MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV EXPERIMENTÁLNÍ BIOLOGIE Habilitační práce RNDr. Sabina Ševčíková, Ph.D. Brno 2015 Á' MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV EXPERIMENTÁLNÍ BIOLOGIE O U"i (TI » NOVE ASPEKTY STUDIA GENOMIKY MNOHOČETNÉHO MYELOMU Poděkování Jirkovi, Sáře a Soně za jejich podporu a lásku prof. MUDr. Romanu Hájkovi, CSc., zakladateli Babákovy myelomové skupiny Mgr. Lence Bešše, Ph.D. a Mgr. Lence Sedlaříkové, mým naprosto skvělým studentkám Ing. Martině Almáši, Ph.D. Prof. MUDr. Anně Vašků, CSc, přednostce Ústavu patologické fyziologie 1 Tuto práci věnuji svým skvělým rodičům, Jarce a Viktorovi 2 Obsah 1 Úvod......................................................................................................................................5 2 Problematika..........................................................................................................................6 2.1 Mnohočetný myelom......................................................................................................6 2.2 Historie MM...................................................................................................................6 2.3 Patofyziologie MM.........................................................................................................7 2.4 Mikroprostředí kostní dřeně u MM................................................................................9 2.5 Klinické projevy MM...................................................................................................10 2.6 Léčba mnohočetného myelomu....................................................................................10 2.6.1 Chemoterapeutika..................................................................................................11 2.6.2 Transplantace hematopoetických kmenových buněk............................................12 2.6.3 Nové léky...............................................................................................................12 2.7 Diagnostika MM...........................................................................................................15 2.8 Prognóza MM...............................................................................................................16 2.8.1 Prognostické systémy............................................................................................16 2.8.2 Cytogenetika..........................................................................................................IV 2.8.3 Genomické analýzy...............................................................................................20 2.9 Flow cytometrie............................................................................................................22 2.10 Detekce minimálni reziduálni choroby.......................................................................23 3 Extramedulární forma mnohočetného myelomu.................................................................25 3.1 Incidence EM................................................................................................................25 3.2 Místa výskytu a typy EM..............................................................................................26 3.3 Prognóza EM................................................................................................................26 3.4 Molekulární mechanismy EM relapsu..........................................................................27 4 MikroRNA...........................................................................................................................31 4.1 Biogeneze mi RNA........................................................................................................32 4.2 Cirkulující mi RNA.......................................................................................................33 4.3 MikroRNA u mnohočetného myelomu........................................................................33 4.4 Resistence na léky a miRNA u mnohočetného myelomu............................................36 4.5 Mechanismus deregulace miRNA u MM.....................................................................37 4.6 miRNA ovlivňující kritické geny u MM......................................................................38 4.7 Cirkulující mikroRNA u MM.......................................................................................41 5 Závěr....................................................................................................................................43 3 6 Reference.............................................................................................................................44 Seznam obrázků a tabulek......................................................................................................58 Seznam příloh.........................................................................................................................59 4 lÚvod Babákova myelomová skupina (BMS) je výzkumné těleso, které se zabývá výzkumem monoklonálních gamapatií. Tato skupina je zapojena do International Myeloma Working Group, což je poradní orgán složený z kliniků i z vědců, který vydává doporučení na léčbu i důležitých výzkumných témat. BMS je také zapojena v rámci České republiky do České myelomové skupiny. Jako skupina jsme se rozhodli zaměřit na několik témat. Především se jednalo o výzkum MGUS (premaligního stavu mnohočetného myelomu), zavedení detekce minimální residuální choroby (MRD), výzkum extramedulárního relapsu mnohočetného myelomu a nových markerů onemocnění, především cirkulujících mikroRNA. Moje práce se v rámci této skupiny rozvíjela právě těmito směry. Jednak jsem se zaměřila na výzkum extramedulárního relapsu, kdy jsme se věnovali především poznání problematiky zvyšující se incidence a komplexní biologie této formy onemocnění. Dále jsme se věnovali zavedení metody detekce MRD pomocí qPCR, která je i nadále zlatým standardem hodnocení účinnosti léčebných strategií u klinických studií. V neposlední řadě jsem se věnovala novým markerům onemocnění, zejména cirkulujícím mikroRNA, které mají potenciál překonat bolestivý postup stanovení diagnózy mnohočetného myelomu, který využívá invazivní odběr kostní dřeně. Rovněž by mohly sloužit jako prognostické či prediktivní markety a mít tak roli i v tzv. „personalizované medicíně", což je u mnohočetného myelomu z důvodu jeho rozsáhlé heterogenity velmi důležité. Také se zdá, že tyto markety mají důležitou roli v patogenezi onemocnění. Tato témata byla pod mým vedením zpracována rovněž studenty bakalářských, magisterských a doktorských studijních programů v rámci jejich závěrečných prací. 5 2 Problematika 2.1 Mnohočetný myelom Mnohočetný myelom (MM) je zhoubné lymfoproliferativní onemocnění které je charakterizováno infiltrací a akumulací patologických klonálních plazmatických buněk (PB) v kostní dřeni (KD), osteolytickými ložisky ve skeletu a prítomností monoklonálního imunoglobulinu (M-Ig) v séru a/nebo moči (Hájek et al, 2012). Jedná se o druhé nej častější hematoonkologické onemocnění, které v České republice vykazuje incidenci okolo 4/100 000 obyvatel. V Evropě je ročně diagnostikováno více než 40 000 nových případů (Adam et al, 2008; Hájek et al, 2012). Celkově MM představuje asi 10 % všech hematologických malignit, 1 až 2 % všech nádorových onemocnění a zhruba 2 % všech úmrtí v důsledku nádorových onemocnění (Avet-Loiseau et al, 2007). MM postihuje zejména starší osoby, medián věku při stanovení diagnózy se pohybuje kolem 65 let a je mírně častější u mužů (Adam etal, 2008). Dnes je prokázané, že MM předchází monoklonální gamapatie nejasného významu (MGUS), ačkoliv okolnosti a důvody zvratu benigního MGUS do maligního MM nejsou dosud objasněny (Kýle et al, 2011). Prvotní příčina vzniku MM je dodnes neznámá a průběh u jednotlivých pacientů velice heterogenní. I když se daří prodlužovat dobu remise, většina pacientů stále relabuje. Skupina vysoce rizikových pacientů (tvoří asi 10 až 15 % všech MM pacientů) relabuje obvykle do 12 měsíců od diagnózy (Shaughnessy et al, 2007). Zároveň je možné za vysoce rizikový MM považovat extramedulární myelom (EM), protože tito pacienti mají také výrazně horší přežití. Důsledkem akumulace genetických změn u EM pacientů je autonomní růst myelomových buněk, který je nezávislý na stromatu KD a umožňuje přechod myelomových buněk do extramedulární oblasti - nejčastěji do podkoží a měkkých tkání (Usmáni etal, 2012). 2.2 Historie MM První řádně zdokumentovaný případ MM byl publikován v roce 1844. Jednalo se o případ 394eté ženy Sarah Newbury, u které se objevily příznaky únavy a bolesti kostí v důsledku mnohonásobných zlomenin (Obr. 1). Teprve po její smrti, která nastala o 4 roky později, pitva prokázala výrazné změny KD (Solly, 1844). 6 Obrázek 1 První popsaný pacient s mnohočetným myelomem - Sarah Newbury (z Kyle et Rajkumar, 2008). A) Destrukce sterna B) Pacientka se zlomeninami C) Destrukce femuru Nejznámnějším případem MM té doby byl však Thomas Alexander McBean, u kterého se ve věku 45 let objevily podobné příznaky jako u Sarah Newbury - bolesti, zlomeniny kostí. I přes veškerou „moderní" léčbu té doby, která zahrnovala například pijavice, McBean zemřel (Macintyre, 1850). Vzorek jeho moče byl poslán Dr. Bence Jonesovi, který popsal přítomnost proteinu v moči. Tento protein se do dnešní doby nazývá Bence-Jonesova bílkovina a stal se jedním ze základních markerů MM (Jones, 1848). Myelom byl také nazýván Kahlerovou chorobou podle známého pražského lékaře Otty Kahlera, který popsal chorobu svého kolegy Dr. Loose. Pacient měl progradující bolesti kostí, proteinurii s typickou charakteristikou Bence-Jonesovy bílkoviny a v době pitvy byly nalezeny velké kulaté buňky konzistentní s buňkami MM (Kýle et Rajkumar, 2008). 2.3 Patofyziologie MM Patogeneze MM je komplexní multifaktoriální proces vedoucí k nádorové transformaci populace B-lymfocytů sérií genetických změn (Hájek et al., 2011). Takto pozměněné buňky dále nekontrolovatelně proliferují a diferencují v buňky plazmablastické, které si zachovávají schopnost migrace a proliferace. Zralá myelomová (plazmatická) buňka je terminálním stadiem vznikajícím z pozměněných plazmablastů nahromaděných v KD, kde přežívá podstatně déle než fyziologické PB, které přibližně po dvou dnech produkce přirozených imunoglobulinů podléhají apoptóze. Proto jsou myelomové buňky označovány za buňky dlouhověké. Ačkoliv se zdá, že buňky v terminálním stádiu se již dále nemnoží, nekontrolovatelná proliferace v KD je hlavním rozdílem mezi vývojovou řadou 7 myelomových buněk a fyziologickým procesem maturace plazmablastů (Obr. 2) (Adam et al, 2001; Shapiro-Shelef et Calame, 2004). Obrázek 2 Srovnání počtu myelomových PB (vlevo) oproti počtu fyziologických PB (vpravo) (Maslak, 2009) Studie provedené během posledního desetiletí přinesly značný posun v porozumění biologických a molekulárních mechanismů, které se patogeneze MM účastní. Zásadní vliv má vhodné mikroprostředí KD umožňující růst nádoru, což úzce souvisí s novotvorbou cév (angiogenezí), poruchou funkce imunitního systému a interakcí buněk myelomových s buňkami stromatu (Uchiyama et al., 1993; Hájek et al., 2011). Kooperace těchto procesů indukuje sekreci mnohých cytokinů a aktivaci vnitrobuněčných signálních drah (pro proliferaci, přežití, lékovou rezistenci a nestabilitu genomu buněk MM). Příkladem může být interleukin-6 (IL-6), který aktivuje signální dráhu JAK2/STAT3 a tím také geny stimulující růst a inhibující apoptózu myelomových buněk (Shain etal., 2009). Zároveň buňky MM vykazují nestabilní genom a v průběhu onemocnění v něm dále dochází k četným mutacím a chromozómovým aberacím (CHA). Poměrně často se jedná o složité komplexní změny karyotypu (Hájek et al., 2011). Běžným cytogenetickým nálezem jsou aneuploidie, monozomie chromozomu 13, translokace zasahující lokus pro těžký řetězec imunoglobulinu (gen IGH, oblast 14q32), ztráta krátkého raménka chromozomu 17, zisk dlouhého raménka chromozomu 1 a další. Některé z těchto změn jsou typické pro vysokorizikový MM a jsou zařazeny mezi nepříznivé prognostické faktory (Sawyer, 2011). MM typicky probíhá v určitých vývojových stadiích (Obr. 3), které je od sebe nutné odlišit pomocí diagnostických kritérií. 8 myeloro MGtiS myi^m ivmplflnigtlclí mytiam A Obrázek 3 Vícestupňový transformační proces MM (Špička et al, 2005) 2.4 Mikroprostředí kostní dřeně u MM Hlavním zdrojem obtíží u MM je osídlování KD myelomovými buňkami, což vede k typickým znakům MM, jako jsou osteolytické léze a patologické zlomeniny. V kostech neustále probíhá proces remodelace, kde resorpce následuje novotvorbu kostní tkáně, přičemž jsou oba děje v rovnováze. U MM je tato rovnováha porušována ve prospěch kostní resorpce (Bataille et al., 1991). MM je vhodný model pro studování interakcí tumoru a mikroprostředí ze tří důvodů: na rozdíl od normálních buněk se maligní PB hromadí pouze v KD, což naznačuje, že stromální buňky poskytují jedinečné mikroprostředí pro růst maligních buněk. Dalším důvodem je přítomnost mnoha adhezivních molekul na povrchu myelomových buněk (Uchiyama et al., 1992), normální buňky povrchové markety téměř neprodukují. Konečně třetím důvodem je růst heterogenních populací adherentních buněk odebraných zKD pacientů s MM v podmínkách in vitro (Caligaris-Cappio et al., 1991). Dochází zde k procesům osídlování („homing") myelomovými buňkami, šíření MM malými cévami a vzniku rozpustných faktorů. Sekrecí těchto faktorů (cytokiny, chemokiny) a fyzickou interakcí podporují stromální buňky růst, přežívání, rezistenci k léčbě a pohyby myelomových buněk (Uchiyama et al., 1992; Pellat-Deceunynck et al., 1995). Mikroprostředí KD je nejen radikálně ničeno přítomností myelomových buněk a naopak také mění jejich chování. Interakce MM buněk s proteiny mezibuněčné hmoty a stromálními buňkami KD je velmi důležitá pro patogenezi a rezistenci k léčbě MM (Obr. 4). Mikroprostředí KD bylo zpracováno do přehledové práce v časopise Klinická onkológie (Fišerová et al., 2012). 9 Obrázek 4 Interakce mezi plazmatickými buňkami a buňkami KD u MM (Palumbo et Anderson, 2011; upraveno) 2.5 Klinické projevy MM Klinické projevy MM zahrnují soubor typických příznaků, z nichž ne všechny se projeví u každého pacienta. Jedná se o příznaky vyvolané produkovaným M-Ig (hyperviskozita, poruchy srážení krve, myelomová nefropatie, motorická a senzitivní polyneuropatie), poruchy imunitního systému (únava, časté infekce a horečky, celková slabost) dále bolesti kostí způsobené osteolýzou a jiné méně časté příznaky jako syndrom zvýšené kapilární propustnosti, metabolické poruchy a kožní projevy (Adam et al., 2008). 2.6 Léčba mnohočetného myelomu Mnohočetný myelom je obtížně léčitelné, ale už ne nevyléčitelné onemocnění. Zatímco v 50. letech minulého století byl medián přežití pacientů se symptomatickým myelomem méně než 1 rok, v současnosti se více než ztrojnásobil, a to především u osob mladších 65 let (Turesson et al., 2010). Cílem primoléčby je dosažení kompletní remise au relabujících pacientů nejméně velmi dobré parciální remise. U většiny pacientů stále dochází k relapsu po různě dlouhé a zkracující se remisi (Hájek et al., 2011). 10 Léčba MM zůstávala dlouhou dobu bez výrazného zlepšení kvality a délky života pacientů. Skutečné výsledky přineslo až zavedení klasických chemoterapeutik melfalanu aprednisonu v 60. letech (Alexanian et al., 1969). Melfalan s prednisonem se v rámci kombinace s terapeutiky nové éry používají dodnes. Léčebnou odpověď a medián přežití až o 12 měsíců prodloužila transplantace multipotentních hematopoetických buněk. První slibné výsledky přinesla intravenózni infuze buněk KD provedená roku 1957 (Thomas et al., 1957). První klasická autologní transplantace s vysokodávkovanou chemoterapií byla zdokumentována McElwainem aPowlesem (McElwain et Powles, 1983). Ačkoliv transplantace zůstává vysoce efektivní léčebnou strategií u MM, nelze ji aplikovat plošně kvůli vysokému věku nemocných. Navíc každá transplantace musí být kombinována s účinnými terapeutiky, mezi nimiž stále větší podíl získávají nové léky. Inhibitory proteazomu (PI) a imunomodulační léky (IMiDs) začaly novou éru v léčbě MM. Jsou to jediné léky proti myelomu s vysokou antimyelomovou aktivitu, jak v monoterapii, kde dosahují léčebné odpovědi u 1/3 předléčených nemocných, tak v kombinaci s konvenčními chemoterapeutiky, kde dosahují léčebné odpovědi u 60 — 100% nemocných (Hájek et al., 2011). Na rozdíl od cytotoxických chemoterapeutik j sou založeny na modulaci signálních kaskád a molekulárních interakcí myelomových buněk s mikroprostředím nádoru, čímž se zvyšuje jejich účinnost a specifita vůči myelomu. V současnosti jsou v preklinických a klinických studiích hodnoceny třetí generace imunomodulačních agens a proteazomových inhibitorů. Očekává se zvýšení efektivity a pokles nežádoucích účinků. 2.6.1 Chemoterapeutika Konvenční protinádorová chemoterapie u myelomu je zaměřena na podávání alkylačních cytostatik a glukokortikoidů (Hájek et al., 2011). Principem působení alkylujících látek je zastavení buněčného cyklu s následnou smrtí buňky. Alkylační činidla působí na rychle proliferující buňky a jsou nezávislá na fázi buněčného cyklu. Mechanismus alkylace spočívá v navázání alkylové skupiny na dusíky dvou guaninových bází sousedních řetězců DNA. Mezi řetězci se vytvoří pevná kovalentní vazba, která je příčinou zastavení replikace, a tím i buněčného cyklu (Spanswick et al., 2002). Mezi nej významnější z nich patří melfalan a cyklofosfamid (Hájek et al., 2011). Glukokortikoidy jsou steroidní hormony, které mimo jiné způsobují apoptózu hematologických buněk pravděpodobně represí 11 transkripce genů kritických pro přežití buňky (Greenstein et al., 2002). Mezi nej významnější kortikosteroidy patří prednison a dexametazon (Hájek et al., 2011). 2.6.2 Transplantace hematopoetických kmenových buněk V České republice bylo v roce 2001 provedeno 330 autologních transplantací, přičemž 90 z nich pro nemocné s MM (Špička et al., 2005). První randomizovanou studií srovnávající autologní transplantační léčbu s kontrolní terapií byla analýza francouzské skupiny, jejímž výsledkem bylo signifikantní zvýšení kompletních remisí a velmi dobrých parciálních remisí o více než polovinu. Medián doby přežití byl prodloužen o 13 měsíců (Attal et al., 1996). Mnoho následujících studií potvrdilo pozitivní přínos autologní transplantace, a to i přesto, že nevede k vyléčení MM, zůstává pro čtvrtinu nemocných způsobem, jak dosáhnout delšího přežití. Léčebný postup s největším kurativním potenciálem představuje alogenní transplantace, která je však díky vysokým rizikům mortality a s tím spojenými restrikčními opatřeními vhodná pouze pro desetinu nemocných s MM (Reynolds et al., 2001). Podle toho, zdaje pacient způsobilý pro transplantaci, byly zavedeny dvě formy iniciální terapie. Klasickou počáteční terapii pro pacienty před transplantací představuje kombinace léků vinkristinu, doxorubicinu a dexametazonu (VAD). Nemocní nezpůsobilí pro transplantaci jsou většinou léčeni kombinací melfalanu, prednisonu (MP) a jedním z nových léků (Alexanian et al., 1990; Palumbo et al., 2008; San Miguel et al., 2008). 