of the CTV. Planning organ-at-risk volume Planning organ-at-risk volumes (PRV) will be defined for each organ at risk defined in Section 14, Radiotherapy Guidelines, and for any other organ that the treating clinical oncologist wishes to limit to a specific dose. The PRV is defined as the volume of the organ at risk plus a margin to account for that organ’s positional uncertainty. Special modifications of GTV and CTV for certain sites ➢Orbit:. For orbit primaries, the CTV will not extend outside the bony orbit, providing there is no bone erosion of the orbit. ➢Thorax:. Tumours which have displaced a significant amount of lung parenchyma which has subsequently returned to normal anatomic position following surgical debulking will have the GTV defined as the preoperative tumour volume excluding the intra-thoracic tumour which was debulked. However, all areas of preoperative involvement of the pleura will be included in the GTV. ➢Bladder/prostate, perineum, pelvis, biliary tree and abdomen:. Tumours which have displaced a significant amount of bowel which has subsequently returned to normal anatomic position following surgical debulking will have the GTV defined as the pre-operative tumour volume excluding the intra-abdominal or intra-pelvic tumour which was debulked. However, all areas of pre-operative involvement of the peritoneum or mesentery, and the site of origin, will be included in the GTV. Timing of radiotherapy:. All patients who require radiation therapy shall begin treatment concurrent with the initiation of chemotherapy after surgery. If surgery is performed up front, radiation therapy should begin as close to the beginning of chemotherapy as possible. If surgery is delayed, radiation therapy should begin after recovery from surgery when chemotherapy is reinitiated. Chemotherapy will be given concurrent with radiotherapy. The regimen is designed so that doxorubicin is avoided during the six weeks following irradiation. Prescribed dose and fractionation The total radiotherapy dose for the various clinical groups are indicated in the table below: B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8280 Interruptions Patients requiring an interruption in radiotherapy (i.e. for low counts, infection, toxicity) will receive a modification in the schedule as shown in the tables below Normal tissue sparing It is important to protect normal vital structures whenever possible. Such shielding must be weighed against the possibility of under treatment of known tumour-bearing tissue. The recommended upper dose limits for different organs are shown in the table below. These limits are the same as, or less than, those used in the previous IRS studies and have not been associated with excessive toxicity when used with chemotherapy. References [1] Stiller C, editor. Childhood cancer in Britain: incidence, survival, mortality. Oxford: Oxford University Press; 2007. [2] Brennan BMD, Stiller C, Bourdeaut F. Extracranial rhabdoid tumours: what we have learned so far and future directions. Lancet Oncology 2013;14:329e36. [3] Tomlinson GE, Breslow NE, Dome J, Adams Guthrie K, Norkool P, Li S, et al. Rhabdoid tumor of the kidney in the National Wilms’ Tumor Study: age at diagnosis as a prognostic factor. Journal of Clinical Oncology 2005;23:7641e5. [4] Sultan I, Qaddoumi I, Rodrı´guez-Galindo C, Al Nassan A, Ghandour K, Al-Hussaini M. Age, stage, and radiotherapy, but not primary tumor site, affects the outcome of patients with malignant rhabdoid tumors. Pediatr Blood Cancer 2010;54: 35e40. [5] Bourdeaut F, Fre´neaux P, Thuille B, Bergeron C, Laurence V, Brugie`res L, et al. Extra-renal non-cerebral rhabdoid tumours. Pediatr Blood Cancer 2008;51:363e8. [6] Waldron PE, Rodgers BM, Kelly MD, Wormer RB. Successful treatment of a patient with stage IV rhabdoid tumor of the kidney:case report and review. J Pediatr Hematol Oncol 1999;21: 53e7. [7] Wagner L, Hill DA, Fuller C, Pedrosa M, Bhakta M, Perry A, et al. Treatment of metastatic rhabdoid tumor of the kidney. J Pediatr Hematol Oncol 2002;24:385e8. [8] Maurer HM, Beltangady M, Gehan EA, Crist W, Hammond D, Hays DM, et al. The Intergroup Rhabdomyosarcoma Study I: a final report. Cancer 1988;61:209e20. [9] Womer RB, West DC, Krailo MD, Dickman PS, Pawel BR, Grier HE, et al. Randomized controlled trial of intervalcompressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children’s Oncology Group. J Clin Oncol 2012 Nov 20;30(33):4148e54. [10] Torchia J, Picard D, Lafay-Cousin L, Hawkins CE, Kim SK, Letourneau L, et al. Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis. Lancet Oncol 2015; 16:569e82. Gross total resection with no residual disease (negative margins) (Group I) 36 Gy in 20 fractions Gross total resection with microscopic residual disease (positive margins) (Group II) 45 Gy in 25 fractions Biopsy only or gross residual disease (Group III) 50.4 Gy in 28 fractions All radiation should be given at 1.8 Gy per fraction with one fraction given per day. Five fractions should be given per week. Patients prescribed 36 Gy (Gp I) Timing Fx size (Gy) # Fx Total Dose (Gy) Total time Normal and/or up to 2-week split 1.8 20 36 4e6 Weeks 2- to 3-week split 1.8 21 37.8 6e7 Weeks >3-week split 1.8 22 39.6 >7 Weeks Patients prescribed 45.00 Gy (Gp II) Timing Fx size (Gy) # Fx Total dose (Gy) Total time Normal and/or up to 2-week split 1.8 25 45 5e7 Weeks 2- to 3-week split 1.8 26 46.8 7e8.4 Weeks >3-week split 1.8 27 48.6 >8.4 Weeks Patients prescribed 50.40 Gy (Gp III) Timing Fx size (Gy) # Fx Total dose (Gy) Total time Normal and/or up to 2-week split 1.8 28 50.4 5.4e7.3 Weeks 2- to 3-week split 1.8 29 52.2 7.4e8.4 Weeks >3-week split 1.8 30 54.0 >8.4 Weeks Normal tissue tolerance Organ Dose limit (Gy) Optic nerve and chiasm 50 Lacrimal gland 41.4 Small bowel 45.0 Spinal cord 45.0 Lung (when >1 /3 but <1 /2 of total lung volume is in the PTV) 18.0 Lung (when >1 /2 of total lung volume is in the PTV) 15.0 Whole kidney 19.8 Whole livera 23.4 a Tolerance for partial liver radiation: when two third of the liver volume is included in the initial radiation port and more than one third of the liver requires a boost beyond the maximum whole liver dose (23.4), the total dose to the boost volume may be limited to a maximum of 30 Gy. The boost volume should not exceed two third of the total liver volume. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e82 81 [11] Morgenstern DA, Gibson S, Brown T, Sebire NJ, Anderson J. Clinical and pathological features of paediatric malignant rhabdoid tumours. Pediatr Blood Cancer 2010;(1):29e34. [12] Kordes U, Bartelheim K, Modena P, Massimino M, Biassoni V, Reinhard H, et al. Favorable outcome of patients affected by rhabdoid tumors due to rhabdoid tumor predisposition syndrome (RTPS). Pediatr Blood Cancer 2014;61(5):919e21. [13] Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci 2013;110(19):7922e7. [14] Lin D, Wei LJ, Ying Z. Checking the cox model with cumulative sums of martingale-based residuals. Biometrika 1993; 80:557e72. B. Brennan et al. / European Journal of Cancer 60 (2016) 69e8282