2.6.3 Nové léky Imunomodulační léky Mezi IMiDs patří thalidomid a jeho analogy, lenalidomid a pomalidomid. Antimyelomové účinky těchto léků, jejichž mechanismy doposud nebyly uspokojivě vysvětleny, jsou pleiotropního charakteru a zahrnují modulace imunitního systému, antiangiogenní, protizánětlivé a antiproliferativní aktivity s přímým vlivem jak na nádorové buňky, tak na jejich mikroprostředí. Jedním z hlavních mechanismů působení IMiDs je modifikace vrozené a adaptivní imunity jedince prostřednictvím buněk imunitního systému. Bylo prokázáno, že IMiDs kostimulují CD4+ a CD8+ T-lymfocyty, které za normálních okolností potřebují ke své plné aktivaci signál zajišťovaný antigén prezentující buňkou prostřednictvím povrchové molekuly CD28. Aktivace T-lymfocytů má za následek jejich zvýšenou proliferaci a produkci prozánětlivých cytokinů třídy Th-1, mezi něž patří interleukin-2 (IL-2) a interferon-y (IFNy) (Haslet et al., 1998). Zvýšená sekrece IL-2 12 následně způsobí aktivaci NK buněk. NK buňky jsou základní složkou vrozené imunity a mají schopnost zabíjet nádorové buňky (Obr. 5). Buněčná cytotoxicita může být indukována buď závisle (ADCC), anebo nezávisle na protilátkách (Davies et al., 2001). Antiangiogenní vlastnosti FMiDs jsou dány inhibicí chemotaktických faktorů podílejících se na migraci endotelových buněk formujících nové cévy. Mezi takové faktory patří zejména tumor nekrotizující faktor a (TNFa), vaskulární endoteliální růstový faktor (VEGF) a bazický fibroblastový růstový faktor (bFGF) sekretované stromálními buňkami KD (Dredge et al., 2002). S antiangiogenním a především s protizánětlivým účinkem je spojena i inhibice enzymu cyklooxygenázy 2 (COX-2). COX-2 hraje roli v transformaci kyseliny arachidonové v prostaglandiny. Produkce enzymu je indukována řadou prozánětlivých stimulů, jako jsou lipopolysacharidy (LPS), TNFa a interleukin-ip (IL-ip), přes signální dráhu jaderného faktoru kB (NFkB). Protizánětlivý efekt FMiDs je zprostředkován interleukinem 10 (IL-10) (Payvandi et al., 2004). Molekulární podstata účinku FMiDs byla zpracována do přehledové práce v časopise Leukémia Research (Sedlaříková etal, 2012). Obrázek 5 Pleiotropní účinek IMiDs u MM (Sedlaříková et al, 2012) 13 Inhibitory proteazomu PI se ukázaly být velice důležitou součástí léčby pacientů s MM. Prvním PI schváleným pro léčbu MM se stal bortezomib, který vykazoval silné antimyelomové účinky (Obr. 6) (Hájek et al, 2011). Bohužel, navzdory jeho vysoké účinnosti se u velkého procenta pacientů s MM po čase objevuje rezistence k tomuto léku. Mechanizmus inhibice proteazomu bortezomibem spočívá v jeho kovalentní vazbě na P5 podjednotku proteazomu, případně LMP7 podjednotku imunoproteazomu. S nižší afinitou se bortezomib váže také na podjednotky pi a P2 (Berkers et al, 2005). Samotná inhibice je zprostředkována farmokoforovou skupinou, v tomto případě zbytkem kyseliny borité (Groll et al., 2006). Jelikož je proteazom zapojen do obratu intracelulárních proteinů, patří mezi primární důsledky jeho inhibice hromadění nefunkčních proteinů a chyby v signálních drahách, které vyúsťují v narušení adheze myelomových buněk, potlačení novotvorby cév, zastavení buněčného cyklu, omezení odpovědi na poškození DNA a indukci apoptózy MM buněk (Richardson et al, 2005). Narušení mechanizmu opravy DNA Indukce stresu ER u MM buněk Aktivace kaspáz 8 a 9 Inhibice PI3K/AKT, MAPK signální dráhy Snížení anti-apoptotických proteinů rodiny BCL-2, stabilizace pro-apoptotických členů rodiny BCL-2 degradace IkB BORTEZOMIB plazmatické buňky 1 stromální buňky kostní dřeně £ i IL-6 Degradace V IGF-1 IkB Aktivace NF-kB dráhy Rust a přežívání ^- a transkripce MM MM buněk buněčných cytokinů Obrázek 6 Účinek bortezomibu u MM (Kubiczková et al., 2012) Úspěch bortezomibu vzbudil obrovský zájem o proteazom ové inhibitory. Optimalizace dávek a kombinace bortezomibu s jinými protinádorovými terapeutiky sice omezily jeho vedlejší účinky a částečně potlačily rezistenci, je však jasné, že druhá generace PI může přinést daleko lepší výsledky. Carfilzomib, Marizomib a MLN9708 reprezentují druhou generaci PI a nabízejí řadu výhod v podobě zvýšené účinnosti, bezpečnosti lékového 14 profilu a překonání rezistence k bortezomibu díky své odlišné chemické struktuře, biologickým vlastnostem, mechanizmu účinku, i/reverzibilitě inhibice proteazomu a způsobu užívání (Lonial et Boise, 2011). Poskytují tak nové možnosti pacientům, kteří se stali rezistentními k bortezomibu. Molekulární dráhy a působení inhibitorů proteazomu byly zpracovány do přehledových prací v časopise Klinická onkológie (Kubiczková et al., 2013a) a v časopise Journal of Cell and Molecular Medicíne (Kubiczková et al., 2014). 2.7 Diagnostika MM Diagnóza se stanovuje na základě srovnání biochemických, cytologických, rentgenologických a histologických nálezů pomocí všeobecně uznávaných diagnostických kritérií publikovaných skupinou International Myeloma Working Group (IMWG) v roce 2003. K diagnóze symptomatického myelomu pak postačí přítomnost M-Ig, klonálních PB a kritéria CRAB (Hájek et al, 2011) (Tab. 1 a 2). Tabulka 1 Diagnostická kritéria MM (IMWG, 2003; upraveno) Symptomatický MM Je přítomen M-Ig v séru a/nebo v moči (bez specifikace koncentrace). V KD jsou přítomny klonální PB (> 10 %). Je přítomno poškození orgánů a tkání myelomem, tak, jak je definováno v níže uvedené tabulce „CRAB". Extramedulární MM Není obvykle přítomný M-Ig, jen zcela výjimečně v nízké koncentraci. Prokázané solitární extramedulární ložisko klonálních PB. Normální KD, není přítomna infiltrace PB. Není přítomna dysfunkce orgánu či tkáně způsobená myelomem. Tabulka 2 CRAB - kritéria poškození orgánů či tkání myelomem (IMWG, 2003) Kritérium dysfunkce orgánu C - calcium Hyperkalcémie, kalcium > 2,75 mmol/1 nebo o 0,25 nad normální limit R - renal Selhání ledvin, kreatinin > 176,8 umol/1 A - anemia Hemoglobin < 100 g/l nebo 20 g/l pod dolní limit B - bone Kostní změny, osteolytická ložiska nebo osteoporóza s kompresivními frakturami 15 Relaps onemocnění je diagnostikován po splnění jednoho či více následujících kritérií: I) znovuobjevení M-Ig v moči nebo v séru; II) počet maligních PB v KD dosáhne či přesáhne 5 %; III) vznikají nová osteolytická ložiska nebo se zvětšují ložiska stávající; IV) hyperkalcémie, pokles hemoglobinu, vzestup sérového kreatininu (bez jiných možných příčin než znovuobnovené aktivity MM) (Adam et al, 2001; Hájek et al, 2012). 2.8 Prognóza MM 2.8.1 Prognostické systémy S využitím nejnovějších léčebných protokolů se nadále zvyšuje celkové přežití (OS) pacientů (5 let u 80 % pacientů, více než 10 let u 30-40 % pacientů). Průběh onemocnění je u jednotlivých pacientů vysoce variabilní - např. vysoce rizikoví pacienti s MM mají natolik negativní prognózu, že i přes využití nových strategií se předpokládaná délka života pohybuje mezi 2 až maximálně 5 roky (Hájek et al, 2011). Z tohoto důvodu musel být kromě diagnostických kritérií zaveden také systém pro stanovení pokročilosti onemocnění a rozdělení pacientů do skupin podle klinického stadia a prognózy. Prvním byl prognostický systém dle Durieho a Salmona (DS) (1975) se třemi stádii, která se vyznačují odlišnými hodnotami vybraných klinických parametrů odrážejících velikost nádorové masy. Prognostický význam tohoto systému v éře nových léků se stále zvažuje, je však nadále použitelný pro určení pokročilosti nádoru a doporučuje se uvádět i toto stádium u diagnózy vzhledem k možnosti srovnání výsledků léčby s dříve diagnostikovanými případy MM (Tuchman et Lonial, 2011; Hájek et al, 2012). V současné době se vedle DS systému používá mezinárodní prognostický systém navržený IMWG nazvaný International Staging System (ISS) (Greipp et al, 2005), který v době diagnózy sleduje pouze dva jednoduché a lehce měřitelné laboratorní ukazatele, sérové koncentrace albuminu a P2-mikroglobulinu (Tab. 3). ISS poskytuje dobrý základ pro budoucí pokročilejší studie, avšak pro vyhledávání vysokorizikových pacientů má určitá omezení. Identifikace pacientů s největším rizikem je zde dosaženo pouze u malé skupiny (5 - 9 %), přesnější vyhodnocení vyžaduje další cytogenetickou a molekulárně-genetickou analýzu. Je zaměřen na prognostiku na populační úrovni, ale nebere v potaz další důležité prognostické parametry, jako je míra proliferace a abnormální genom. Jeho význam a uplatnění v éře nových léků bude muset být upřesněn (Avet-Loiseau, 2010). Vysoce 16 rizikoví pacienti přestavují v DS systému i ISS klinické stadium III, které je v DS systému charakterizováno zejména poruchou funkce ledvin s hodnotou sérového kreatininu (> 2 mg/100 ml) (Durie et Salmon, 1975) a v ISS vysokou hladinou sérového P2-mikroglobulinu (> 5,5 mg/l) (Greipp etal., 2005). Tabulka 3 International Staging System (Greipp et al., 2005; upraveno) Klinické stadium Kritéria Medián přežití I sérový p2-mikroglobulin < 3,5 mg/l sérový albumin > 3,5 g/dl 62 II A: sérový p2-mikroglobulin < 3,5 mg/l a sérový albumin < 3,5 g/dl B: sérový p2-mikroglobulin 3,5 - 5,5 mg/l bez ohledu na hladinu sérového albuminu 44 III sérový p2-mikroglobulin > 5,5 mg/l 29 Ideální prognostický systém by měl podle Avet-Loiseau (2010) kombinovat sledování hladiny sérového P2-mikroglobulinu (jakožto odrazu nádorové masy), poruchy funkce ledvin, obecného stavu pacienta, proliferace PB a genetických změn. Je více než zřejmé, že stávající prognostické faktory nemohou být považovány za univerzální, zejména z důvodu již zmíněného nejistého uplatnění a významu v době nových léků. Identifikace rizikových skupin s vysokou prediktivní hodnotou by mohla přispět k lepšímu výběru pacientů pro personalizovanou léčbu (Decaux etal., 2008). 2.8.2 Cytogenetika Detekce cytogenetických změn u MM představuje významný prognostický faktor, ať se jedná o početní odchylky či strukturní přestavby chromozomů (ztráty, zmnožení i přemístění genetického materiálu). PB se v KD v rané fázi onemocnění vyskytují v nízkých počtech a mají nízkou proliferační aktivitu. Využití konvenčních cytogenetických metod, při kterých je zapotřebí analyzovat dělící se buňky, je proto značně omezeno a některé významné změny jimi není možné odhalit. Pomocí modernějších molekulárně-cytogenetických metod (interfázní fluorescenční in situ hybridizace - iFISH, komparativní genomové hybridizace - CGH) bylo však prokázáno, že CHA je možné nalézt u téměř všech MM pacientů (medián 8 až 10 změn karyotypu u jednoho pacienta v době diagnózy). Dnes 17 se proto tyto metody rutinně využívají k detekci všech vyšetřovaných aberací (Kuglík et al, 2008; Chen et al, 2007). Početní chromozómové aberace Jedním z nej důležitějších prognostických faktorů jsou početní aberace chromozomů (aneuploidie) a s nimi spojené výrazné rozdíly v přežívání pacientů. Jako hypodiploidní se označuje karyotyp s méně než 46 chromozomy, častými monozomiemi a IGH translokacemi. Hypodiploidie má nepříznivý dopad na prognózu a představuje hlavní nezávislý faktor pro vyhodnocení OS (pouze 10 % pacientů přežívá dobu 5 let). Abnormální hyperdiploidní karyotyp se vyznačuje zmnožením jednoho či více chromozomů, a tudíž celkovým počtem chromozomů vyšším než 46. Hyperdiploidie a trizomie představují pozitivní prognostické nálezy a mají lepší OS (medián OS 33,8 měsíců u hyperdiploidie vs. 12,6 u hypodiploidie) (Smadja et al, 2001; Debes-Marun et al, 2003). Kromě uvedených se u MM vzácně vyskytuje také pseudodiploidie a hypotetraploidie - souhrnně s hypodiploidií tyto stavy označujeme jako nonhyperdiploidní karyotyp (Wuilleme et al, 2005). Částečné delece v oblasti 13q nebo monozomie chromozomu 13 jsou spojeny s negativním dopadem na prognózu i přežívání. Na tomto chromozomu je v oblasti 13ql4 lokalizován tumor-supresorový gen RB1, který kóduje jaderný protein Rb regulující buněčný cyklus. RB1 má důležitou úlohu v patogenezi solidních nádorů, a přestože jeho role v patogenezi MM není dosud objasněna, jeho delece je považována za významný negativní molekulární marker kvůli ztrátě kontroly buněčného cyklu (Avet-Loiseau et al, 2000). Strukturní chromozómové přestavby Hlavním typem strukturních aberací jsou IGH translokace v oblasti 14q32, které jsou často spojeny s nonhyperdiploidním karyotypem. Transkripci genu IGH v oblasti 14q regulují tři zesilovače, které jsou v důsledku reciproké translokace přemístěny na partnerský chromozom, kde zvyšují expresi přítomných onkogenů, jako např. CCND1 (llql3), FGFR3 a MMSET (4pl6), c-MAF (16q23), MAFB (20ql2) a CCND3 (6p21) (Mohamed et al, 2007). Nejčastější je translokace t(ll;14)(ql3;q32), která je přítomna u 15-20 % pacientů a vede k nadměrné expresi cyklinu Dl (gen CCND1). Na rozdíl od ostatních 14q32 přestaveb je považována spíše za příznivý či neutrální prognostický faktor. U 10-15 % pacientů je detekována translokace t(4;14)(pl6;q32) (často ve spojení se částečnou delecí/monozomií chromozomu 13), jejíž přítomnost má negativní dopad na přežívání. Důsledkem této přestavby je zvýšení exprese dvou genů, které jsou lokalizovány v oblasti 18 4pl6 - FGFR3 a MMSET. Translokace t(14; 16), která ovlivňuje expresi transkripčního faktoru kódovaného genem c-MAF, je sledována u 5 -7 % pacientů a má negativní dopad na prognózu a přežívání (Fonseca et al, 2003; Fonseca et al, 2009). Zisk/amplifikace dlouhého raménka chromozomu 1 v oblasti lq21 je nej častější strukturní přestavbou nacházenou u MM (40 % nově diagnostikovaných a až 70 % relabujících pacientů). Často je detekována v asociaci s delecí genu RB1 v oblasti 13ql4 a její dopad na prognózu a OS je (stejně jako u této abnormality) nepříznivý. Jedním z genů lokalizovaných v této oblasti je CKS1B (oblast lq21.3, gen pro regulační podjednotku 1B kinázy CDC28), jehož zvýšená exprese zrychluje proliferaci a hraje tak roli v progresi MM (Němec et al, 2010; Zhan et al, 2007). Delece na krátkém raménku chromozomu 17 v oblasti 17pl3 je asociována s progresí MM, zkrácením OS a celkově zhoršenou prognózou v důsledku delece supresorového genu TP53. Ten kóduje protein p53, který reguluje buněčný cyklus, iniciuje opravu DNA či apoptózu buňky při neopravitelném poškození (Chang et al, 2005). Vysokorizikový MM jez hlediska cytogenetických změn charakterizován nálezem hypodiploidie, monozomie 13 nebo delece genu RB1, del(17)(pl3) a IGH translokace zasahující 4pl6 nebo 16q23 (Tab. 4) (Fonseca et al, 2003). Decaux et al. (2008) v jejich studii později potvrdili, že skupina vysoce rizikových pacientů měla častější výskyt delece 13ql4, delece 17pl3, zisku/amplifikace lq21 a IGH translokace, zatímco karyotyp pacientů s nízkým rizikem relapsu (low-risk) byl často hyperdiploidní. Přestože cytogenetické analýzy jsou pro určení prognózy stále významné, genomické techniky využívající analýzu genové exprese se pro identifikaci pacientů s vysokým rizikem ukázaly jako účinnější (Sawyer, 2011). Tabulka 4 Srovnání mediánu OS pacientů rozdělených podle přítomnosti či nepřítomnosti specifické cytogenetické abnormality a vliv na prognózu (Fonseca et al, 2003; upraveno) Cytogenetická Medián OS s nalezenou Medián OS bez nalezené Vliv na abnormalita abnormalitou (měsíce) abnormality (měsíce) prognózu t(ll;14)(ql3;q32 50 (37-60) 39 (36-44) poz. t(4;14)(pl6;q32) 26 (21-33) 45 (39-50) neg. t(14;16)(q32;q23 16 (13-22) 41 (37-48) neg. del(17)(pl3) 23 (20-36) 44 (39-49) neg. monozomie 13 35 (29-41) 51 (41-57) neg. 19 2.8.3 Genomické analýzy Zjišťování profilu genové exprese (GEP, z angl. gene expression profiling) umožnilo analýzu různých genů, které mohou být zapojeny v patogenezi MM a tím mohou přispívat k přežívání pacientů. Prvním molekulárním klasifikačním systémem byla tzv. TC klasifikace, kde T v názvu představuje translokace a C představuje cykliny D. Pacienti jsou zde rozděleni do osmi skupin na základě přítomnosti IGH translokace, specifické trizomie a odlišné exprese cyklinů D. Tímto způsobem bylo zjištěno, že nejhorší prognózu mají pacienti s IGH translokacemi zasahujícími 4pl6 nebo 16q32 a deregulovanou expresí cyklinu D2 (Bergsagel et al., 1996). Zhan et al. (2006) vytvořili klasifikaci, která je založena na kombinaci GEP, přítomnosti translokace nebo hyperdiploidie. V tomto systému byly dvě ze sedmi skupin spojeny se špatnou prognózou a vysoce rizikovými proměnnými - skupina PR vyznačující se zvýšenou expresí genů řídících buněčnou proliferaci a progresi buněčného cyklu a skupina MS se zvýšenou expresí genůMMSETa FGFR3 (tj. translokace zasahující 4pl6). První validovaný prognostický GEP model byl vytvořen dle hypotézy, že extrémní změny v genové expresi určité podmnožiny genů jsou spojeny s kratším přežíváním pacientů, a profil genové exprese těchto genů tak může představovat významný nezávislý prognostický znak. Skupina Dr. Shaughnessyho z University of Arkansas na základě této hypotézy identifikovala panel 70 genů, jejichž exprese je pozměněna (snížena nebo zvýšena) právě u skupiny pacientů s vysokorizikovým MM (zde 13-14 % všech MM pacientů). Zajímavým rysem tohoto modelu je vysoké zastoupení genů ležících na chromozomu 1 -téměř 50 % z 19 genů se sníženou expresí a 30 % z 51 genů vykazujících její zvýšení. V souladu s dříve publikovanými cytogenetickými nálezy je poloha genů se zvýšenou expresí v oblasti dlouhého raménka chromozomu 1, tedy v oblasti s častým výskytem amplifikace genetického materiálu, která je zároveň spojena s nepříznivým dopadem na prognózu a přežívání (Shaugnessy et al., 2007). Procento buněk s amplifikací oblasti lq21 by podle některých studií mohlo být spojeno s progresí onemocnění (Hanamura et al., 2006). Kromě této spojitosti má skupina vysokorizikových pacientů souvislost s dalšími známými klinickými a prognostickými parametry - vysoká hladina sérového P2-mikroglobulinu a kreatininu, delece chromozomu 13 a jiné cytogenetické abnormality s negativním vlivem indikující kratší přežívání této skupiny pacientů. Zjednodušený model využívá pouze 17 genů z původních 70 (s přesností 97,7 %) (Shaughnessy etal., 2007). 20 Model využívající pro předpověď přežívání nově diagnostikovaných pacientů jen 15 genů byl vytvořen francouzskou skupinou The Intergroupe Francophone du Myélome (IFM). U pacientů s vysokým rizikem je sledováno zvýšení exprese u genů účastnících se řízení rozličných fází celého buněčného cyklu (např. geny řídící kontrolu buněčného cyklu, replikaci, opravu a sbal ování DNA, mitózu a vytvoření dělícího vřeténka). Ve spojení s dalšími prognostickými faktory (IGH translokace a sérový P2-mikroglobulin) může být využit k identifikaci nejvíce rizikové skupiny pacientů (Decaux et al, 2008). Sledování homozygotních delecí (ztráta obou alel) u genů zapojených do patogeneze MM může také sloužit k nalezení specifických GEP s prognostickým významem. Na základě hypotézy, že změny na úrovni DNA musí být spojeny se změnami na úrovni genové exprese, bylo identifikováno celkem 97 genů spojených s nepříznivým dopadem na přežívání pacientů. Z tohoto seznamu byly vybrány tři páry genů {BUB1B versus HDAC3, CDC2 versus FIS1, RAD21 versus 1TM2B), které jsou po vzájemném srovnání schopny odlišit jedince s horší prognózou (Dickens etal, 2010). Moreaux et al. (2011) získali pomocí srovnání exprese genů v lidských myelomových liniích (HMCL, z angl. human myeloma cell lineš) a klasifikace do skupin dle Zhan et al. (2006) sedm genů, jejichž pozměněná exprese by mohla být znakem pro negativní prognózu (TEAD1, CLEC11A, LRP12, MMSET, FGFR3, NUDT11 &KIAA1671). Tyto geny využili k vytvoření jednoduchého systému bodování pacientů od 0 do 7 a jejich rozdělení do tří skupin s odlišným dopadem na přežívání. S nejhorší prognózou je spojena třetí skupina, která vykazuje změnu exprese u 5 nebo více z těchto genů. Avšak žádný z nich nebyl v doposud publikovaných GEP modelech využit. Celkově mají dodnes prezentované modely pouze málo společných genů (zejména kvůli vysoké heterogenite onemocnění spojené s heterogenitou v expresi genů), což pro přesné určení prognózy a rozdělení pacientů do skupin, které by byly navzájem srovnatelné, představuje značnou komplikaci. Doporučení pro stávající modely rozdělující pacienty do odlišných rizikových skupin je následující: vyšetření klinického stadia ISS dle sérového albuminu a p2-mikroglobulinu, FISH vyšetření cytogenetických abnormalit t(4; 14), t(14; 16), del(17) a zisk/amplifikace lq21, histologie a doplňkové vyšetření např. GEP (Munshi etal., 2011). Dalšími kritérii jsou v případě relabujících pacientů typ odpovědi na terapii a délka bezpříznakového období (Hájek etal, 2012). Tématika vysoce rizikového MM byla zpracována do přehledové práce v časopise Clinical Lymphoma, Myeloma and Leukémia (Paszeková etal, 2014). 21 2.9 Flow cytometrie Flow cytometrie u MM patří mezi hlavní vyšetřovací metody a identifikace imunofenotypu a stanovení počtu PB se používá v diferenciální diagnostice (Kovářová et al., 2008). Své uplatnění nachází i při stanovení množství cirkulujících PB u pacientů s nově diagnostikovaným onemocněním, tato hodnota je nezávislým prognostickým faktorem pro celkové přežití (Nowakowski et al., 2005). Dále se uplatňuje také při určování pravděpodobnosti progrese asymptomatické monoklonální gamapatie, v hodnocení minimální residuální nemoci nebo účinnosti léčby MM (Kovářová et al., 2008). Vzhledem k tomu, že většina MM pacientů relabuje i v současné době, jsou analýza odpovědi na léčbu a detekce minimální residuální choroby velice důležité. Zde se ukazuje velká výhoda vícebarevné flowcytometrie, která poskytuje rychlé a přesné informace o stavu pacienta (Silvennoinen et al., 2014). PB jsou charakterizovány především expresí cytoplazmatického imunoglobulinu a povrchových membránových antigénu, jako jsou membránový glykoprotein 1 plazmatických buněk (PC-1), antigén 1 rakoviny prostaty (PCA-1), CD38 a CD 138. Po dlouhou dobu byl fenotyp myelomových buněk považován za totožný s fenotypem normálních PB. Posléze se ovšem začaly odhalovat odlišnosti v expresi některých povrchových znaků (Paiva et al., 2010). CD138 (Syndekan-1) je transmembránový heparan sulfátový proteoglykan typicky exprimovaný na PB a je považován za nejvíce specifický marker pro PB (Lin et al., 2004). K jeho expresi dochází jak na fyziologických PB, tak i patologických, a to již ve stádiu prekurzorů. Pokud dojde ke ztrátě exprese tohoto markeru a jeho uvolnění do cytoplazmy, dochází k apoptóze PB (Kovářová et al., 2008). Dalším zásadním znakem pro identifikaci všech typů PB je CD38. Tento marker je nespecifický a může být detekován na hematopoietických kmenových, T a B buňkách. Neoplastické PB jej typicky exprimují s nižší intenzitou než normální (Lin et al., 2004). Dalším důležitým markerem je CD45. Rané PB tento znak exprimují, ale v průběhu diferenciace na zralé PB jej ztrácí a stávají se CD45- (Paiva et al., 2010). Pro určení rozdílu mezi patologickými a normálními PB hraje klíčovou roli exprese markeru CD19 a CD56 (Kovářová et al., 2008). CD56 je adhezivní molekula, jejíž ztráta může fungovat jako transmigrační signál a umožnit uvolnění maligních buněk z KD (Paiva et al., 2010). Zatímco zralé MM buňky jsou jasně odlišné od normálních díky fenotypu 22 CD19-CD56+, popřípadě v menší míře se vyskytující i CD19- CD54- a CD19+56+, normální PB vykazují fenotyp CD19+CD56- (Paiva et al, 2010). Další markery, jejichž exprese je omezena především na maligní PB jsou B lymfocytární marker CD20, kostimulační molekula T-lymfocytů CD28, CD117 a CD200. Naopak markety spojované s fyziologickými PB jsou CD81 a CD27, jejichž exprese se pojí s lepší prognózou pacientů (Kovářová etal., 2008). Klonální PB u MM tedy vykazují zvýšené hladiny CD56, CD86, CD126 a sníženou hladinu u CD38 a CD40 (Pérez-Andrés et al., 2005). Analýza cytoplazmatické exprese lehkých řetězců k a X je potřebná k potvrzení klonality PB (Kovářová et al., 2008). 2.10 Detekce minimální reziduálni choroby I když jev současné době MM již léčitelným onemocněním, velkým problémem je relaps onemocnění, ke kterému dochází u velké většiny pacientů. Minimální residuální choroba (MRD, minimal residual disease) je stav, kdy u pacientů v klinické remisi stále přetrvávají klonogenní buňky, jejichž proliferace vede ke klinickému relapsu. Současné přístupy pro zhodnocení přítomnosti nádorových buněk jsou založeny na morfologickém hodnocení vzorku KD a elektroforetických metodách, které detekují změny v séru a hladině paraproteinu v moči. Citlivost těchto metod je velice limitovaná. I u pacientů dosahujících kompletní remise nakonec dochází k relapsu onemocnění jako důsledku přítomnosti MRD. Většinou standardních metod jsou tyto klonogenní buňky nezachytitelné, ale včasný záchyt by však měl přímý vliv na přežívání pacientů. V současné době se k detekci MRD využívá flow cytometrie a PCR, které mají uplatnění pro zjištění účinnosti léčby, porovnání efektivity různých léčebných strategií, monitorování pacientů a relapsu onemocnění. Flow cytometrie je jednodušší, rychlejší, levnější, ale méně citlivá. PCR je citlivější a umožňuje provádět retrospektivní studie s využitím zamražené DNA. Detekce MRD u MM pomocí PCR využívá amplifikace tumor-specifického molekulárního markeru, který je detekován v nádorových ale ne ve zdravých buňkách. U MM je takovýmto markerem přestavba těžkého řetězce imunoglobulinu, ke které dochází v pre-B lymfocytech a dále je tato oblast modifikovaná v terminálni ch centrech pomocí mechanismu somatické hypermutace. Při detekci MRD pomocí PCR je využíváno především právě takto vzniklé hypervariabilní oblasti těžkého řetězce imunoglobulinu, jejíž sekvence slouží pro návrh primerů a sond specifických pro pacienty, které jsou pak v následné PCR reakci schopny detekovat přítomnost MRD s citlivostí až 10"6. PCR produkt 23 je pak možné kvalitativně vyhodnotit jako přítomnost residuální choroby. Včasný záchyt klonogenních buněk a zvýšení citlivosti detekčních metod povede k lepšímu přežívání pacientů, rychlejší detekci relapsu a rychlejšímu nástupu účinku léčby. Sledování residuální choroby na molekulární úrovni pomocí PCR se v současné době využívá u leukémií i různých typů lymfomů pro zjištění prognózy onemocnění a je rovněž důležitým faktorem při volbě terapie. U MM je však situace složitější z důvodu vysoké heterogenity tohoto onemocnění. Z toho důvodu se detekci MRD na bázi PCR prozatím podařilo zavést a optimalizovat pouze na několika pracovištích. Naše skupina byla první skupinou v ČR, která zavedla detekci MRD na bázi PCR. V rámci studie byly zavedeny jednotlivé kroky detekce MRD na bázi PCR u pacientů s MM, a to: kvalitativní PCR s využitím již známých rodin primerů a práce se sekvencemi a detekce specifických přestaveb IgH pomocí bioinformatického nástroje IMGT/V-QUEST, kdy se podařilo stanovit nádorově specifický marker u 80 % pacientů. Dále design ASO primerů pro následné kvalitativní zhodnocení přítomné MRD. A identifikace a návrh specifických sond pro kvantitativní zhodnocení přítomnosti myelomového klonu jednotlivých pacientů v 50 % případů. Citlivost metody dosahovala až 10"6. Další snahou optimalizace PCR detekce MRD u MM bude standardizace celého postupu práce vzhledem k odběru vzorků (při diagnóze, mezi cykly terapie, u pacientů vremisi), definice prognostické hladiny MRD a zhodnocení její prediktivní hodnoty pro přežití. Na základě takto získaných informací by bylo vhodné vyvinout databázi genetických map, primerů, sond a protokolů pro detekci myelomových klonů a MRD. Tato metoda by tak měla velkou šanci stát se nezávislým prognostickým faktorem s širokým využitím v multicentrických studiích. Tato práce byla opublikována v časopise Biomedical Papers (Sedlaříková et al., 2014). 24 3 Extramedulární forma mnohočetného myelomu Extramedulární MM (EM) představuje agresivní formu tohoto onemocnění s velmi špatnou prognózou (Varettoni et al, 2010; Usmáni et al, 2012, Pour et al, 2013). EM bývá diagnostikován v době stanovení diagnózy nebo v průběhu MM (Varettoni et al, 2010). Zdá se, že EM je spojen se sekundárními změnami v myelomovém klonu, progresí agresivního onemocnění, špatnými prognostickými faktory a rezistencí k léčbě (Katodritou et al., 2009; Sheth et al., 2009). I když je EM v současné době velice aktuálním tématem, první zprávy pocházejí ze 40. a 50. let minulého století, kdy Churg et Gordon (1942) a Hayes (1952) uvedli výskyt EM u pacientů s MM. Z jejich poznatků vyplývá, že EM byl přítomen ještě před érou vysokodávkovaných cytostatik a není tedy novou diagnózou. 3.1 Incidence EM Existují různé zprávy o incidenci EM. Prvními zmínkami o tomto onemocnění jsou pitevní protokoly, které ukázaly, že přibližně u 70 % pacientů s MM se vyskytovalo extraskeletální ložisko (Churg et Gordon, 1942; Hayes et al., 1952). Tyto nálezy byly popsány ještě před zavedením chemoterapie, ale i tak se výskyt EM zdá být velmi vysoký. Thomas et al. (1957) uvádí 40% výskyt EM v játrech při pitvě v sérii 64 pacientů s MM. S vývojem lepších zobrazovacích systémů byl EM diagnostikován v průběhu života a uváděný výskyt nálezu byl mnohem nižší. V nedávných studiích byl výskyt EM popsán u 6 - 20 % pacientů v průběhu onemocnění (Varretoni et al., 2010) a až 37 % u pacientů po alogenní transplantaci (Perez-Simon et al., 2006), ale existují i zprávy udávající nízkou incidenci a to kolem 9 - 14 % (Alegre et al, 2002). Novější studie (Wu et al, 2009; Varretoni et al, 2010) uvádějí, že EM se v 68 až 85 % objevuje při diagnóze a vyskytuje se jako infiltrace měkkých tkání spojená s kostními lézemi. Další rozsáhlá studie, kterou zveřejnila skupina z University of Arkansas, analyzovala pacienty mezi lety 2000 - 2010, kteří vykazovali EM v době diagnózy MM nebo při progresi onemocnění/relapsu. Výzkumná skupina analyzovala 936 pacientů podle protokolu „total therapy" (TT), 240 pacientů podle „non - TT" protokolu a 789 pacientů léčených mimo protokol (n = 1965). Celkově byl primární EM (v době diagnózy) zdokumentován u 2,41 % TT pacientů, 4,35 % u non-TT pacientů a 4,5 % pacientů léčených mimo protokol. Výskyt sekundárního (při progresi onemocnění/relapsu) EM u pacientů po 5 letech od transplantace autologními kmenovými buňkami byl dokumentován v případě 3,43 25 % TT pacientů, 5,2 % non - TT pacientů a 7,24 % pacientů léčených mimo protokol (Usmáni etal, 2012). 3.2 Místa výskytu a typy EM Většina autorů rozlišuje dvě skupiny EM: první skupina je charakterizována přímým rozšířením extramedulárního nálezu z kosterního nádoru, zatímco druhá je výsledkem hematogenního šíření (Blade et al., 2011). EM může ovlivnit jakoukoliv tkáň, ale mezi nejčastěji zasažené orgány patří pohrudnice, lymfatické uzliny, měkké tkáně, játra, kůže, plíce, urogenitální trakt, prsa a pankreas (Varettoni et al., 2010). Usmáni et al. (2012) jako primární EM označují extramedulární ložisko nalezené při diagnóze MM a sekundární jako extramedulární ložisko diagnostikováno v době relapsu MM. Jako nej častější místa výskytu primárního EM uvádí hrudní stěnu, lymfatické uzliny, kůži, měkké tkáně a paraspinální prostor, zatímco sekundární EM byl nejčastěji nalezen v játrech. 3.3 Prognóza EM Přítomnost EM je spojen s agresivním typem onemocnění, což vede ke zkrácení OS a přežití bez progrese (PFS). Varretoni et al. (2010) ukázali změny celkového přežití u MM pacientů v letech 1971 až 1999, které se měnilo následovně: 32 měsíců v letech 1971-1993, 45 měsíců v letech 1994-1999 a 54 měsíců v letech 1994-1999. Nicméně pacienti s EM měli kratší PFS než pacienti s MM bez EM relapsu (18 vs. 30 měsíců, p = 0,003), ale medián OS nebyl statisticky významně odlišný. Incidence EM měla negativní prognostický dopad na OS a PFS i po úpravě na věk, pohlaví a stupeň stádia nemoci (Varettoni et al., 2010). Existuje stále více důkazů, že EM je spojen se sekundárními změnami, progresí agresivního onemocnění, špatnými prognostickými faktory a rezistencí k léčbě (Oriol, 2011). Nová data také naznačují, že EM představuje subklon nádoru, který získal sekundární mutace, zejména delece genu TP53, což způsobilo, že se tento klon stal více rezistentní k léčbě (Oriol, 2011). Usmáni poukázal na kratší OS u pacientů s primárním EM než u pacientů s MM (31 % vs. 59 % po 5 letech, p < 0,001), jakož i kratší PFS ve všech třech léčebných skupinách (50% vs. 21% po 5 letech, p < 0,001). Kumulativní incidence EM 5 let po transplantaci (kdy data o primárním a sekundárním EM byla spojena) byla zvýšena u vysoce rizikových pacientů definovaných pomocí metody GEP (10,8 % vs. 2 %, p < 0,001) a u pacientů s před transplantačními cytogenetickými abnormalitami (7 % vs. 4,1 %, p = 0,004). Nízká hladina 26 hemoglobinu a trombocytů stanovená před transplantací byla spojena se zvýšeným výskytem EM ve všech třech skupinách pacientů (8,9 % vs. 3,4 % a 8,6 % vs. 3,4 % , p < 0,001). 3.4 Molekulární mechanismy EM relapsu V současné době existuje jen velmi málo publikací, které se snaží charakterizovat molekulární mechanismy EM. Většina vědeckých pracovišť publikuje své poznatky na základě testování malého množství vzorků, z těchto důvodů nejsou přesné molekulární mechanismy stále známy. Je zřejmé, že při EM relapsu je výrazně snížená závislost PB na mikroprostředí KD, protože PB jsou schopny přežívat i mimo KD. Na přežívání PB mimo KD se podílejí zejména změny v signální dráze chemokinového CXC receptoru 4 (CXCR4), která je důležitá pro usídlení a expanzi buněk MM. Během adheze PB ke stromatu KD, dochází k expresi molekul very late antigen-4 (VLA-4), CD56 a CD44, které interagují s receptory na povrchu endotelových buněk KD, jako např. adhezivní molekula vaskulárních buněk 1 (VCAM-1). Existuje také několik chemokinů a chemokinových receptoru, jako jsou CCR1, CCR2 a CXCR4, které jsou důležité pro migraci a adhezi buněk MM. Zdá se tedy, že existuje několik mechanismů, které umožňují extramedulární šíření PB, jako je snížení exprese adhezivních molekul, snížená regulace chemokinových receptoru, změny týkající se angiogeneze VEGF, matrixové metaloproteinázy (MMP-9) a dalších faktorů a mutace v NF-kB signální dráze (Blade et al, 2011). V poslední době bylo prokázáno, že thalidomid indukuje sníženou regulaci CXCR4 a jeho Ugandu, které jsou kritické pro usídlení buněk MM - je tedy možné, že thalidomid by mohl usnadnit extramedulární růst buněk MM prostřednictvím ztráty stromálních buněčných interakcí. Thalidomid také snižuje regulaci CD56, dalšího faktoru důležitého pro usídlení MM buněk v KD (Ali et al, 2007). EM je pravděpodobně dále spojen s biologickými změnami v samotném klonu buněk MM. Sheth et al. (2009) ukázali asociaci mezi expresí TP53, CD56 a Ki-67 a EM relapsem. Inaktivace p53 je spojena s více agresivním onemocněním, s rezistencí na chemoterapii a horším OS pacientů s MM (Neri et al, 1993) a navíc byla popsána akumulace p53 v jádru PB EM. CD56 je adhezivní molekula nervových buněk, která hraje důležitou roli v buněčné adhezi a migraci. U EM byla zjištěna zvýšená exprese Ki-67, snížená regulace CD56 a delece TP53 (Shethet et al, 2009). Také další výzkumná skupina potvrdila sníženou regulaci CD56 ve vzorcích EM (Dahl et al, 2002), nicméně v jiné studii byla CD56 společně s CCND1 nadměrně exprimovány v EM vzorcích ve srovnání s MM vzorky (Kremer et al, 27 2005). Dále lze předpokládat, že aktivační mutace genu RAS hrají roli v šíření EM nádorů (Rasmussen et al., 2005). Ve velké studii 764 nově diagnostikovaných pacientů s MM byly identifikovány komplexní genomové přestavby u 1,3 % pacientů a tito pacienti vykazovali medián OS 12 měsíců (Magrangeas et al., 2011). Zejména oblasti lq a 16q měli největší počet kopií přestaveb. K chromotrypsis (tříštění chromozómů) dochází při diagnóze, což u těchto pacientů značí špatnou prognózu. Je možné, že pacienti s chromotrypsis při diagnóze představují odlišnou biologickou entitu na rozdíl od pacientů, kteří přežijí více než 10 let (Wirkeřor/., 2013). Existuje pouze několik studií, které pečlivě analyzovaly morfologii a cytogenetiku PB v extramedulárních ložiscích. Většina článků ukazuje, že tyto buňky mají nezralou nebo plasmablastickou morfologii (Katodritou et al., 2009). Jiné skupiny neukázaly žádné rozdíly mezi cytogenetickými abnormalitami nalezenými v PB buňkách KD a v extramedulárních ložiscích (Rosinol et al., 2009). Tématika extramedulárního relapsu byla zpracována do práce Pour et al. (2013) v časopise Haematologica, který se zabýval incidencí, přežitím a aberacemi u pacientů s EM relapsem. Přítomnost EM relapsu byla prospektivně hodnocena u všech pacientů léčených na IHOK FN Brno pro relaps MM od roku 2005 do roku 2008. Z analýzy byli vyloučeni všichni pacienti, kteří měli EM ložiska nebo prokázanou infiltraci parenchymatózních orgánů klonálními PB již při stanovení diagnózy MM. Celkem bylo hodnoceno 226 relabovaných pacientů. Medián věku činil 60,8 let (27,9 - 83,5 let), medián sledování pacientů byl 3,7 let (0,1-22 let). EM relaps byl verifikován pomocí zobrazovacích metod (ultrazvuk, počítačová tomografie (CT), magnetická rezonance (MRI). Pokud se jednalo o ložisko dostupné odběru v lokální anestézii, bylo provedeno i cytologické hodnocení aspirátu z ložiska nebo histologické hodnocení bioptického vzorku k průkazu klonálních PB. Za EM relaps byl považován nález patologických měkkotkáňových hmot na MRI či CT nejčastěji v oblasti související s osovým skeletem u pacientů s jinými projevy relapsu/progrese MM, nebo nález klonálních PB v cytologickém nebo histologickém hodnocení vzorku z ložiska. Pacienti s EM relapsem byli rozděleni do dvou skupin: 1) EM-S (soft-tissue related) - pacienti s EM relapsem, u nichž nebyla prokázána souvislost měkkotkáňových ložisek s kosterní tkání či difuzní infiltrací parenchymatózních orgánů. 2) EM-B (bone-related) -pacienti s EM relapsem s ložiskem klonálních PB, které vyrůstalo ze skeletu. 28 Zhodnoceno bylo celkové přežití pacientů, doba výskytu a léčba předcházející EM relapsu v obou skupinách pacientů. Léčba pacientů byla velice heterogenní, nicméně všichni pacienti byli v průběhu choroby léčeni thalidomidem nebo bortezomibem. Pro léčbu EM relapsu byl standardně použit léčebný režim, který obsahoval nový lék, který nebyl dosud použit standardně v kombinaci s cytostatikem a kortikoidy. Pokud byl k dispozici štěp a stav pacienta to umožňoval, byl podán vysokodávkovaný melfalan. Pro léčbu EM byly tedy použity thalidomid (v 33 %), bortezomib (v 38 %) nebo lenalidomid (v 5 %) obsahující režimy a u 42 % patientů byl použit i vysokodávkovaný melfalan s podporou autologního štěpu. Hodnoceno bylo 226 relabovaných MM pacientů. EM relaps byl prokázán u celkem 24 % (55/226) nemocných. U 58 % (32/55) z těchto pacientů nebyla prokázána souvislost EM s kosterní tkání. Zaznamenali jsme postižení téměř všech orgánů. Nejčastěji bylo pozorováno solidní nádorové ložisko zasahující do kůže a podkoží u 40 % (22/55) pacientů. Celkem 42 % (23/55) pacientů mělo EM myelomová ložiska související s kosterní tkání, nejčastěji byla takto postižena páteř a to u celkem 33% (18/55) pacientů. EM relaps/progrese se vyskytl překvapivě časně v průběhu choroby. V prvním relapsu bylo zaznamenáno EM postižení u 53 % (29/55) pacientů, ve druhém relapsu u 33 % (18/55). A později v průběhu choroby pouze u 14 % (8/55) pacientů. Mezi skupinami EM-S and EM-B nebyl pozorován rozdíl v době, kdy se EM postižení objevilo, p = 0,868. U obou skupin bylo více než 50 % pacientů postiženo již v prvním relapsu. Před vznikem EM relapsu/progrese bylo konvenčními chemoterapeutickými režimy léčeno 20 % (11/55) pacientů, režim obsahující thalidomid byl použit u 38 % (21/55) pacientů, režim s bortezomibem mělo 29 % (16/55) pacientů a vysokodávkovaný melfalan s autologní transplantací periferních kmenových buněk byl před vznikem EM postižení použit jako léčebná varianta u celkem 53 % (29/55) pacientů. Ve skupině pacientů EM-B byly pozorované numerické rozdíly v podaném typu léčby (konvenční terapie 26 % vs 16 %; bortezomibem 22 % vs 34 %), rozdíly však nebyly statisticky signifikantní (p = 0,669). Medián OS všech 226 pacientů činil 89,5 měsíců a rozdíl mezi nemocnými bez EM relapsu/progrese (76 %; 171/226) a s EM relapsem (24 %; 55/226) byl statisticky významný (medián 109 vs. 38 měsíců; p < 0,001). Ve skupině 55 nemocných s EM měla signifikantně kratší medián OS podskupina nemocných s EM-S (medián 30 vs. 45 měsíců, p = 0,002). Medián OS od stanovení diagnózy EM relapsu/progrese činil u všech 55 pacientů 8 měsíců a byl významně nižší v podskupině nemocných s EM-S (4 vs. 12 měsíce, p = 0,006). U pacientů s EM byla zaznamenána léčebná odpověď (ORR, z angl. overall response rate) 29 pouze u 24 % (13/55) nemocných, z toho kompletní remise u 5 % (3/55) a u 19 % (10/55) parciální remise. Doba do progrese u těchto pacientů však činila pouhých 5,4 měsíců. Léčebné výsledky nezávisí na podané léčbě a jsou velmi špatné, nebyl nalezen signifikantní rozdíl (p = 0,412), výpovědní hodnota je však limitována velikostí souboru. Dalším tématem této části byly možné změny v expresi 15 vybraných genů u pacientů s EM relapsem ve srovnání s pacienty s vysokorizikovým MM podle publikace Dr. Shaughnessyho, která vycházela z toho, že EM relaps je možbé považovat za nejvíce rizikovou formu MM. Devět pacientů s EM relapsem, u kterých byl k dispozici jak nádor (TU) tak KD, a 9 pacientů s vysokorizikovým (HR) MM bylo zahrnuto do této části práce. Jako vysokorizikoví byli identifikováni ti pacienti, kteří prodělali relaps během 2 let od zjištění diagnózy. Pacienti s EM relapsem byli identifikováni v rámci předchozí studie. Statisticky významné rozdíly byly nalezeny mezi třemi skupinami vzorků - PB KD u vysokorizikových pacientů (HR) oproti PB KD u pacientů s EM, PB KD u HR pacientů oproti PB TU u pacientů s EM a PB KD u pacientů s EM oproti PB TU u pacientů s EM. V prvním srovnání genové exprese PB KD u HR pacientů proti PB KD u pacientů s EM byl významný rozdíl v expresi stanoven pouze u 4 genů. Ve druhém srovnání byly nalezeny také 4 geny se signifikantními změnami exprese mezi PB KD u HR pacientů oproti PB TU u pacientů s EM. Třetí srovnání genové exprese mezi PB KD u pacientů s EM oproti PB TU u pacientů s EM odhalilo nej větší podíl statisticky významných rozdílů a to hned u 9 z 15 genů. Ikdyž je tento soubor pacientů velice malý, ukazuje na rozdíly v genové expresi mezi skupinami myelomových pacientů a s nimi související heterogenitu MM. EM relaps představuje pokročilý stav maligní transformace onemocnění a ztráta závislosti na mikroprostředí KD značí další změny genomu. Vzorky EM z TU nacházející se mimo prostředí KD vykazují odlišnou genovou expresi oproti vzorkům získaným u EM pacientů přímo z KD, zejména z důvodu vyšší agresivity tohoto „klonu" buněk. Jelikož byl nalezen poměrně velký počet statisticky významných rozdílů v expresi mezi skupinami PB z KD a PB z TU u pacientů s EM, zdá se, že se opravdu jedná o odlišné klony. Tato část práce byla zpracována v článku v časopise Biomedical Papers (Ševčíková etal, 2015). 30 4 MikroRNA MikroRNA (miRNA) jsou krátké, nekódující, 21-25 nukleotidů dlouhé jednořetězcové molekuly RNA, které regulují genovou expresi u rostlin i živočichů (Clarke etal., 2007) Aby mohla být krátká RNA označena za miRNA, musí splňovat následující kritéria: musí být jednoznačně identifikovatelná pomocí Northern blotu, musí se vyskytovat v kmenové části vlásenkové, asi 70 nukleotidů dlouhé prekurzorové struktury, sekvence krátké RNA a jejího prekurzoru musí být fylogenetický konzervovaná a inhibice klíčových ribonukleáz v biogenezi miRNA musí vést k poklesu hladin krátké RNA a k akumulaci její prekurzorové struktury (Ambros, 2000; Slabý et Svoboda, 2012). MiRNA jsou negativní regulátory a fungují dvěma způsoby v závislosti na stupni komplementarity mezi miRNA a cílovou mRNA. Za prvé, miRNA se mohou vázat s dokonalou nebo téměř dokonalou komplementaritou k protein-kódujícím mRNA sekvencím a tím indukují RNA-zprostředkovanou interferenční (RNAi) dráhu. Stručně řečeno, mRNA transkripty jsou štěpeny ribonukleásami v multiproteinových RNA-indukovaných komplexech (miRISC), což vede k degradaci cílových mRNA. Tento mechanismus miRNA-zprostředkovaného umlčování exprese genů je běžný u rostlin (Llave et al., 2002), ale vyskytuje se i v savčích buňkách (Yekta et al., 2004). Nicméně u většiny živočišných buněk převládá druhý mechanismus genové regulace, který nezahrnuje štěpení cílové mRNA, ale pouze její umlčení. Tyto miRNA se vážou s neúplnou komplementaritou na 3' nepřekládané oblasti (UTRs) jejich mRNA cíle a cíleně potlačují genovou expresi post-transkripčně, zřejmě na úrovni translace, prostřednictvím komplexu RISC, který je podobný (nebo možná totožný) s komplexem dráhy RNAi (Olsen et Ambros, 1999). miRNA pak využitím tohoto mechanismu snižují hladiny proteinů bez ovlivnění hladiny mRNA. Nicméně se ukazuje, že i neúplně komplementární miRNA mohou také indukovat mRNA degradaci (Bagga et al., 2005; Lim et al., 2005). MiRNA byly objeveny u Caenorhabidtis elegans roku 1993 (Lee et al., 1993). Množství popsaných miRNA u všech organismů roste exponenciálně a jejich počet je obsažen v internetové databázi miRBase. Doposud bylo popsáno 28 645 miRNA (nejnovější verze 21, červen 2014, www.mirbase.org). Přibližně polovina z anotovaných lidských miRNA je mapována do fragilních míst chromozomů, což jsou oblasti genomu, které jsou spojeny s různými lidskými nádory. Nedávné důkazy naznačují, že miRNA mohou fungovat jako nádorové supresory 31 aonkogeny, a proto jsou označovány jako "oncomirs". Faktory, které jsou potřebné pro biogenezi miRNA, jsou rovněž spojovány s různými typy nádorů a mohou samy fungovat jako nádorové supresory a onkogeny. Dále bylo zjištěno, že subtypizace a klasifikace nádorů do určitých podskupin pomocí jejich miRNA profilů je přesnější než pomocí expresních profilů protein-kódující genů. Právě rozdíly v expresi určitých miRNA v různých typech nádorů by se mohly stát velice užitečným nástrojem při diagnóze a léčbě rakoviny. Genové terapie využívající miRNA by mohly být posléze využity k zamezení progrese nádoru. Příkladem slibných kandidátů pro léčbu rakoviny mohou být let-7, která negativně reguluje Ras nebo miR-15 a miR-16, které negativně regulují BCL-2 (Esquela-Kerscher et Slack, 2006). V současnosti začíná být význam miRNA plně doceňován, probíhá klinické testování miRNA jako nových léků pro léčbu hepatitídy C (Miravirsen). Miravirsen je specifický inhibitor miR-122, což je specifická jaterní miRNA, která je nezbytně nutná pro replikaci viru hepatitídy C. Miravirsen tedy odstraňuje molekulu, kterou virus potřebuje k replikaci. Předpokládá se, že tato terapie zabrání vzniku resistence viru k tomuto novému typu terapie. Virus hepatitídy C je známý vysokou rychlostí vzniku mutací (www.santaris.com). 4.1 Biogeneze miRNA MiRNA jsou převážně transkribovány pomocí RNA-polymerázy II jako pri-miRNA (primární miRNA), dlouhé prekurzory, které na koncích obsahují čepičku a polyadenylový konec (Obr. 7). Pri-miRNA jsou zpracovány v jádře pomocí enzymu Drosha a proteinu Pasha (také známý jako DGCR8), do zhruba 70nukleotidových pre-miRNA, které se skládají do nedokonalých struktur vlásenek se smyčkou (Basyuk et al., 2003; Lee et al., 2003). Pre-miRNA jsou pak exportovány do cytoplazmy pomocí GTPdependentního transportérového proteinu exportin 5 (Lund et al., 2004; Yi et al., 2003) a podstupují další zpracování. Během dalších procesuje z pre-miRNA odstraněna vlásenka pomocí enzymu Dicer a zůstává pouze dvouřetězcová RNA o velikosti zhruba 22 nukleotidů (duplex 5p a 3p) (Ketting et al., 2001). Následně je duplex miRNA:miRNA* začleněn do miRISC komplexu. Zralý řetězec (vedoucí řetězec) miRNA je přednostně udržen ve funkčním miRISC komplexu a negativně reguluje své cílové geny, kdežto druhý řetězec je uvolněn a degradován (Esquela-Kerscher et Slack, 2006). O osudu řetězců rozhoduje stabilita párování na 5' konci duplexu miRNA: miRNA* (Slabý et Svoboda, 2012). 32 Obrázek 7 Biogeneze miRNA (Esquela-Kerscher et Slack, 2006). 4.2 Cirkulující miRNA Nová skupina cirkulujících miRNA byla nalezena prakticky ve všech lidských tělních tekutinách, jako je plazma, sérum, sliny, moč aj. Tyto miRNA jsou vysoce stabilní a odolné vůči působení RNáz. Je možné, že se tyto extracelulární miRNA účastní mezibuněčné komunikace, což by znamenalo, že miRNA mohou obsahovat specifickou informaci a mohou být exportovány dovnitř nebo ven z buněk jako odpověď na biologické podněty (Wang et al., 2012). Hladiny specifických cirkulujících miRNA by mohly sloužit jako biomarkery různých patologických stavů, včetně nádorových onemocnění, u kterých specifické profily cirkulujících miRNA jsou schopny odlišit zdravé jedince od pacientů (Wittmann et Jáck, 2010) nebo přímo korelují s progresí a stádiem nádoru (Menéndez et al., 2012). Jako markery mají tyto cirkulující miRNA několik předností: jsou jednoduché, snadno dostupné a snadno měřitelné standardními laboratorními metodami (Wang et al., 2012). Zejména u solidních nádorů byly cirkulující miRNA popsány jako molekulární marker nádorové diagnostiky i prognózy (Ng et al., 2009; Wittmann et Jáck, 2010). 4.3 MikroRNA u mnohočetného myelomu První abstrakta zabývající se úlohou miRNA v patogenezi MM byla prezentována v roce 2005 na setkání Americké hematologické společnosti (ASH). Jako první byly popsány 33 expresní profily miRNA u myelomových linií a vzorku pacientů a bylo zjištěno, že jak buněčné linie, tak maligní, CD138+ plazmatické buňky pacientů mají odlišnou expresi některých miRNA (miR-125b, miR-133a, miR-1 nebo miR-124a) ve srovnání s PB zdravých dárců (Masri et al, 2005). Další práce, ve které byla použita kvantitativní PCR (qRT-PCR), popisuje zvýšenou expresi let-7a, miR-16, miR-17-5p a miR-19b a naopak sníženou expresi miR-372, miR-143 a miR-155 u MM pacientů a buněčných linií ve srovnání se zdravými kontrolami (Bakkus et al, 2007). Exprese miR-15 a miR-21 se v této studii významně nelišila mezi zdravými dárci a nemocnými, což je v rozporu s pozdější studií, která identifikovala miR-21 jako onkogen s antiapoptotickou funkcí (Lóffler et al., 2007). Pomocí chromatinové imunoprecipitace bylo zjištěno, že se STAT3 podílí na regulaci exprese miR-21 v IL-6 závislých PB po přídavku IL-6. Zdá se, že u těchto buněk je transkripce miR-21 kontrolována pomocí IL-6 a zprostředkovaná aktivací STAT3, což napomáhá přežívání maligních buněk. Navíc ektopická exprese miR-21 za nepřítomnosti IL-6 vedla ke snížení apoptózy buněk, což potvrzuje účast miR-21 v procesu apoptózy, která je zprostředkovaná pomocí STAT3 (Lóffler et al, 2007). V pilotní studii, zabývající se úlohou miRNA v maligní transformaci PB, byla pomocí miRNA mikročipů a následné qRT-PCR srovnávána exprese miRNA jak u zdravých dárců, tak u osob s MGUS, pacientů s MM a u buněčných linií (Obr. 8). Byly identifikovány specifické profily miRNA popisující PB v MM, MGUS a MM liniích, tak transformaci z MGUS do MM. U MGUS bylo nalezeno 48 miRNA, u MM pacientů již 96 odlišně exprimovaných miRNA ve srovnání se zdravými dárci. U obou skupin, MM i MGUS, byla pozorována zvýšená exprese miR-21, klastru miR-106-25 a miR-181a/b, nicméně pouze u MM byla stanovena zvýšená exprese miR-32 a klastru miR-17-92. Zdá se tedy, že se tyto miRNA podílejí na progresi onemocnění a napomáhají transformaci z MGUS do MM (Pichiorri etal, 2008). 34 B-lymfocyt germinálního centra tmiR-106-25 tmiR-181a/b tmiR-1 fmiR-181a/b tmiR-17-92 tmiR-32 tmiR-133a tmiR-193b-365 ♦ miR-192-194-215 *miR-1Sn/1B_ Obrázek 8 Schematické znázornění transformace plazmatické buňky. Reprezentativní miRNA a geny významně deregulované u jedinců s MGUS a MM ve srovnání se zdravými jedinci. (Kubiczkova et al., 2012) V návaznosti na získané poznatky byla provedena (u PB zdravých dárců a MM pacientů) srovnávací analýza expresního profilu miRNA a expresního profilu kódujících genů (Gene Expression Profiling - GEP), která prokázala souvislost mezi globální zvýšenou expresí miRNA a špatnou prognózou high-risk MM pacientů (Zhou et al., 2010). Další studie by mohly podpořit tuto souvislost, jelikož bylo pozorováno, že vyšší viabilita MM buněk souvisí s vyřazením z funkce Argonaut (EIF2C2/AG02) komplexu, který je hlavním regulátorem maturace a funkce miRNA a jehož exprese je zvýšená u high-risk MM (Diederichs et Haber, 2007; Liu et al., 2004). EIF2C2/AG02 se navíc podílí na diferenciaci B-lymfocytů (O'Carroll et al., 2007) a je znám jako marker nádorové progrese u MM (Shaughnessy et al., 2007). V této studii byla také navržena hypotéza, že miRNA mohou působit synergisticky a tím významně přispívat k progresi MM. Jiná miRNA mikročipová srovnávací studie odhalila zvýšenou expresi klastru miR-193b-365 u PB MM pacientů (Unno et al., 2009). Dále byly porovnány expresní miRNA profily PB MM pacientů s profily normálních PB a byla zjištěna významně zvýšená exprese miR-222, miR-221, miR-382, miR-181a a miR-181b a snížená exprese miR-15a a miR-16 (Roccaro et al., 2009). Gutiérrez et al. (2010) ve své práci porovnali miRNA expresní profil PB 60 MM pacientů s PB zdravých dárců a pozorovali sníženou expresi 11 miRNA (miR-375, miR-650, miR-214, miR-135b, miR-196a, miR-155, miR-203, miR-95, miR-486, miR-10 a miR-196b), z nichž pouze miR-155 byla již dříve popsána v souvislosi s lymfoidními buňkami. Nedávno publikovaná práce popisuje 40 miRNA se sníženou expresí v PB MM pacientů ve srovnání se zdravými dárci, z nichž 6 miRNA (miR-214, miR-135b, miR-196a, 35 miR-155, miR-203 a miR-486) se shoduje s miRNA publikovanými skupinou Gutiérrez et al.(2010). Navíc výsledky klastrovací analýzy 54 MM pacientů poukázaly na 3 miRNA, a to miR-296, miR-194 a let-7f, jejichž zvýšená exprese souvisí s lepším přežíváním pacientů (Corthals et al, 2011). Stanovené expresní profily PB MM pacientů nejsou jednotné, nicméně některé miRNA byly potvrzeny ve více studiích. 4.4 Resistence na léky a miRNA u mnohočetného myelomu Přítomnost miRNA je také spojována s rezistencí vůči některým lékům. Jak bylo výše zmíněné, bortezomib patří do skupiny inhibitorů proteasomu. Jedná se o dipeptid kyseliny borité vykazující protinádorové účinky (Adams et al, 1999). Bortezomib byl schválen k léčbě MM v relapsu i pro léčbu nově diagnostikovaných pacientů (Hájek, 2009). V roce 2009 byly popsány expresní dráhy miRNA, které souvisí s léčebnou odpovědí k bortezomibu. Srovnání expresních profilů linií rezistentních a citlivých k bortezomibu odhalilo 22 deregulováných miRNA, z toho zvýšenou expresi měly miR-155, miR-342-3p, miR-181a, miR-181b, miR-128 a miR-20b, naopak snížená exprese byla pozorována u let-7b, let-7i, let-7d, let-7c, miR-222, miR-221, miR-23a, miR-27a a miR-29a. Mezi predikované cíle těchto miRNA patří geny zapojené do buněčného cyklu, buněčného růstu, apoptózy a ubikvitinace. Následně, pro stanovení klinického významu uvedených miRNA, byly korelovány expresní profily miRNA PB pacientů rezistentních a citlivých k bortezomibu s jejich odpovědí na léčbu. Bylo zjištěno, že pacienti citliví k terapii bortezomibem měli stejný profil deregul ováných miRNA jako linie citlivé k bortezomibu a stejně tak profil pacientů rezistentních k bortezomibu inklinoval k profilu stanovenému na liniích (Neri et al, 2009). V další studii, zabývající se změnou expresních profilů miRNA během získané lékové rezistence, byly srovnány modelové expresní profily miRNA mezi MM buněčnými liniemi (RPMI-8226 a U266) se získanou rezistencí k doxorubicinu a melfalanu a jejich parentálními liniemi. Výsledky expresní analýzy byly validovány pomocí qRT-PCR a významné změny byly pozorovány u miR-21 a miR-181a a miR-181b. Exprese miR-21 byla zvýšená u obou klonů linií rezistentních k melfalanu. Překvapivě bylo zjištěno, že exprese miR-181a a miR-181b byla snížená u U266 doxorubicin rezistentní linie, ale zvýšená u RPMI-8226 doxorubicin rezistentní linie. Zdá se, že změny vedoucí k lékové rezistenci jsou náhodné a efekt miRNA je závislý na kontextu (Munker et al, 2010). 36 4.5 Mechanismus deregulace miRNA u MM Nové studie, navazující na předchozí objevy, částečně vysvětlují mechanismus deregulace miRNA u MM. Srovnávací mikročipová analýza miRNA a analýza počtu kopií (Copy Number Variations - CNV) DNA nebo GEP MM linií objasnily deregulaci 16 miRNA, jejichž geny leží v oblastech chromozomů, které jsou často předmětem různých alelových změn u MM. Mezi nejčastější změny patřily zisky chromozomů. Bylo zjištěno, že miR-548-1 se vyskytovala s nejvyšší četností (94 %) v oblastech zisku chromozomu, zatímco miR-130b, miR-185, miR-648 a miR-649 (všechny leží v oblasti 22qll.21) jsou zastoupeny v oblastech ztráty chromozomu. Mezi další často deregulované miRNA patřily miR-22 ležící v oblasti 17pl3.3, miR-106b a miR-25 v oblasti 7q22.1, miR-15a v oblasti 13ql4.3, miR-21 v oblasti 17q23.1 a miR-92b, která se nachází v oblasti lq22 (Lionetti et al., 2009a). Klastr miR-15a/16-l byl dále podrobněji studován a bylo zjištěno, že u pacientů s delecí chromozomu 13 zcela chybí miR-15a a miR-16, nicméně u pacientů bez delece chromozomu 13 byla exprese miR-15a a miR-16 také významně snížená (Roccaro et al., 2009). Další studie, srovnávající CNV s čipy mapující jednonukleotidové polymorfismy (Single Nucleotide Polymorphism - SNP) ukázala, že exprese miR-15a a miR-16 není závislá na statutu chromozomu 13, ale obecně je u MM pacientů exprese zmíněných miRNA zvýšená oproti normálním PB (Corthals et al., 2010). Byla také nalezena korelace mezi šesti intragenovými miRNA a geny, uvnitř kterých se miRNA nacházejí. Tyto geny jsou deregulovány u MM linií a pacientů, a některé jsou důležité v patogenezi MM, jako mesoderm specific transcript (MEST) a miR-335 nebo Ena/vasodilator-stimulated phosphoprotein-like (EVL) a miR-342-3p (Ronchetti et al., 2008). V jiné práci byla nalezena souvislost mezi 32 intragenovými miRNA a geny, uvnitř kterých leží, některé z těchto genů jsou opět významně deregul ovány u MM pacientů. Studie potvrdila již výše zmíněné korelace, navíc byla zjištěna souvislost mezi genem coatomer protein complex, subunit zeta 2 (COPZ2) a miR-152 (Lionetti et al., 2009a). Získané výsledky naznačují, že změna počtu kopií genu souvisí se zvýšenou expresí jeho intragenových miRNA, což by částečně vysvětlovalo mechanismus změněné exprese miRNA u MM. Jelikož je myelom velmi heterogenní onemocnění, pro které jsou charakteristické komplexní cytogenetické aberace, je velmi pravděpodobné, že tyto aberace ovlivňují také expresi miRNA. V nedávné studii bylo rozděleno 60 MM pacientů do různých 37 cytogenetických podskupin na základě traslokačních partnerů IgH genu a statutu RB genu a tyto podskupiny pacientů byly srovnány s jejich expresí 365 miRNA. Výsledky klastrovací analýzy poukázaly na zvýšenou expresi miR-1 a miR-133a, které souvisí s translokací t(14; 16) (Gutiérrez et al., 2010). Změněná exprese jiných miRNA byla dále popsána v souvislosti s translokacemi t(4; 14), t(l 1; 14) nebo t(14; 16) (Gutiérrez et al., 2010; Lionetti et al., 2009b). Nově bylo popsáno 5 miRNA, které byly zvýšený u pacientů s t(l 1; 14) a to miR-122a, miR-33, miR-489, miR-519 a miR-555 (Corthals et al., 2011). Další možností deregulace miRNA je změna v jejich zpracování nebo maturaci. Již dříve zmíněná studie EIF2C2/AG02 komplexu uvádí, že úbytek AG02 souvisí se zástavou růstu a apoptózou u MM buněk (Zhou et al., 2010). V souladu s tím bylo prokázáno, že změněná hladina enzymu Dicer, ale ne enzymu Drosha, může souviset s progresí MM. Autoři pozorovali podobnou hladinu enzymu Dicer u PB zdravých dárců a pacientů sMGUS, která je však významně zvýšená oproti SMM (Smouldering MM) a MM pacientům. Navíc bylo pozorováno, že skupina pacientů s vyšší hladinou enzymu Dicer měla delší dobu do progrese (Sarasquete et al., 2011). Zmíněné výsledky jsou však v rozporu s nedávno provedenou studií, ve které nižší exprese genu DICER1 u skupiny MM pacientů souvisí s delší dobou do progrese nemoci (Corthals et al., 2011). Zdá se tedy, že regulační mechanismy ovlivňující jak miRNA maturaci tak jejich funkci se mohou podílet na změněné expresi miRNA, další studie určitě pomohou objasnit zmíněné nesrovnalosti. 4.6 miRNA ovlivňující kritické geny u MM Mnoho vědeckých skupin se zabývalo otázkami, jak důležité jsou z funkčního hlediska změny v expresi miRNA a jak tyto změny souvisí s patogenezí MM. Pro zodpovězení těchto otázek jsou využívány různé přístupy od predikce cílových genů pomocí in silico modelů až po pokusy s transgenními zvířaty. Je známo, že kódující geny, které se podílejí na procesu kancerogeneze u MM, jsou cílem pro deregulováné miRNA. Bylo prokázáno, že klastr miR-17-92, nacházející se v oblasti 13q31-32, ovlivňuje expresi genu PTEN, genu pro transkripční faktor E2F1 a BJJV1 (Ventura et al., 2008; Xiao et al., 2008). U transgenních myší se zvýšenou expresí tohoto klastru v lymfocytech byly pozorovány lymfoproliferativní a autoimunitní onemocnění a časná úmrtí. Dále bylo zjištěno, že purifikované myší CD4+ lymfocyty se zvýšenou expresí miR-17-92 obsahovaly snížené množství proteinů Pten a Bim, což naznačuje, že 38 miR-17-92 klastr ovlivňuje tyto nádorové supresory (Xiao et al., 2008). Brzy nato byla publikována další studie, ve které bylo prokázáno, že zmíněný klastr je nezbytný pro vývoj B-lymfocytů. Nepřítomnost miR-17-92 vedla ke zvýšené hladině pro-apoptotického proteinu BIM a tím k zástavě vývoje z pro-B do pre-B stádia (Ventura et al., 2008). Zdá se tedy, že zvýšená exprese miR-17-92 negativně reguluje zmíněné nádorové supresory a přispívá k transformaci PB a progresi MM. Predikce in silico také ukázala, že cílem miR-21 a klastru miR-106-25 jsou mezi jinými nádorové supresory PTEN, BIM a p21, a proto je pravděpodobné, že se tyto miRNA mohou podílet na vývoji plně rozvinutého myelomu (Pichiorri et al., 2008). Jiná miRNA, miR-19a/b, ovlivňuje dráhu STAT-3/IL-6, která je důležitá vpatogenezi MM. Bylo prokázáno, že miR-19a/b přímo ovlivňuje suppressor of cytokine signaling-1 (SOCS-1, negativní regulátor IL-6), a tím přispívá k jeho časté deregulaci u MM buněk (Pichiorri et al., 2008). Také miR-21 zmíněná výše působí jako onkogen a podílí se na regulaci této dráhy (Lóffler etal., 2007). Jak již bylo zmíněno dříve, miR-15a a miR-16-1 leží v oblasti chromozomu 13ql4.3, která je deletována u více než 50 % pacientů s MM. Tato delece je považována za primární mutaci, která se podílí na patogenezi MM (Fonseca et al., 2004). miR-15a/16 jsou považovány za nádorové supresory podílející se na proliferaci MM buněk in vitro i in vivo tím, že inhibují AKT serin/treonin proteinovou kinázu (AKT3), ribosomální protein S6, MAP kinázy a NFkB aktivátor MAP3KIP3 (Roccaro et al., 2009). Dále bylo prokázáno, že miR-15a/16 nejen regulují expresi genů buněčného cyklu, jako jsou cykliny Dl a D2, dále CDC25A, ale rovněž ovlivňují expresi genů spojených s apoptózou: BCL-2 nebo MCL-1 (Aqeilan et al., 2010). Navíc ektopická exprese miR-15a/16 negativně reguluje angiogenezi pomocí VEGF (Roccaro et al., 2009). Nedávno byla popsána úloha miR-15a/16 v mikroprostředí kostní dřeně. Bylo zjištěno, že exprese miR-15a/16 je v MM buňkách po ovlivnění cytotoxickými látkami vyšší. Nicméně, po interakci těchto buněk se stromálními buňkami kostní dřeně odvozenými od MM (MM-BMSC) pacienta, byla pozorována snížená exprese miR-15a/16 u myelomových buněk. Důvodem byla zvýšená produkce IL-6 stromálními buňkami, který inhiboval expresi zmíněných miRNA. Zdá se tedy, že mikroprostředí je důležité pro přežití MM buněk a chrání je před působením léků pomocí sekrece IL-6, který inhibuje expresi miR-15a/16 (Hao et al., 2011). Nově publikované práce se dále zaměřují na vztah miRNA k nádorovému supresoru p53. Výsledky screeningové metody umožňující identifikovat miRNA, které negativně regulují signalizaci p53 pomocí přímé interakce s genem TP53 naznačily, že miR-25 a miR- 39 30d mohou ovlivňovat p53. Navíc byla exprese miR-25 a miR-30d zvýšená v PB MM pacientů a u miR-25 zvýšená exprese korelovala se sníženou expresí mRNA TP53 (Kumar et al., 2011). Také miR-181a byla popsána jako negativní regulátor exprese genu TP53, což potvrzuje spojitost mezi p53 a aberantní miRNA expresí (Pichiorri et al., 2010). Je známo, že miR-34a je transkripčním cílem p53 zprostředkovávajícím apoptózu (Lodygin et al., 2008). U MM pacientů byla pozorována hypermetylovaná miR-34a v oblasti lp36. Jelikož se krevních nádorových onemocnění nevyskytuje mutace TP53 tak často, jako u solidních nádorů, mohla by hypermetylace miRNA částečně vysvětlit dysregulaci p53 signalizace (Chim et al., 2010). V další studii byla nalezena snížená exprese miR-192, miR-194 a miR-215 u části nových diagnóz MM pacientů. Další pokusy in vitro prokázaly, že při použití molekulárních inhibitorů MDM2 mohou být tyto miRNA transkripčně aktivovány pomocí p53 a posléze modulovat expresi MDM2. Je tedy patrné že miR-192, miR-194 a miR-215 ovlivňují MDM2/TP53 regulační osu a kontrolují rovnováhu mezi MDM2 a p53. Navíc miR-215 a miR-192 ovlivňují signální dráhu IGF a tím zabraňují zvýšené migraci PB do KD (Pichiorri etal, 2010). Během posledních let bylo provedeno mnoho studií srovnávajících globální profil CD 13 8+ PB MM pacientů a zdravých dárců pomocí různých high-throughput screeningových metod, od oligonukleotidových čipů až po qRT-PCR profilování. Každá z metod má své silné a slabé stránky poskytující rozdílné výsledky, ke kterým navíc přispívá velká heterogenita onemocnění. Obecně bylo doposud ve většině prací u myelomu identifikováno více miRNA se zvýšenou expresí u PB než se sníženou expresí. Výjimkou je práce Guttiérez et al.{20\0), která popisuje více miRNA se sníženou expresí. Dále můžeme říci, že ani identifikace jednotlivých miRNA není jednotná, což může být způsobeno několika faktory. Za prvé je v každé studii rozdílný soubor pacientů a kontrol a rozdílná velikost souboru. Jak již bylo zmíněno, je MM velmi heterogenní onemocnění a každý pacient má jinou kombinaci genetických mutací a cytogenetických aberací, což se může projevit na rozdílné sub klasifikaci do skupin ve srovnání se zdravými dárci. Za druhé, pacienti mohou mít v různých stádiích onemocnění odlišné profily miRNA. Na příklad miR-15 byla popsána jako zvýšená u nových diagnóz, ale snížená u relapsů (Pichiorri et al., 2008; Zhou et al., 2010). V neposlední řadě se na odlišnostech podílejí rozdíly ve zpracování vzorku, purifikaci PB, extrakci miRNA a dále rozdílné mikročipové platformy a různé verze čipů. Dnes již víme, že změněná exprese miRNA u MM může být z příčin genetických, cytogenetických nebo epigenetických. Byly také popsány specifické miRNA charakterizující 40 progresi MM, lepší prognózu nebo rezistenci k lékům. Mechanismus deregulace není zatím přesně známý, víme již, že v pozadí stojí jak změna v cílovém genu pro miRNA, tak změny v počtu kopií lokusů, ve kterých se nachází miRNA, defekty v biogenezi miRNA a epigenetické změny. Snahou dalších studií by mělo být objasnění komplexity regulace miRNA a identifikace terapeutických cílů. Tato část práce byla zpracována do kapitoly v knize MikroRNA v onkológii (Kubiczková et al. in: Slabý et al., 2012). 4.7 Cirkulující mikroRNA u MM Naše práce se v současné době soustřeďuje na cirkulující miRNA u MM. Vzhledem k tomu, že většina vyšetření pro detekci relapsu nebo onemocnění se provádí z kostní dřeně, je nutné najít markety, které by byly snadno dostupné, možnost odběru by minimálně zatěžovala pacienta a byly by opakovatelně odebíratelné. Naše první práce se zaměřila na několik miRNA, které by mohly být důležité z hlediska patogeneze MM - miR-29a, miR-142-5p, miR-410 a miR-660. Pro tyto pokusy bylo použito vzorků séra pacientů s MM (91 pacientů) při diagnóze ve srovnání se zdravými dárci (bez hematologických malignit). Pomocí real-time PCR upravené pro miRNA byly stanoveny hladiny jednotlivých miRNA ve vzorcích MM pacientů a bylo zjištěno, že hladiny miR-29a, 660 a 142-5p jsou zvýšeny v séru pacientů s MM, ale jen hladina miR-29a je schopna odlišit pacienty s MM od zdravých dárců se specificitou 70 % a senzitivitou 88 %. Naše práce o cirkulující formě miR-29a jako markeru mnohočetného myelomu byla mezi prvními pracemi o cirkulujících miRNA v oblasti výzkumu MM a byla opublikována v časopise Leukémia & Lymphoma (Ševčíková et al., 2012). Role miR-29a v hematologických malignitách byla shrnuta v přehledovém článku v časopise Biomedical Papers (Fišerová etal., 2014). Naše další práce se zaměřila na odlišení pacientů s MM a s MGUS od zdravých dárců a využila jiný postup. Nejdříve byl vytvořen expresní profil miRNA pomocí Taq Man Low Density Arrays, který byl potom ověřen pomocí real-time PCR. Expresní profil označil 14 deregulováných miRNA, které byly detekovány na větším souboru pacientů. Multivariační analýza ukázala, že kombinace miR-34a a let-7e odliší MM pacienty od zdravých kontrol se specificitou 80,6 % a sensitivitou 86,7 % a od MGUS se specificitou 91,1 % a sensitivitou 96,7 %. Další analýzy prokázaly korelaci hladiny let-7e a miR-744 s přežitím pacientů s MM. 41 Tato práce o cirkulujících formách mikroRNA u MM a MGUS byla opublikována v časopise Haematologica (Kubiczková et al., 2013b). Byla vybrána pro tzv. grafický abstrakt daného čísla časopisu (Obr. 9). Circulating serum microRNAs as novel and prognostic biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance diagnostic prognostic multiple myeloma miR-34a t let-7e 1 miR-744 ♦ let-7d ( sensitivity 80.6% specificity 86.7% -low expression: overall survival-! miR-130a4 remission* healthy I donor miR-34a let-7e miR-744 let-7d miR-130a sensitivity 91.1% specificity 96.7% monoclonal I gammopathy of undetermined significance miR-34a t let-7e I miR-744 * let-7d ♦ miR-130a 1 Kubiczková etal., Haematologica, 20V Obrázek 9 Grafický abstrakt článku Kubiczková et al., 2014 Dále jsme se zabývali také cirkulujícími formami miRNA u Waldenstromovy markoglobulinemie (Kubiczková-Bešše et al., 2014), která byla opublikována v časopise American Journal of Hematology. Tato práce potvrdila naše výsledky u pacientů s MM a MGUS na nezávislé skupině pacientů ve srovnání s pacienty s Waldenstromovou makroglobulinémií. V současnosti připravujeme manuskript o roli mikroRNA v EM relapsu pacientů s MM (Bešše et al., in preparation). 42 5 Závěr Mnohočetný myelom je krevní nádorové onemocnění. Jde o velice heterogenní nemoc, což je z hlediska výzkumu i kliniky problematické. Naše práce se zaměřuje na nové aspekty studia funkční genomiky této choroby a současně se snaží o lepší pochopení patogeneze MM včetně extramedulárního relapsu tohoto onemocnění. Extramedulární progrese myelomu je krajně nepříznivou variantou MM. Bohužel se jeho incidence výrazně zvyšuje, což je pravděpodobně dáno prodlužujícím se přežíváním pacientů a podle nových studií i novými léky, které sice dramaticky prodlužují přežití i kvalitu života pacientů, ale zdá se, že mění myelomové buňky a umožňují jejich přežití mimo kostní dřeň. I naše výsledky ukazují, že klon PB vKD a EM ložisku je odlišný. Změna biologického chování myelomových buněk ve smyslu umožnění vzniku extramedulárního ložiska není způsobena pouhou změnou exprese genů či CD markerů na povrchu buňky, ale zdá se, že se jedná se o změny komplexní, zahrnující bezesporu i změny v mikroprostředí celé KD. Dále je naší snahou přispět ke zlepšení diagnostiky onemocnění jako takového, i diagnostiky v rámci monoklonálních gamapatií a aplikovat získané poznatky a metodiku v rámci ČR. V současné době se pozornost obrací na cirkulující miRNA. Ty se zdají být vhodnými kandidáty pro biomarkery onemocnění díky své vysoké stabilitě a souvislosti s onemocněním. Především u MM má možnost využití cirkulujících miRNA jako biomarkerů potenciál překonat bolestivý postup stanovení diagnózy MM, který využívá invazivního odběru KD. I když je v současnosti MM již léčitelným onemocněním, relaps zůstává stale velkým problémem u většiny pacientů. 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Proc Natl Acad Sci USA. 107: 7904-9. 57 Seznam obrázků a tabulek Obrázek 1 První popsaný pacient s mnohočetným myelomem - Sarah Newbury (z Kýle et Rajkumar, 2008).......................................................................................................................7 Obrázek 2 Srovnání počtu myelomových PB oproti počtu fyziologických PB (Maslak, 2009).........................................................................................................................................8 Obrázek 3 Vícestupňový transformační proces MM (Špička et al, 2005)..............................9 Obrázek 4 Interakce mezi plazmatickými buňkami a buňkami KD u MM (Palumbo et Anderson, 2011; upraveno)....................................................................................................10 Obrázek 5 Pleiotropní účinek FMiDs u MM (Sedlaříková et al, 2012).................................13 Obrázek 6 Účinek bortezomibu u MM (Kubiczková et al, 2012).........................................14 Obrázek 7 Biogeneze miRNA (Esquela-Kerscher et Slack, 2006).......................................33 Obrázek 8 Schématické znázornění transformace plazmatické buňky. Reprezentativní miRNA a geny významně deregulováné u jedinců s MGUS a MM ve srovnání se zdravými jedinci. (Kubiczková et al, 2012)..........................................................................................35 Obrázek 9 Grafický abstrakt článku Kubiczková et al, 2014................................................42 Tabulka 1 Diagnostická kritéria MM (IMWG, 2003; upraveno)...........................................15 Tabulka 2 CRAB - kritéria poškození orgánů či tkání myelomem (IMWG, 2003)..............15 Tabulka 3 International Staging System (Greipp et al, 2005; upraveno)..............................17 Tabulka 4 Srovnání mediánu OS pacientů rozdělených podle přítomnosti či nepřítomnosti specifické cytogenetické abnormality a vliv na prognózu (Fonseca et al, 2003; upraveno). 19 58 Seznam příloh Článek 1: Význam mikroprostředí kostní dřeně v patogenezi mnohočetného myelomu Název časopis: Klinická onkologie IF v roce 2012: 0 Článek 2: TGF-beta — an excellent servant but a bad master Název časopisu: Journal of Translational Medicine IF v roce 2012: 3,459 Článek 3: Mechanism of immunomodulatory drugs in multiple myeloma Název časopisu: Leukemia Research IF v roce 2012: 2,764 Článek 4: Serum miR-29a as a marker of multiple myeloma Název časopisu: Leukemia and Lymphoma. IF v roce 2012: 2,301 Článek 5: Soft-tissue extramedullary multiple myeloma prognosis is significantly worse in comparison to bone-related extramedullary relapse Název časopisu: Haematologica IF v roce 2013: 5,935 Článek 6: Inhibitory proteazomu v léčbě mnohočetného myelomu Název časopisu: Klinická onkologie IF v roce 2013: 0 Článek 7: High-Risk Multiple Myeloma: Different Definitions, Different Outcomes? Název časopisu: Clinical Lymphoma Myeloma and Leukemia IF v roce 2013: 1,880 59 Článek 8: Circulating serum microRNAs as novel diagnostic and prognostic biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance Název časopisu: Haematologica IF v roce 2013: 5,935 Článek 9: Detection of tumor-specific marker for minimal residual disease in multiple myeloma patients Název časopisu: Biomedical Papers. IF v roce 2014: 1,661 Článek 10: Proteasome inhibitors - molecular basis and current perspectives in multiple myeloma Název časopisu: Journal of Cellular and Molecular Medicine IF v roce 2014: 4,75 Článek 11: The miR-29 family in hematological malignancies Název časopisu: Biomedical Papers IF v roce 2014: 1,661 Článek 12: Combination of serum microRNA-320a and microRNA-320b as a marker for Waldenstrom macroglobulinemia Název časopisu: American Journal of Hematology IF v roce 2014: 3,477 Kapitola: MikroRNA u mnohočetného myelomu. In Slabý et al. MikroRNA v onkológii. Praha (ČR). Galén, 2012. s. 271-280. 60 Přílohy Význam mikroprostředí kostní dřeně v patogenezi mnohočetného myelomu Fišerová B, Kubiczková L, Ševčíková S, Hájek R. Klin Onkol. 2012;25(4):234-40. Review. IF v roce 2012: - PŘEHLED Význam mikroprostředí kostní dřeně v patogenezi mnohočetného myelomu Implication of Bone Marrow Microenvironment in Pathogenesis of Multiple Myeloma Fišerová B., Kubiczková L., Ševčíková S., Hájek R. Babákova myelomová skupina, Ústav patologické fyziologie, LF MU Brno Souhrn Mnohočetný myelom je hematoonkologické onemocnení charakterizované maiigní proliferací plazmatických buněk. Tyto buňky se hromadí v kostní dřeni, kde potlačují fyziologickou krve-tvorbu a zároveň interagují s celou škálou cytokinů, růstových faktorů a adhezivních molekul. Je zřejmé, že právě mikroprostředí kostní dřeně hraje velkou roli v patogenezi onemocnění, ale i v rezistenci k léčbě. Klíčová slova mnohočetný myelom - kostní dřeň - IL-6 Summary Multiple myeloma is a hematooncological disease characterized by malignant proliferation of plasma cells. These cells accumulate in the bone marrow where they suppress physiological hematopoiesis; at the same time, these cells interact with a wide variety of cytokines, growth factors and adhesion molecules. It is obvious that the bone marrow microenvironment plays an important role in disease pathogenesis as well as treatment resistance. Key words multiple myeloma - bone marrow - IL-6 Práce byla podpořena výzkumným záměrem MSMT CR i. MSM0021622434, grantem GA CR GAP304/10/1395 a granty IGA MZ CR č, NT111S4aNT12130. This study was supported by scientific program of the Czech Ministry of Education, Youth and Sports No. MS M0021622434, by grant of Czech Science Foundation No. GAP304/10/1395 and by grant of Internal Grant Agency of the Czech Ministry of HeaIth No. NT 1 U 54 and r-ÍTl 2130. Autoři deklaruji, že v souvislosti s předmětem studie nemají rádné komerční zájmy. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. Redakční rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro publikace zasílané do biomeóicinských časopisů. The Editorial Board declares that the manuscript met the ICMJE 'uniform requirements" for biomedical papers. m Mgr. Sabina Ševčíková, Ph.D. Babákova myel omová skupina Ústav patologické fyziologie LF MU Brno Kamenice 5, A3 625 00 Brno e-mail: sevcik@med.muni.cz Obdrženo/Submitted: 26.4.2012 Přijato/Accepted: 10.5.2012 234 Klin Onkol 2012; 25(4}: 234-240 VÝZNAM MIKROPROSTŘEDÍ KOSTNÍ DŘENĚ V PATOGENEZI MNOHOČETNÉHO MYELOMU Osteoklast Obr. 1. Interakce v m i kro pro středí kostní dřené mrtohočetrtého myelomu. Myelomové buňky interagují s ostatními buňkami a mimobunéčnou hmotou prostřednictvím adhe-zivních molekul (CD44, RHAMM, VCAM-1) a po adhezi je podporováno vylučování cyto-kinů a růstových faktorů. Některé faktory působí i autokrinně (IL-6) a tím se ješté více stimuluje jejich produkce. Úvod Mnohočetný myelom (MM) je maligní hematologické onemocnění charakterizované monoklonální expanzí plazmatických (myelomových) buněk. Toto onemocnění se týká hlavně starších pacientů s mediánem věku stanovení diagnózy 69 let, incidence v CR je 4/100 000. Myelomové buňky jsou lokalizovány v kostní dřeni, kde narušují fyziologický proces hematopoezy a zároveň narušuji strukturu kosti, což vede ke vzniku osteolytických lézí, které jsou pro pacienty s MM hlavním zdrojem obtíží [1,2]. MM je vhodným modelem pro studium interakcí tumoru a mikroprostředí ze tří důvodů: na rozdíl od normálních buněk se maligní plazmatické buňky hromadí zejména v kostní dřeni, což znamená, že stroma! ní buňky poskytují jedinečné mikroprostředí pro růst maligních buněk. Dalším důvodem je přítomnost mnoha adhezivních molekul na povrchu myelomových buněk [3], normální buňky povrchové markery téměř neprodukují. Třetím důvodem je bezesporu možnost kultivace heterogenních populací adherentních buněk odebraných z kostnídřeně pacientů s MM v podmínkách in vitro [4]. Interakce adhezivních molekul Adhezivní molekuly umožňují přímé propojení mezibuněčné hmoty s myelo-movými buňkami a buňkami navzájem. Po navázání dalších molekul se spouští řada dějů, které mohou ovlivňovat vývoj buněčných složek nebo aktivovat signalizační kaskády. Je také podporována aktivace osteoklastů a růst maligních buněk, které jsou ješté více zadržovány v kostní dřeni [5] {obr. 1). Jedním ze specifických povrchových markerů myelomových buněk je synde-kan-1 (CD138). Je to transmembránový proteoglykan, který se může přímo vázat na proteiny mezibuněčné hmoty, u MM se váže na kolagen typu I [6]. V kostní dřeni je syndekan-1 detekován pouze na buňkách z B lymfoidní linie a jeho exprese se mění se stupněm diferenciace. U myší byl nalezen na povrchu pre-B buněk, u zralých 8 buněk se nevyskytoval a opět byl produkován u plazmatických buněk [7]. U pacientů s MM se vyskytuje pouze na povrchu myelomových buněk [8], inhibuje osteoklas-togenezi a pozitivně ovMvňuje diferenciaci osteobtastů [9]. U myelomových buněk, které procházejí apoptózou, se však rychle ztrácí [10]. Jelikož je syndekan-1 produkován na povrchu životaschopných myelomových buněk, byly vyvinuty specifické protilátky, které dnes umožňují identifikaci a purifikaci myelomových buněk ze vzorků pacientů [8]. Za interakci myelomových buněk s hyaluronanem jsou zodpovědné dva receptory, a to CD44 [11] a RHAMM [12]. Standardní forma receptoru CD44 se na povrchu myelomových buněk vyskytuje zřídka, ovšem některé nestandardní receptorové varianty jsou zde velmi časté. Byly detekovány například varianty 3v, 4v, 6v a lOv, které nejsou přítomné u zdravých jedinců [13], Receptor RHAMM napomáhá pohybu myelomových buněk po hyaluronovém substrátu. U MM jsou vylučovány tři formy: RHAMMFL, což je běžný typ, a dále dva deleční mutanti, RHAMM-48, (delece 48 bp) a RHAMM-147 (delece 147 bp). Oba deleční mutanti jsou přítomni jen u B buněk a plazmatických buněk MM, ale ne u zdravých jedinců. Výskyt deleč-ních mutantů mění intracelulární signalizaci v buňkách MM [12], Cytokiny, růstové faktory Myelomové i stromální buňky produkují látky, které ovlivňují vývoj MM. Mezi tyto látky se řadí cytokiny a chemokiny, které mohou působit jako promotory nádorového vývoje, růstové faktory nebo chemoatraktanty. Váží se na receptory, a tím aktivují různé signální kaskády. Pacienti s MM vykazují typické rozpustné faktory v mikroprostředí kostní dřeně: IL-16, IL-2R, MCP-1, HGF, IL-1RA, MIG, IP-10, EGF[14]. Stěžejním cytokinem MM je inter-leukin-6 (IL-6), který je produkován mnoha buňkami včetně osteoblastů, monocytů, makrofágů a stromálních buněk kostní dřeně. Za fyziologických podmínek je jeho hladina nízká nebo nedetekovatelná, ale bylo prokázáno, že u MM pacientů s osteolytickými lézemi je hladina IL-6 zvýšená Í151. U MM je IL-6 hlavním cytokinem, který zprostředkovává růst, přežívání a lékovou rezistenci myelomových buněk (obr. 2). I když některé myelomové buňky produkují IL-6 autokrinně [16], primárně je produkován stromálními buňkami kostnídřeně a působí parakrinně na růst a diferenciaci myelomových buněk [17]. Produkce IL-6 ve stromálních buňkách je tedy indukována buďadhezí myelomových buněk [18], nebo prostřednictvím jiných cytokinů, jako tumor nekrotizující Klin Onkol 2012; 25(4): 234-240 235 VÝZNAM Ml KRO PROSTŘEDÍ KOSTNÍ DŘENÉ V PATOGENEZI MNOHOČETNÉHO MYELOMU Obr. 1. IL-6 a signálni kaskády v mnohočetném myelomu. Navázáním IL 6 na jeho receptor se aktivují čtyři signální dráhy důležité pro vývoj, růst a lékovou rezistenci v MM. Fosfo-rylace Jak spustí dráhu Ras/Ras/MEK/ERK a STAT. ERK a STATse přesunou do jádra, kde aktivují cílové geny. Jak také spouští signální dráhu Pl 3K/Akt, Akt dáie může aktivovat mTOR, který působí na STAT a NF-kB, který se opět přesouvá do jádra a přepisuje dané geny. faktor a{TNFa) [19] a vaskulární endote-liá lni růstový faktor (VEGF) [20]. Zmíněné faktory následně aktivují např. signální dráhu jaderného faktoru kB (NF-kB), která má vliv na přežívání a růst myelo-mových buněk [21]. Osteolýza, datší patologický proces podporovaný přítomností IL-6, je navozena hned několika mechanizmy. Za prvé, IL-6 indukuje produkci RANKL v mezenchymálních buňkách kostní dřeně a osteoblastech. Vazbou RANKL na RANK je navozeno dozrávání os-teokíastů a aktivace signálních drah [22]. Za druhé, IL-6 indukuje zvýšení hladin proteinů zapojených do procesu kostní resorpce, např. peptidů vázajících paraty roidní hormon PTHrP [23]. Za třetí, IL-6 in h i buje osteogenezi zprostředkovanou Wnt, ještě více ruší homeostázu v kosti a posouvá rovnováhu směrem k degradaci kosti [24]. Hladina IL-6 odráží stupeň rozvoje mo-noklonálních gamapatit, jak bylo prokázáno ve studii, do které bylo začleněno 131 pacientů. Ze skupiny 22 nemocných s MGUS, což je prekancerózní stadium předcházející MM, byl IL-6 detekován pouze u jednoho jedince, u MM to bylo už 35% pacientů a u nejagresiv-nějšího stadia zvaného plazmocytární leukémie se IL-6 vyskytoval ve vysoké koncentraci u všech pacientů. Navíc se hodnoty IL-6 lišily i mezi MM pacienty, podíl pacientů s vyšší hladinou IL-6 byl jiný při diagnóze (37%), během intenzivního vývoje (60%) a během stabilní fáze (13%) [251. Z druhé strany bylo prokázáno, že agresivní mimokostní stadia MM mohou být nezávislá na hladině IL-6 [26]. V klinických studiích byl testován účinek anti-IL-6 mAB (např. CNTO 328), který ale neprokázal zásadní vliv na léčbu MM. RANKL, ligand z rodiny TNF, je produkován nezralými osteoblasty, stromál-ními buňkami a T lymfocyty [27,28], Os-teoklasty a jejich prekurzory produkují jeho receptor RANK. Vazbou RANKL na RANK se aktivují signální dráhy, které jsou důležité při diferenciaci osteoklastů z jejich prekurzorových buněk, RANKL také reguluje diferenciaci, funkci a přežívání osteoklastů [22,29]. Osteoprote-gerin (OPG) je antagonistou RANKL, in-hibuje vazbu RANK-RANKL, inhibuje diferenciaci a aktivaci osteoklastů, a tím brání degradaci kosti [30]. U MM je RANKL hlavním osteoklasto-genním faktorem podílejícím se na ly-tické kostní nemoci. Jeho vysoká produkce ve stromálních buňkách pacientů s MM má důležitou úlohu v patoge-nezi MM [31]. Některé studie ukázaly, že RANKL je produkován i myelomo-vými buňkami [32], V mikroprostředí kostní dřeně MM se interakcí stromálních a myelomových buněk produkce RANKL zvyšuje a OPG snižuje, což podporuje kostní resorpci a osteolýzu [33]. Hladiny těchto molekul korelují s klinickou aktivitou MM a závažností kostní nemoci [34]. Denosumab, monoklo-nální protilátka proti RANKL, byla schválena FDA k ochraně kostí před dalším poškozením u pacientů s nádory prostaty a prsu s kostními metastázami. V současné době probíhají klinické studie fáze III u pacientů s MM. Myelomové buňky také vylučují transformující růstový faktor p (TGFp), pleiotropní cytokin, který za normálních podmínek mimo jiné inhibuje imunitní odpověď tím, že brání proliferaci a diferenciaci B lymfocytů a sekreci Ig [35]. Na rozdíl od účinku na B lymfocyty TGFp nesnižuje proliferaci myelomových buněk, a při vysokých koncentracích dokonce podporuje vylučování IL-6 těmito buňkami. Tímto zčásti zprostředkovává růst myelomu a podporuje patogenezi [36]. Navíc je TGFp důležitý v nerovnovážné kostní remodelaci u MM, je aktivní při zvýšené kostní resorpci a inhibuje osteoblasty. InhibiceTGFp podporuje diferenciaci osteoblastů, které pak in-hibují růst a přežívání myelomových buněk. Naopak potlačení diferenciace osteoblastů urychluje ztrátu kostní tkáně [37], Signální dráhy důležité v mikroprostředí kostní dřeně MM V patogenezi MM je mnoho regulátorů, proteinových kináz a růstových faktorů. 236 Klin Onkol 2012; 25(4): 234-240 VÝZNAM MlKROPROSTŘED! KOSTNÍ DŘENĚ V PATOGENEZI MNOHODETNÉHO MYELOMU Obr. 3. Dráha Wnt a její inhibitory v mnohočetném myelomu. V pravé části obrázku je zobrazena situace bez inhibitorů, kdy po navázáni Wnt není B-katenin degradován. Přesunutím do jádra se spoušti přepis genů pro normální vývoj osteoblastů. V levé části obrázku myelomová buňka produkuje inhibitory DKK-1 a sklerostin, které se vážou na receptor LRP. a sFRP. který blokuje vazbu na Frizzled receptor. B-katenin není přenesen do jádra, aie degradován v proteazomu, a tak je potlačena osteoblastogeneze. pomocí kterých buňky komunikují. Tyto procesy nejsou jednoduché, právě přímým kontaktem strukturních a buněčných složek nebo podporou autokrinní a parakrinní produkce cytokinů se v mi-kroprostředí kostní dřeně aktivuje široké spektrum signálních drah [18,21,38,39]- Wnt/B-katenin Důležitou roli při růstu, vývoji a fungování osteoblastů hraje signální dráha Wnt/fS-katenin (obr. 3). Glykoproteiny Wnt se váží na koreceptory LRP-S nebo LRP-6 a Frizzled receptor a aktivují Wnt dráhu. Přenos signálu stabilizuje fl-kate-nin, který je translokován do jádra a zde stimuluje expresi genů zodpovědných za diferenciaci osteoblastů. Bez přítomnosti signálu je fi-katenin fosforylován a degradován v proteazomu. Existují dvě funkční skupiny antago-nistů Wnt signalizace, a to sFRP a Dick-kopf (DKK), po jejichž navázání se naruší funkce osteoblastů [40,41]. Významným inhibitorem u MM je DKK-1, který se váže na LRP-5. Je vylučován myelo-movými buňkami a vjejich přítomnosti také stromálními buňkami a osteoblasty. DKK-1 se vyskytuje především u pacientů s osteolytickými lézemi a jeho hladina koreluje s rozšířením osteolytic-kých ložisek [24,42,43]. Kromě potlačení diferenciace osteoblastů také podporuje osteoklastogenezi zvýšenou expresí RANKL a sníženou expresí OPG [44]. V současnosti probíhají klinické studie fáze l/II, které testují anti-DKK-1 mono-klonáiní protilátku (BHQ880) u MM. Další skupina inhibitorů sFRP blokuje vazbu k receptoru Frizzled. Myelomové buňky produkují sFRP-2 a sFRP-3 a ty významné potlačují diferenciaci osteoblastů a tvorbu kostí [45,46]. Existuje však i studie, která ukazuje, že hla-diny DKK-1 a sFRP u pacientů s MM nepotlačuj! diferenciaci lidských osteoblastů. To znamená, že nemusí být jedinými faktory zodpovědnými za inhibici osteoblastů [47], Sklerostin je produkován osteocyty a působí na dráhu Wnt podobně jako DKK-1, tedy navázáním na LRP-5 [48]. Inhibuje aktivitu osteoblastů a indukuje jejich apoptózu, je negativním regulá- torem tvorby kostní tkáně [49]. Teprve nedávno bylo potvrzeno, že sklerostin je vylučován i myelomovými buňkami, a tak přispívá k patogenezi MM [50]. Dráha NF-kB Obecně je tato dráha důležitá pro pro-liferaci, přežívání a vývoj nádorových buněk. U MM působí aktivace signalizace NF-kB pozitivně na růst, rezistenci k lékům a přežívání myelomových buněk v mi kro prostředí kostní dřené [SI], Aktivace může probíhat jak klasickým způsobem, tak i alternativně. Bylo prokázáno, že m i kro prost ředí kostní dřeně u MM spouští tuto signalizaci prostřednictvím adheze i vylučováním cytokinů a chemokinů. Například růstový a anti--apoptotický faktor IL-6, jehož produkce je vyšší při adhezi myelomových a stro-málních buněk, může indukovat dráhu NF-kB [21]. Také TNFct aktivuje NF-kB: za prvé u stromálnřch buněk, čímž podporuje vylučování IL-6 těmito buňkami, a za druhé u myelomových buněk, kde podporuje adhezi buněk a zvyšuje produkci intracelulární adhezivní molekuly 1 (ICAM-1) a vaskulární adhezivní molekuly 1 (VCAM-1) [19]. Aktivace je u MM možná oběma způsoby. Již dříve bylo zjištěno, že klasická dráha může být blokována inhibici IKKp" proteinu [51], ovšem to neplatí u alternativního způsobu aktivace. Proto byla také zjišťována inhibicejiné molekuly, a to IKKa, která se vyskytuje u obou způsobů aktivace. Růst buněk byl sice zpomalen, ale aktivita signalizace NF-kB byla vyšší než u kontroly, což naznačuje, že inhibiční efekt IKKa je nezávislý na aktivitě NF-kB [52]. Dráha PI3K/Akt Fosfatidylinositol-3-kináza (PI3K)/Akt je jedna z nejčastěji aktivních drah u lidských nádorů. Mnoho proteinových kináz a transkripčních faktorů, které se účastní této signalizace, ovlivňuje rezistenci myelomových buněk k léčbě. Dráha PI3K/Akt reguluje průběh buněčného cyklu a apoptózu, indukuje syntézu DNA a působí na přežívání a migraci myelomových buněk. Je propojena s dráhou NF-kB přes kinázu Akt, která, podobně jako IKK u dráhy NF-kB, fosfo-ryluje a degraduje kBa. To vede k přesunutí NF-kB do jádra, kde může induko- Klin Onkol 2012; 25(4): 234-240 237 VÝZNAM MIKROPROSTŘEDÍ KOSTNÍ DŘENĚ V PATOGENEZI MNOHOČETNÉHO MYELOMU vat transkripci anti-apoptotických genů. Tedy Akt může inhibovat apoptózu aktivací NF-kB [53], Aktivátory této dráhy jsou iL-6 a růstový faktor podobný inzulínu 1 (IGF-1); aktivace PI3K je důležitá při prolife-raci a anti-apoptotické odpovědi mye-lomových buněk na tyto cytokiny [54]. IL-6 nejen spouští dráhu PÍ3K/Akt, ale tato interakce má navíc regulační účinky na buněčný cyklus, chrání buňky před apoptózou způsobenou léky a ovlivňuje růst MM [55]. Důležitou součástí této dráhy je ki-náza mTOR, která se u savců vyskytuje ve dvou rozdílných komplexech, mTORCI a mTORC2, které ovšem mají rozdílné funkce. Akt aktivuje mTORCI, který fosforyluje další molekuly regulující syntézu proteinů, a tak kontroluje buněčný růst. Naopak mTORC2 v odpovědi na růstové faktory aktivuje Akt a reguluje přežívání buněk [56]. V souvislosti s MM byl identifikován DEPTOR, který za fyziologických podmínek inhi-buje mTORCI a mTORC2.1 když jeho vysoká produkce u MM inhibuje mTORCI, překvapivě také vede k aktivaci dráhy PI3K/mTORC2/Akt. Tento nepřímý způsob aktivace je důležitý ku příkladu u myelomových buněk, kterým chybí mutace aktivující PI3K [57]. V současnosti probíhají klinické studie, ve kterých jsou testovány kombinace lenalidomidu a everolímu (RAD001), inhibitoru mTOR jak v solidních nádorech, tak u refraktor-ního MM. Dráhy Ras/Raf/MEK/MAPK a Jak2/STAT3 Dalšími důležitými drahami jsou Ras/Raf/MEK/MAPK a Jak2/STAT3, které mohou být aktivovány IL-6. Inhi-bice IL-6R sice blokuje fosforylaci STAT3. ale neovlivňuje aktivaci dráhy MAPK, z čehož vyplývá, že v mikroprostředí kostní dřeně se dráha STAT3 aktivuje prostřednictvím IL-6 a dráha MAPK mechanizmy nezávislými na IL-6 [58]. K aktivaci dráhy Ras/Raf/MEK/MAPK, která stimuluje angiogenezi, proliferaci buněk a jejich apoptózu, jsou potřebné jak adhezivní interakce, tak vylučování růstových faktorů. Hlavními aktivačními faktory této dráhyjsou IL-6 a IGF-1, které aktivují Ras [59,60]. Následně dojde k ak- tivaci i ostatních složek- Raf, MEKa ERK. Tuto dráhu spouštějí také další faktory, jako VEGF [61]. Byto dokázáno, že inhi-bicí aktivity ERK se sníží produkce VEGF, která vede ke snížené tvorbě nových cév v kostní dřeni indukované myelomo-vými buňkami [62]. Dráha Jak2/STAT3 má vliv na přežívání myelomových buněk, aktivace této dráhy indukuje proliferaci a inhibici apo-ptózy. STAT3 přímo přispívá k malignímu rozvoji MM tím, že chrání myelomové buňky před apoptózou a podporuje přežívání [63]. Stimulace buněk IL-6 vede k signalizaci přes IL-6R a spouští fosforylaci STAT3 přes Jak, STAT3 je přenesen do jádra, kde aktivuje transkripci daných anti-apoptotických genů. Aktivace pomocí IL-6 tedy reguluje přežívání myelomových buněk vylučováním anti--apoptotických proteinů z rodiny Bcl-2, například Bcl-XL, Mcl-1 [63,64]. Bylo zjištěno, že v myelomových buňkách je STAT3 neustále aktivován a inhibice této dráhy indukuje apoptózu in vitro [58,65]. Rezistence k lékům způsobená m i kro pro st ředí m MM Rezistentní fenotyp způsobený mikro-prostředím MM může být dvojího typu: léková rezistence zprostředkovaná interakcemi cytokinů (cytokine mediated drug resistance - CM-DR), nebo léková rezistence zprostředkovaná adhezivním kontaktem buněk (cell adhesion-me-diated drug resistance - CAM-DR). Oba mechanizmy mají zásadní význam v pa-togenezi MM. Výraz CAM-DR byl poprvé použit ve studii MM, kde byla pozorována zvýšená produkce c^, p, a f3; inte-grinů, které jsou vylučovány myelomo-vými buňkami. Adheze maligních buněk kfibronektinu může přispět ke vzniku rezistence de novo, chrání buňky před apoptózou způsobenou léky [38]. S adhezí myelomových buněk k fíbro-nektinu je také asociována zvýšená hladina p27kipl. Tento protein je důležitý pro udržení rezistentního fenotypu, má význam v buněčném cyklu, kde zadržuje buňky ve fázi Gr Bylo prokázáno, že přerušením adheze se hladina p27kicl sníží, buňky pokračují v S fázi buněčného cyklu a opět se stávají citlivými k lékům. Pokusy s inhibici produkce p27*'p' neměly vliv na adhezi, ale zvrá- tily lékovou rezistenci. To dokazuje, že vyšší produkce tohoto proteinu přispívá k CAM-DR [66]. U MM je také nadměrně vylučována adhezivní molekula P-selektin a její li-gand PSGL-1. Kromě jiných funkcí, jako jsou adheze a osídlování myelomových buněk v mikroprostředí kostní dřeně, je PSGL-1 důležitý v rozvoji lékové rezistence myelomových buněk in vivo a in vitro. Inhibice interakcí tohoto li-gandu k selektinu podporuje citlivost myelomových buněk k bortezomibu, inhibitoru proteazomu [67]. Hladina HSP70jetaké zmíněnými interakcemi zvýšena, navíc podporuje vyšší produkci IL-6, který pak napomáhá přežívání myelomových buněk pomocí aktivace signálních drah. Inhibice HSP70 potlačuje adhezi myelomových buněk k fibronektinu a způsobuje apoptózu rezistentních buněk. Fenotyp lékové rezistence je tak změněn a buňky se stávají citlivými k lékům. Tato studie ukázala, že inhibice HSP70 může způsobit apoptózu buněk, které vykazují rezistenci de novo i rezistenci získanou během léčby [68]. S rezistencí je také asociována dráha NF—kB, jejíž aktivace je stimulována adhezí k fibronektinu [39]. Neustálá aktivita této dráhy vzdoruje inhibujícím účinkům bortezomibu, i v jeho přítomnosti přispívá jinými mechanizmy k rezistenci. V této studii bylo navíc zjištěno, že pokud se myelomové buňky kultivují se stromálními buňkami pacienta s MM, aktivita dráhy NF-kB, a tím i rezistence k bortezomibu, je ještě více podporována [69], Pozdější výsledky potvrdily, že aktivace dráhy NF-kB je významně vyšší ve stromáSních buňkách a tyto buňky se významně liší od fyziologicky normálních buněk [701. Závěr V posledních letech bylo provedeno mnoho studií týkajících se mikroprostředí kostní dřeně u pacientů s MM, které dostávají toto téma do popředí zájmu a ukazují na jeho význam v pato-genezi nemoci. Nicméně mechanizmy probíhající v tomto mikroprostředí nebyly doposud zcela objasněny, proto je nutné pokračovat v dalším výzkumu. 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ČESKO-SLOVENSKÁ HEMATOLOGICKÝ KONFERENCE T -u-"" ATRANSFUZIOLOGICKÝ SJEZD LABOFiATORNÍ HEMATOLOGIE 6.-8. září 2012 5.-6. září 2012 ^ ČESKÁ REPUBLIKA BRNO VÝSTAVIŠTĚ - PAVILON E www.hematotogy2012.cz 240 Klin Onkol 2012; 25(4): 234-240 TGF-beta - an excellent servant but bad master Kubiczkova L, Sedlarikova L, Hajek R, Sevcikova S. J Transl Med. 2012 Sep 3;10(1):183. PMID: 22943793 IF vroce 2012: 3,459 Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 JOURNAL OF TRANSLATIONAL MEDICINE REVIEW Open Access TGF-(3 - an excellent servant but a bad master Lenka Kubiczkova, Lenka Sedlarikova, Roman Hajek and Sabina Sevcikova* Abstract The transforming growth factor (TGF-(3) family of growth factors controls an immense number of cellular responses and figures prominently in development and homeostasis of most human tissues. Work over the past decades has revealed significant insight into the TGF-(3 signal transduction network, such as activation of serine/threonine receptors through ligand binding, activation of SMAD proteins through phosphorylation, regulation of target genes expression in association with DNA-binding partners and regulation of SMAD activity and degradation. Disruption of the TGF-(3 pathway has been implicated in many human diseases, including solid and hematopoietic tumors. As a potent inhibitor of cell proliferation, TGF-(3 acts as a tumor suppressor; however in tumor cells, TGF-(3 looses anti-proliferative response and become an oncogenic factor. This article reviews current understanding of TGF-(3 signaling and different mechanisms that lead to its impairment in various solid tumors and hematologica malignancies. Keywords: TGF-(3, SMAD proteins, Oncogene, Suppressor, Solid tumors, Leukemia, Multiple myeloma Introduction Although our understanding of molecular mechanisms that underlie cancer development and progression has increased, cancer remains a significant health concern in many developed countries. There is a strong requirement for new diagnostic and treatment options as well as elucidation of how cells acquire the six essential phe-notypes, or hallmarks, necessary to become fully malignant [1]. Pharmacological targeting of cancer hallmarks may offer new possibilities of effectively treating development and/or metastases of human tumors (reviewed in [2]). Transforming Growth Factor-13 (TGF-|3) is a key player in cell proliferation, differentiation and apoptosis. The importance of this regulation is apparent from the role of TGF-|3 in development and consequences of aberrant TGF-|3 signaling in cancer [3]. Nevertheless, it is still not elucidated how malignant cells overcome the cytostatic functions of TGF-|3 or how TGF-|3 stimulates the acquisition of cancer hallmarks of developing and progressing human cancers. In this paper, we review different molecular and cellular mechanisms that lead to impairment of TGF-|3 signaling in various solid tumors and hematological malignancies. * Correspondence: sevcik@med.muni.cz 3abak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno 625 00, Czech Republic (3 BioMed Central History of TGF-0 discovery In the early 1980s, it had become apparent that cell growth is controlled by many polypeptides and hormones. A new hypothesis of 'autocrine secretion' was postulated, which suggested that polypeptide growth factors are able to cause malignant transformation of cells [4]. A new polypeptide called SGF (Sarcoma Growth Factor) was discovered in cultures of transformed rat kidney fibroblasts [5]; soon it became apparent that this factor is a mixture of at least two substances with different functions. They were called Transforming Growth Factor-a (TGF-a) and Transforming Growth Factor-|3 (TGF-|3) [6]. TGF-|3 was further described by Roberts and Sporn as a secreted polypeptide capable of inducing fibroblast growth and collagen production [7]. Soon after its discovery, TGF-|3 was found to inhibit cell proliferation as well; thus, a dual role of this cytokine was recognized [8,9]. TGF-p family and isoforms The TGF-|3 superfamily is composed of a large group of proteins, including the activin/inhibin family, bone mor-phogenetic proteins (BMPs), growth differentiation factors (GDFs), the TGF-|3 subfamily, and the glial cell line-derived neurotrophic factor (GDNF) family. This review will focus solely on the TGF-|3 family. ©2012 Kubiczkova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.Org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 2 of 24 The TGF-|3 proteins have been discovered in a variety of species, including invertebrates as well as vertebrates. TGF-|3 superfamily is fundamental in regulation of various biological processes, such as growth, development, tissue homeostasis and regulation of the immune system [10,11]. Beta-type subfamily growth factors are homodimeric or heterodimeric polypeptides with multiple regulatory properties depending on cell type, growth conditions and presence of other polypeptide growth factors. Since their expression is also controlled by distinct promoters, their secretion is temporal and tissue specific [12]. There are three known isoforms of TGF-|3 (TGF-|3l, TGF-|32 and TGF-|33) expressed in mammalian tissues; they contain highly conserved regions but diverge in several amino acid regions. All of them function through the same receptor signaling pathways [13,14]. TGF-|3l, the most abundant and ubiquitously expressed isoform, was cloned from human term placenta mRNA [15]. In mouse development, Tgf-|3l mRNA and/or protein have been localized in cartilage, endochondral and membrane bone and skin, suggesting a role in the growth and differentiation of these tissues [16]. TGF-|32 was first described in human glioblastoma cells. It was found that TGF-|32 is capable of suppressing inter-leukin-2-dependent growth of T lymphocytes. Thereby, it was named glioblastoma-derived T cell suppressor factor (G-TsF). Physiologically, TGF-|32 is expressed by neurons and astroglial cells in embryonic nervous system [17]. It is also important in tumor growth enhancing cell proliferation in an autocrine way and/or reducing immune-surveillance of tumor development [18]. Their mature forms, which consist of the C-terminal 112 amino acids, TGF-|3l and TGF-|32 share 71% sequence similarity [19]. The third isoform, TGF-|33, was isolated from a cDNA library of human rhabdomyosarcoma cell line; it shares 80% of amino acid sequence with TGF-|3l and TGF-|32. Studies on mice demonstrated essential function of Tgf-(33 in normal palate and lung morphogenesis and implicate this cytokine in epithelial-mesenchymal interaction [20,21]. Its mRNA is present in lung adenocarcinoma and kidney carcinoma cell lines; interestingly, umbilical cord expresses very high level of TGF-|33 [19]. TGF-p synthesis and activation Mature dimeric form of TGF-|3, composed of two monomers stabilized by hydrophobic interactions and disul-phide bridge, initiates intracellular signaling [22]. The three TGF-|3s are synthesized as pro-proteins (pro-TGF-|3s) with large amino-terminal pro-domains (called latency associated proteins - LAPs), which are required for proper folding and dimerization of carboxy-terminal growth-factor domain (mature peptide) [23]. This complex is called 'small latent complex' (SLC). After folding and dimerization, TGF-|3 dimer is cleaved from its propeptides in trans-Golgi apparatus by furin type enzymes; however, it remains associated with its pro-peptide through noncovalent interactions, creating large latent complex' (LLC). Most cultured cell types release latent TGF-|3 into extracellular matrix as LLC which in addition includes a 120-240 kDa glycoprotein called latent TGF-|3 binding protein (LTBP) [24]. LTBP is composed primarily of two kinds of cysteine-rich domains: EGF-like repeats (most of which are calcium-binding) and eight-cysteine domains [25]. LTBP participates in the regulation of latent TGF-|3 bioavailability by addressing it to the extracellular matrix (ECM) [26]. Non-active TGF-|3 stays in ECM; its further activation is a critical step in the regulation of its activity (Figure 1). A number of papers have reported TGF-|3 activation by retinoic acid and fibroblast growth factor-2 (FGF-2) in endothelial cells [27,28], or by endotoxin and bleomycin in macrophages [29]. Further, a variety of molecules is involved in TGF-|3 activation. Proteases including plasmin, matrix metaloproteases MMP-2 and MMP-9, are TGF-|3 activators in vitro [30,31]. Other molecules involved in the mechanism of activation are thrombospondin-1 [32], integrins, such as aV|36 or aV|38 [33,34], or reactive oxygen species (ROS). Moreover, latent TGF-|3 present in conditional medium is activated by acid treatment (pH 4.5) in vitro [35]. In vivo, a similar pH is generated by osteoclasts during bone resorption. Since the bone matrix deposited by osteoblasts is rich in latent TGF-|3, the acidic environment created by osteoclasts in vitro might result in latent TGF-|3 activation [36]. TGF-P receptors In most cells, three types of cell surface proteins mediate TGF-|3 signaling: TGF-|3 receptor I (T|3RI), II (TBRII) and III (TBRIII) [13,37]. Out of these three receptors, TBRIII, also called betaglycan, is the largest (250-350 kDa) and most abundant binding molecule. This cell-surface chondroitin sulfate / heparan sulfate proteoglycan is expressed on both fetal and adult tissues and most cell types [38]. Endoglin (CD 105) was shown to act as type III receptor for TGF-B as well [39]. Endoglin is a membrane, an RGD-containing glycoprotein, which is expressed in a limited set of cell types, primarily vascular endothelial cells, several hematopoietic cell types, bone marrow stromal cells and chondrocytes. Its expression strongly increases in active vascular endothelial cells upon tumor angiogenesis [40-42]. Moreover, in normal brain, it was found to be expressed in the adventitia of arteries and arterioles, and it is expressed on several types of tumor cells, such as invasive breast cancers and cell lines or renal cell carcinoma [43-45]. Although betaglycan and endoglin are co-receptors not directly Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 3 of 24 Figure 1 TGF-P synthesis and activation. TGF-fSs are synthesized as inactive precursors that contain pre-region (Signal peptide) and pro-region (N terminal peptide - LAP). Processing of inactive form starts with proteolytic cleavage that removes signal peptide from pre-pro-TGF-(3s form. After dimerization, TGF-fSs are cleaved by proteases (eg. Furin) into C-terminal mature peptides and N-terminal LAP (Latency Associated Peptide). TGF-fSs with LAP form small latent complexes (SLP) that are transported to extracellular matrix where can further covalently bind to latent TGF-(3 oinding protein (LTBP) to form a large latent complexes (LLC). LTBP is able to connect inactive TGF-p" forms to ECM proteins. This interaction is further supported by covalent transglutaminase-induced (TGase) crosslinks. Activation of TGF-p" starts with release of LCC from ECM by proteases. Then, the mature protein is cleaved from LTBP, which is provided in vitro by acidic condition, pH or plasmin or in vivo by thrombospondin (TSP). Once the active TGF-p" family member is released from the ECM, it is capable of signaling. involved in intracellular TGF-|3 signaling due to lack of kinase domain, they can control access of TGF-|3 to TGF-|3 receptors and consequently modulate intracellular TGF-|3 activity [46,47]. Betaglycan binds all three isoforms of TGF-|3, with higher affinity for TGF-|32; however, endoglin binds TGF-|3l and -|33 with constant affinity and has only weak affinity for TGF-|32 [39,48]. T|3RI and T|3RII mediate signal transduction. Both receptors are transmembrane serine/theronine kinases, which associate in a homo- or heteromeric complex and act as tetramers. They are organized sequentially into an N-terminal extracellular ligand-binding domain, a transmembrane region, and a C-terminal serine/threonine kinase domain. The type II receptors range from 85 to 110 kDa, while the type I receptors are smaller and their size ranges from 65 to 70 kDa [49]. Moreover, T|3RI contains a characteristic, highly conserved 30 amino acids long GS domain in the cytoplasmic part, which needs to be phosphorylated to fully activate T|3RI [36]. T|3RII contains 10 bp polyadenine repeat in the coding region of the extracellular domain. This region is frequently a target of changes leading to frameshift missense mutations or early protein terminations that result in truncated or inactive products [50]. TGF-p receptors activation Bioactive forms of TGF-|3s are dimers held together by hydrophobic interactions and, in most cases, by an inter-subunit disulfide bond as well. The dimeric structure of these ligands suggests that they function by bringing together pairs of type I and II receptors, forming heterote-trameric receptor complexes [51]. Binding of TGF-|3 to extracellular domains of both receptors also induces proper conformation of the intracellular kinase domains. These receptors are subject to reversible post-transla-tional modifications (phosphorylation, ubiquitylation and Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 4 of 24 sumoylation) that regulate stability and availability of receptors as well as SMAD and non-SMAD pathway activation. Receptor phosphorylation activates TGF-|3 signaling pathway - the ligand binds to T|3RII first, followed by subsequent phosphorylation of a Gly-Ser regulatory region (GS-domain) within T|3RI. This leads to incorporation of T|3RI and formation of a large ligand-receptor complex that consists of dimeric TGF-|3 ligand and two pairs of T|3RI and T|3RII [52]. The TGF-|3 receptor complex is extremely stable upon solubilization [53]. TGF-|3l and TGF-|33 bind to T|3RII without participation of type I receptor, whereas TGF-|32 interacts only with combination of both receptors (reviewed in [54]). Although ligand binding may induce autophosphorylation of T|3RII cytoplasmic domain, signaling in the absence of T|3RI has not been reported [49]. TpRIII betaglycan promotes binding of TGF-|32 to T|3RII, since the affinity of TGF-|32 to T|3RII is low in the absence of betaglycan [46]. Endoglin binds TGF-|3l, TGF-|33 but not TGF-|32 in the presence of the T|3RI and T|3RII. In some cell types, endoglin was found to inhibit TGF-|3 signaling - for example in chondrocytes, it enhances TGF-|3l-induced SMAD1/5 phosphorylation but inhibits TGF-|3l-induced SMAD2 phosphorylation [55]. Ubiquitylation and ubiquitin-mediated degradation define stability and turnover of receptors. Ubiquitylation occurs through sequential actions of El, E2 and E3 ubi-quitin ligases that provide specificity in the ubiquitylation process [56]. The E3 ubiquitin ligases such as Smurfl and SmurfiZ (SMAD ubiquitylation-related factor 1 and 2) regulate the stability of T|3RI and heteromeric TGF-|3 receptor complex [57,58]. Sumoylation, similarly to ubiquitylation, requires El, E2 and E3 ligases which results in SUMO polypeptide attachment. Although sumoylation has not been observed for any other transmembrane receptor kinases, it was shown to modify T|3RI function by facilitating the recruitment and phosphorylation of SMAD3 [59]. TGF-|3 receptors are also constitutively internalized via clathrin-dependent or lipid-raft-dependent endocytic pathways (reviewed in [60]). TGF-P signaling SMAD proteins The SMAD proteins are the only known latent cytoplasmic transcription factors that become directly activated by serine phosphorylation at their cognate receptors. SMADs can be classified into 3 groups based on their function: the receptor-regulated SMADs (R-SMADs), SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8; the common SMAD (Co-SMAD), SMAD4, and the inhibitory SMADs (I-SMADs), SMAD6 and SMAD7 (reviewed in [61]). R-SMADs and Co-SMAD consist of a conserved MH1 domain (Mad-homology-1) and C-terminal MH2 domain (Mad-homology-2), which are connected by a linker' segment. The C-terminal domain promotes transcriptional activity, when fused to a heterologous DNA binding domain [62]. On the contrary, I-SMADs contain only the highly conserved MH2 domain. The MH1 domain is responsible for binding to DNA; however, the MH2 domain contains hydrophobic patches also called hydrophobic corridors that allow binding to nucleopor-ins, DNA-binding cofactors and various cytoplasmic proteins, as well as interaction with receptors. Both domains can interact with sequence-specific transcription factors. SMAD3 and SMAD4 bind with their MH1 domain to SMAD-binding elements (SBE) on DNA, whereas the common splice form of SMAD2 does not bind to DNA (reviewed in [63]). I-SMADs function as intracellular antagonists of R-SMADs. Through stable interactions with activated serine/threonine receptors, they inhibit TGF-|3 family signaling by preventing the activation of R- and Co-SMADs. I-SMADs regulate activation of R-SMADs via binding with their MH2 domain to T|3RI, thereby competing with R-SMADs and preventing R-SMADs phosphorylation [64]. SMAD6 is also able to compete with SMAD4 for heteromeric complex formation with activated SMAD1 [65]. Whereas SMAD6 appears to preferentially inhibit BMP signaling, SMAD7 acts as a general inhibitor of TGF-|3 family signaling. Another possible mechanism of inhibition signaling transduction by I-SMADs is facilitated by HECT type of E3 ubiquitin lig-ase Smurfl and SmurfiZ [57,58]. Canonical signaling The SMAD pathway is the canonical signaling pathway that is activated directly by the TGF-|3 cytokines (Figure 2). T|3RI recognizes and phosphorylates signaling effectors - the SMAD proteins. This phosphorylation is a pivotal event in the initiation of TGF-|3 signal, followed by other steps of signal transduction, subjected to both positive and negative regulation. R-SMAD binding to the type I receptor is mediated by a zinc double finger FYVE domain containing protein SARA (The SMAD Anchor for Receptor Activation). SARA recruits non-activated SMADs to the activated TGF-|3 receptor complex [66]. However, TMEPAI (TransMembranE Prostate Androgen-Induced gene/pro-tein), a direct target gene of TGF-|3 signaling, perturbs recruitment of SMAD2/3 to T|3RI and thereby participates in a negative feedback loop to control the duration and intensity of SMADs activation [67]. Receptor-mediated phosphorylation of SMAD2 decreases the affinity of SMAD2 to SARA, leading to dissociation from SARA [68]. Afterwards, phosphorylated complex of Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 5 of 24 SMAD2/3 forms a higher-order complex with SMAD4 and moves to the nucleus. At this point, Smurfl interacts with R-SMADs in order to trigger their ubiquityla-tion and degradation and hence their inactivation [69]. Further, it was found that Smurfl and Smurf2 facilitate the inhibitory effect of I-SMADs. Smurf2 binding in the nucleus to SMAD7 induces export and recruitment to the activated T|3Rs, where it causes degradation of receptors and SMAD7 via proteasomal and lysosomal pathways [57]. Smurfl (specific for BMP-SMADs) also interacts with SMAD7 and induces SMAD7 ubiquityla-tion and translocation into the cytoplasm [58]. For proper translocation to the nucleus, the SMADs contain a nuclear localization-like sequence (NLS-like; Lys-Lys-Leu-Lys) that is recognized by importins [70]. Interestingly, the nuclear translocation of SMADs was also described in vitro to occur independently of added importin-like factors, because SMAD proteins can directly interact with nucleoporins, such as CAN/Nup214 [71,72]. Complex of SMAD2/3 and SMAD4 is retained in the nucleus by interactions with additional protein binding partners and DNA. Dephosphorylation and dissociation of SMAD transcriptional complexes are thought to end this retention, allowing export of R-SMADs out of the nucleus [73]. Different protein binding partners provide another venue for regulatory inputs controlling the activity of SMADs. Each SMAD-partner combination targets a particular subset of genes and recruits either transcriptional co-activators or co-repressors. Members of many DNA-binding protein families participate as SMADs cofactors, such as FOX, HOX, RUNX, E2F, API, CREB/ATF, Zinc-finger and other families. The SMAD cofactors differ in various cell types, thereby determining the cell-type dependent responses [63]. By association with DNA-binding cofactors, SMADs reach target gene specificity and target specificity. Stimulation of various cells by TGF-|3 leads to rapid activation or repression of a few hundred genes; possibly, the pool of activated SMAD proteins is shared among different partner cofactors [74,75]. On chromatin level, SMADs can recruit histone acet-yltransferases. Several studies revealed that TGF-|3 proteins influence transcription of different genes through interaction of the MH1 domain of SMADs with sequence-specific transcription factors and co-activators CBP and p300. CBP and p300 interact with SMAD1, SMAD2, SMAD3 and SMAD4 in vitro and in vivo, and the interaction between the SMADs and CBP/p300 is stimulated in response to TGF-|3 [76-79]. Moreover, histone deacetylases and chromatin remodeling complexes are also involved in SMAD regulation. In this way, SMADs functionally interact with a variety of transcription factors and regulate diverse signaling pathways as well (reviewed in [80]). SMADs act as sequence specific transcription factors; however, they can regulate cell fate by alternative mechanisms. Recent data indicate that R-SMADs Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 6 of 24 associate with the p68/Drosha/DGCR8 miRNA processing complex to regulate miRNA processing in a ligand-dependent and RNA-sequence specific manner. So far, more than 20 TGF-|3/BMP-regulated miRNAs (T7B-miRs) have been described [81,82]. Non-SMAD signaling Diversity of TGF-|3 signaling in cells is determined not only by various ligands, receptors, SMAD mediators or SMAD-interacting partners, but also by the ability of TGF-|3 to activate other signaling pathways (Figure 3). TGF-|3 can indirectly participate in apoptosis, epithelial to mesenchymal transition, migration, proliferation, differentiation and matrix formation (reviewed in [83]). It activates various branches of mitogen-activated protein kinases (MAPK) pathway, such as ERK1/ERK2, Jun-N terminal kinase (JNK) and p38 and PI3K kinases [84]. In response to TGF-|3, both SMAD-dependent and SMAD-independent JNK activations are observed [85]. SMAD-independent activation of p38 was observed in mouse mammary epithelial NMuMG cells with mutant T|3RI [86]. Other pathways influenced by TGF-|3 are the growth and survival promoting pathway AKT/PKB, the small GTP-binding proteins RAS, RHOA, RAC1 as well as CDC42 and mTOR [87-89]. TGF-|3 participates in mediating activation of protein tyrosine kinases FAK, SRC and ABL, particularly in mesenchymal or dedifferentiated epithelial cells [90-92]. TGF-|3 also influences NF-kB signaling and Wnt/|3-catenin pathway [93]. Role of TGF-P in tumors In tumors, TGF-|3 can be either a proto-oncogene or a tumor suppressor, depending on cell context and tumor stage [94]. Cancer cells often evade growth inhibition effects of TGF-|3, while leaving intact TGF-|3-mediated cellular responses that promote tumor progression. Importantly, the use of mouse models has enabled the elucidation of the dual role of TGF-|3 in cancer (reviewed in [95]). As homozygous deletions of Tgf-fil, Tgf-02, Tgf-03, TfiRI and TfiRII are lethal in mice, manipulation of TGF-|3 pathway was achieved mainly through transgene expression or conditional null mutations in vivo [96]. The dual role of TGF-|3 was shown on a set of experiments with mice skin cancer. The first study demonstrated that TGF-|3l expression targeted to keratinocytes inhibits benign tumor outgrowth; however, later it enhances malignant progression rate and pheno-type of the benign papillomas [97]. Study on transgenic mice overexpressing a dominant negative T|3RII in the basal cell compartment and in follicular cells of the skin complemented previous results. In non-irritated epidermis of transgenic mice, proliferation and differentiation were normal; however, during tumor promotion, transgenic mice showed an elevated level of proliferation in the epidermis [98]. Furthermore, using mice with inducible expression of TGF-|3l in epidermis confirmed the dual role of TGF-|3 [99,100]. TGF-p as a tumor suppressor The most critical effect of TGF-|3 on target cells is suppression of proliferation. Its growth inhibitory function Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 7 of 24 is based on the ability to suppress expression and function of c-Myc and cyclin-dependent kinases (CDKs) and to enhance expression of the CDK inhibitors pl5 [101] [102] and p27KIP1 [103]. Cellular responses to TGF-|3 depend on cell type and physiological conditions. TGF-|3 stimulates various mesenchymal cell types, including fibroblasts; however, it is a potent inhibitor of epithelial, endothelial, neural cells and hematopoietic cells, including immune cells [10]. Central function of TGF-|3 is inhibition of cell cycle progression by regulating transcription of cell cycle regulators (Figure 4). Anti-proliferative responses can be induced at any time during cell cycle division; yet, they are effective only in Gl phase. Once a cell is committed to enter replication, it will continue to double its DNA, divide and then arrest when entering the following Gl phase. At this point, TGF-|3 mediates cell cycle arrest by suppressing expression and function of c-Myc, members of the Id family inhibitors and CDKs and enhancing expression of CDK inhibitors, such as pl5INK4B, p21CIP1 and p27KIP1 [104,105]. TGF-|3 induces the expression of the CDK inhibitor pl5 in a variety of cell types. pl5 is a member of the INK4 family of CDK inhibitors, which binds to CDK4 and CDK6 subunits, inactivates their catalytic activity and prevents cyclin D-CDK4/6 complex formation [101,106]. Furthermore, TGF-|3 can induce expression of p21CIP1 in several cell types [107,108]. Other CDK inhibitory responses, observed in several cell types after exposure to TGF-|3, are inhibition of CDK4 expression and down-regulation of CDC25A expression [109]. Low levels of c-Myc allow for TGF-|3 induced transcription of pl5 and p21 genes. Decreased expression of c-Myc in keratinocytes is mediated by SMAD3 in association with transcription factors E2F4 and E2F5, pl07 co-repressor and SMAD4 [110]. On the other hand, down-regulation of Id proteins in epithelial cells is due to activated SMAD3 that induces activating transcription factor (ATF) expression and then together with ATF directly represses the Id promoter [104]. TGF-(J as a tumor promoter TGF-|3 acts as tumor suppressor in normal epithelium; it inhibits cell proliferation and induces apoptosis. Yet, during tumor progression, sensitivity to these effects of TGF-|3 is frequently lost and, in later stages, TGF-|3 signaling has pro-oncogenic function. Several activities have been described to TGF-|3 that would favor tumor progression [111]. Mutations in signaling components Malignant cells become resistant to suppressive effects of TGF-|3 either through mutation and/or functional in-activation of TGF-|3 receptors or by downstream alterations in the SMAD-signaling pathway. During late stages of tumor progression, TGF-|3 acts as tumor promoter and is often over-expressed in many cancers. Elevated plasma level of TGF-|3l was observed in hepatocellular Figure 4 Role of TGF-0 in regulation of cell cycle. Physiologically, TGF-p" is a potent inhibitor of cell cycle; it induces expression of p15 4 and represses expression of c-Myc. p15INK4B js able to prevent cyclin D-CDK4/6 complex formation; moreover, it displaces p21clpl and p27KIP from cyclin D-CDK4/6 complexes. These CIP/KIP inhibitors are subsequently able to inactivate other complexes of G1 and S phase and thereby inhibit cell cycle. Moreover, low levels of c-Myc allows for TGF-p" induced p15INK4B and p21clpl transcription. Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 8 of 24 carcinoma, colon, HCC, prostate, lung and breast cancers and correlates with poor prognosis [112]. Mutations in downstream TGF-|3 signaling components cause variable attenuations or complete loss of expression; these mutations, which have been detected in many common tumors, affect TGF-|3 signal transmission that potentially results in human cancer development and progression. In particular, T|3RI, T|3RII, SMAD2 and SMAD4 are frequently lost, mutated or attenuated (gene/LOH/expression). Inactivation of T|3RII leads to increased tumor spreading and metastasis in a variety of carcinomas, including colon [113], breast [114], pancreatic [115], intestinal [116] or head and neck squamous cell carcinoma (HNSCC) [117]. Also, deregulated expression or aberrant function of Smurfl and 2 was described. Several human carcinoma cell lines such as colon HT-29, breast MDA-MB-231, gastric MKN-1 and ovarian OVCAR-5 display high levels of one or more E3 ligases, including Smurf2 [118,119]. Moreover, in esophageal squamous carcinoma, high expression levels of Smurf2 associated with low levels of SMAD2 phosphorylation were detected [120]. Furthermore, TGF-|3 pathway is modulated by epigenetic mechanisms, such as transcriptional repression of TjiRII, DNA methylation of TjiRI and TjiRII and histone modifications [121-123]. TGF-B in tumor microenvironment and metastases Tumor metastases accounts for the majority of cancer associated deaths. Recent evidence strongly suggests that tumor microenvironment is essential in this process. It consists of tumor cells and a variety of immune cells, which infiltrate into tumors. This dynamic microenvironment is not only important for cross-talk with tumor cells or escape of tumor from host immune surveillance, but it also induces formation of new blood vessels and invades the vasculature. Areas of hypoxic tissue are thought to drive genomic instability and alter DNA damage repair [124]. Recent studies suggest that TGF-|3 is one of the critical regulators of inflammation; it is thought that tumor metastasis is a coordinated process between tumor cells and host cells through inflammation [125]. However, it seems that different mechanisms are implemented in different tumor type. TGF-|3 as a proto-oncogene is important in stromal-epithelial cross-talk, as was shown for the first time in mouse experiments, where deletion of the T|3RII in stromal fibroblasts resulted in transformation of adjacent epithelia of prostate and forestomach. Moreover, in this model, hepatocyte growth factor (HGF) was up-regulated and complementary activation of the HGF receptor MET was detected in tissues where T|3RII had been ablated, which implicates this paracrine signaling network as a potential mechanism for regulation of carcinoma development [126]. Further experiment performed on these mice revealed that mice fibroblasts have up-regulated expression of growth factors and increased proliferation of mammary cancer cells [127]. Together, it indicates that TGF-|3 responses mediated by stromal fibroblasts can regulate carcinoma initiation and progression of adjacent epithelium in vivo and in vitro. Interestingly, it was found that TGF-|3 in breast cancer favors metastasis to lungs. TGF-|3 stimulation of mammary carcinoma cells in tumor microenvironment, before they enter circulation, primes these cells for seeding of lungs through a transient induction of angiopoetin-like4 (Angptl4) via canonical signaling pathway [128]. TGF-|3 is involved in regulation of chemokines and che-mokine receptors which take part in inflammatory cells recruitment. The loss of T|3RII in breast cancer cells can enhance recruitment of F4/80+ cells to tumor micro-environment and increase the expression of proinflammatory genes, including CXCL1, CXCL5 and PTGS2 (cyclooxygenase-2). Further, in vitro treatment of carcinoma cells with TGF-|3 suppressed the expression of CXCL1, CXCL5 and PTGS2 [129]. Different mechanism was observed in gastric carcinoma, where SMAD-dependent TGF-|3 pathway, in collaboration with PKC-S expression and phosphorylation and integrin expression and activation, regulates cell invasion and cell spreading [130]. Beside the effects already mentioned, TGF-|3 is broadly implemented in induction of epithelial-to-mesenchymal transition [131]. The NBT-II cell line, derived from a chemically induced rat bladder carcinoma, forms epithelial colonies that can be converted into migratory mesenchymal cells within a few hours by adding Tgf-|3 and other factors, such as Fgfl, Fgf7, FgflO, Egf, Igfl, Igf2 or Hgf [132]. TGF-B as a regulator of immune cells The tumor microenvironment is filled with various inflammatory cells, including myeloid cell subpopulations, T cells and B cells. TGF-|3 is one of the most potent endogenous negative regulators of hematopoiesis. It modulates proliferation, differentiation and function of all types of lymphocytes, macrophages and dendritic cells, thus regulating the innate, non-antigen-specific as well as antigen-specific immunity [133]. TGF-|3 is involved in normal B cells maturation and differentiation, such as regulation of expression of cell-surface molecules, inhibition of IgM, IgD, CD23 and the transferrin receptor and induction of MHC class II expression on pre-B cells and mature B cells [134]. In T cells, TGF-|3 regulates maturation; for example, it is released by regulatory T cells and inhibits the Ag-specific proliferation of naive CD4+ cells from T cell receptor (TCR) [135]. TGF-|3l also inhibits aberrant T cell expansion by maintaining intracellular calcium Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 9 of 24 concentration levels low enough to prevent mitogenic response by Ca ^independent stimulatory pathways [136]. In myeloid cells, such as macrophages and monocytes, TGF-|3l is mostly suppressive, it inhibits cell proliferation and down-regulates production of reactive oxygen and nitrogen intermediates; however, it is able to enhance some other activities of myeloid cells. TGF-|3l can be recognized by monocytes and macrophages as a chemotactic factor; it induces direct monocytes migration in vitro [137]. TGF-|3 pro-metastatic and pro-inflammatory effects are regulated via nuclear factor kappa B (NF-kB), the master regulator of inflammation and a regulator of genes that controls cell proliferation and cell survival. TGF-|3l is a negative regulator of NF-kB activation, as was shown in the gut; it directly stimulates IkB-cc promoter transcriptional activity in vitro. However, SMAD7 maintains high NF-kB activity by blocking TGF-|3l signaling [138]. Targeting the TGF-0 signaling pathway As the signaling pathway deregulations are responsible for cancer initiation and progression, interrupting the tumor promoter properties of TGF-|3 signaling would be an attractive therapeutic strategy, without altering physiologic tumor suppressor functions exhibited in early stages of tumorigenesis. Strategies such as using monoclonal TGF-|3-neutralizing antibodies, large molecule ligand traps, reducing translational efficiency of TGF-|3 ligands using antisense technology and antagonizing TGF-|3 receptor I/II kinase function by small molecule inhibitors are the most prominent methods being explored today [139,140]. Furthermore, studies have shown that combined treatment with tumor cell vaccines and antisense TGF-|3 therapy reduced tumor size and increased survival benefit [141,142]. Preclinical studies also show that TGF-|3 inhibition can augment therapeutic efficacy of cytotoxic agents [143]. However, as there are still potential limitations and risks of TGF-|3 targeted therapy, caution must be given as to when, how and how much therapy would be beneficial or how much toxicity will be induced by chronically administered therapy. However, daily administration of a high dose of neutralizing TGF-|3 antibody in adult mice for 12 weeks and a lifetime exposure to soluble T|3RII (sT|3RII) in transgenic mice did not significantly affect their health. This suggests that anti-TGF-|3 treatments are likely to be safe [144]. TGF-P in solid tumors Brain tumors TGF-|3 has a suppressive role in physiological development of the central nervous system (CNS): all TGF-|3 isoforms and receptors necessary for TGF-|3 signal transduction are detected in developing as well as adult CNS [145]. The most aggressive type of primary brain tumors, glioblastoma multiforme (GBM), is characterized by poorly differentiated and highly proliferating cells that originate from glial cells [146,147]. Here, the release from cytostatic TGF-|3 effect is explained by a broad range of inactivating mutations in the TGF-|3 signaling pathway. Several studies describe mutations in T|3RI and T|3RII in adenomas and gliomas [148,149] as well as correlation between higher expression of T|3RI and T|3RII with more aggressive glioma cell lines and tumors [150,151]. Moreover, high levels of TGF-|3 indicate that TGF-|3 is able to induce its own expression and thereby create a malignant autocrine loop and control glioma-cell proliferation [152]. Alterations of SMAD protein levels and activation were reported in brain tumor cell lines and patient samples. In glioma cell lines, SMAD3 level and SMAD2 nuclear translocation was lower in 9 out of 10 cell lines [153]. Kjellman et al. reported that SMAD2, SMAD3 and SMAD4 mRNA levels were reduced in GBM samples in comparison to normal brain samples, astrocytomas and anaplastic astrocytomas [150]. Nevertheless, these data are controversial to a study in which higher phospho-SMAD2 (p-SMAD2) level correlated with higher grade of glioma [154]. Further analysis of cell lines and patient samples would elucidate such discrepancies. Urogenital tumors TGF-|3 is a crucial molecule in the genesis of urogenital tumors, such as urinary bladder carcinoma, renal cell carcinoma, ovarian and prostate cancers [155]. The TGF-|3 pathway is involved in urinary bladder cancer progression. The amount of secreted TGF-|3l correlates with more aggressive phenotype of cell lines. In addition, deregulated TGF-|3 signaling led to enhanced migration and invasiveness of bladder cancer cells [156]. Silencing of T|3RI expression by siRNA led to significant inhibition of TGF-|3-induced signal transduction and thereby reduced invasiveness of bladder cancer cells [157]. Clear cell renal cell carcinoma (CCRCC) is the most common malignancy of the kidney; it accounts for 2-3% of all malignant diseases in adults [158]. In CCRCC patient samples, sequential loss of TpRIII and T|3RII expression was associated with renal cell carcinogenesis and progression [155]. Cross-talk between Notch signaling and TGF-|3 pathway contributes to aggressiveness of CCRCC. Recently, it was described that inhibition of Notch signaling leads to attenuation of basal TGF-|3-induced signaling in CCRCC cells; it also influenced genes involved in cancer migration [159]. Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 10 of 24 Ovarian cancer In advanced ovarian tumors, low expression of TGF-|3l mRNA is connected to better prognosis. It was found that TGF-|3l mRNA expression was significantly lower in tumors of patients who had optimal surgery than in patients with suboptimal surgery. TGF-|3l mRNA expression was also significantly lower in tumors with high sensitivity to chemotherapeutics than in those with low sensitivity [160]. Alterations in the TjiRI gene occur in ovarian cancer and account, at least in part, for the frequent loss of TGF-|3 responsiveness of these cancer cells. Presence of TjiRI 6*A allele in about 27% of human ovarian cancers suggests that it acts as a low penetrating tumor marker in the development of ovarian cancer [161-163]. Mutations in the TjiRII allele that cause loss or decrease in T|3RII protein level are also present, BAT-RII mutations (mutations in polyadenine tract in exon 3) were found in 22% of ovarian tumors [161]. Although this mutation is connected to microsatellite stability, in ovarian cancers this association remains controversial [164]. Mutations in SMAD4 are not very common in ovarian cancer but were reported in primary cultures or cell lines [165]. Reduced expression or loss of SMAD4 protein leads to decreased ability to bind DNA; SMAD4 in-activation is involved in the acquisition of a more aggressive tumor [161]. It has been suggested that SMAD4 and SMAD3 are involved in metastatic potential of ovarian cancers [166,167]. In ovarian cancer cell lines, TGF-|3 supported metastatic activity at least partly through activation of MMPs [168]. Deregulation in TGF-|3/SMAD4 signaling leads to epigenetic silencing of a putative tumor suppressor, RunXlTl, during ovarian carcinogenesis [169]. Recently, genome-wide screening done by ChlP-seq of TGF-|3-induced SMAD4 binding in epithelial ovarian cancer revealed that SMAD4-dependent regulatory network was strikingly different in ovarian cancer compared to normal cells and was predictive of patients survival [170]. Prostate cancer In prostate cancer, high level of TGF-|3l expression is linked to tumor progression, cell migration and angio-genesis [171]. In some prostate cell lines, even low level of TGF-|3l induced its own expression in an autocrine manner. However, only in benign cells, higher concentration of TGF-|3l leads to recruitment of protein phosphatase 2A (PP2A) by activated T|3RI, which terminates the induction of TGF-|3l. On the contrary, in malignant cells, incorrect recruitment of PP2A by T|3RI is responsible for protruded production of TGF-|3l [172]. When compared to other types of cancer, such as breast and colon, down-regulation of T|3Rs is found more often than mutations in SMADs. Kim et al. compared protein levels of T|3RI and T|3RII in benign and malignant prostate tissues and observed that loss of receptors expression correlated with more advanced tumor [173]. Decreased level of receptor protein is accompanied with decreased mRNA expression; thereby, loss of receptor expression is a potential mechanism to escape the growth-inhibitory effect of TGF-|3 [174]. However, mutations are present in only some cases of prostate cancer, which suggests that other mechanisms are involved. For example, in a study by Turley et al, loss of TpRIII expression correlated with disease progression [175]. In some cases of prostate cancer, insensi-tivity to TGF-|3 is caused by promoter methylation in T|3RI [176]. So far, mutations in SMAD2 proteins were not found in prostate cancer. However, studies in vitro revealed that SMAD2 functions as a tumor suppressor of prostate epithelial cells. It is possible that tumor suppressor function of SMAD2 could be lost during differentiation of normal tissues or during prostatic carcinogenesis [177-179]. Breast cancer In normal mammalian breast development, all TGF-|3s isoforms are functionally equivalent; they are all involved in establishing proper gland structures and apoptosis induction. However, they have distinct roles in mammary growth regulation, morphogenesis and functional differentiation [180-182]. In breast cancer, results evaluating TGF-|3 as a prognostic factor are controversial. On the one hand, analysis demonstrated TGF-|3l expression to be significantly higher in patients with a favorable outcome as compared to patients with a poor prognosis [183]. On the other hand, several studies showed that TGF-|3 over-expression is related to worse outcome [184,185]. Elevation of TGF-|3 has been shown to participate in breast cancer metastasis [186]. Alterations of TGF-|3 signaling molecules are relatively rare, except for T|3RII down-regulation. No specific mutations were found in the coding or in the regulatory region of the T|3RII gene promoter in breast cancer [187,188]. However, the loss of T|3RII expression has been linked to tumor progression and metastasis, principally in HER2-negative patients [114]. In addition, resistance of breast cell lines to TGF-|3 may be due to reduced expression of T|3RII [189]. Mutations of T|3RII are rare among breast cancer patients, while changes in receptor expression may take part in tumor progression [187]. Opposite to T|3RII, intragenic mutations occur in T|3RI and are associated with metastatic breast cancer [190]. Although the role of TpRIII remains unclear, it seems that this receptor is a suppressor of breast cancer. Loss of TpRIII through allelic imbalance is a frequent genetic Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 11 of 24 event during human breast cancer development that increases metastatic potential; moreover, decreased TBRIII expression correlates with decreased recurrence-free survival in breast cancer patients [191]. Mutations in downstream signaling pathway including SMAD proteins are not very common in breast cancer; however, inactivating mutations or loss of expression in SMAD4 have been described [164,192]. Tumors of the digestive tract Gastric cancer Resistance to TGF-B is a hallmark of gastric cancer. The relationship between TGF-B resistance and up-regulated level of miR-106b-25 cluster (miR-106b, miR-93, and miR-25) has been recently elucidated [193]. The cluster is an intronic part of the Mcm7 gene and thus is regulated by E2F1. Conversely, miR-106b and miR-93 control E2F1 expression thus establishing negative feedback that prevents E2F1 self-activation. Over-expression of miR-106b, miR-93 and miR-25 decreases response of gastric cancer cells to TGF-B since they interfere with synthesis of TGF-B downstream effectors that promote cell cycle arrest and apoptosis, such as p21 and BIM, respectively [193] (Figure 5). Mutations in TBRII that lead to insensivity of cell lines to TGF-B mediated growth inhibition have been previously described [194]. It has been shown that conditional loss of TGF-B signaling due to dominant negative mutation in TBRII leads to increased susceptibility to gastrointestinal carcinogenesis in mice [195]. Epigenetic changes in TBRI are another important mechanism of escape from TGF-B physiological function. Hypermethylation of a CpG island in the 5' region of the TBRI was found in 80% of gastric cancer cell lines and 12.5% of primary tumors. Treatment with demethy-lating agent increased expression of TBRI and transient transfection of TBRI into TGF-B resistant cell line restored TGF-B responsiveness [123]. Effects of TGF-B on gastric cancer invasiveness and metastasis are mediated by activation of JNK and ERK pathways which support expression of fascin-1, an actin-binding protein. Moreover, signaling pathway based on SMAD proteins is not involved in this process because transitional repression of SMADs did not alter fascin-1 expression [196]. Nevertheless, impaired signaling based on SMAD proteins also occurs in gastric cancer. Shinto et al. found a correlation between expression level of p-SMAD2 and patients prognosis. P-SMAD2 protein expression level was significantly higher in patients with diffuse form of carcinoma and metastatic tumors and is associated with worse outcome [197]. TGF-B signaling is also abrogated by decreased expression of SMAD3. Low or undetectable level of SMAD3 was observed in 37.5% of human gastric cancer tissues. In cell lines, which showed deficient expression of SMAD3, introduction of SMAD3 gene led to growth inhibition caused by TGF-B [198]. Sonic hedgehog (Shh), a member of the hedgehog signaling pathway, promotes invasiveness of gastric cancer through TGF-|3-mediated activation of the ALK5-SMAD3 pathway. Higher concentrations of N-Shh (human recombinant form of Shh) enhanced cell motility and invasiveness in gastric cancer cells; moreover, treatment of cells with N-Shh led to enhanced TGF-Bl secretion, TGF-B-mediated transcriptional response, expression of ALK5 protein and phosphorylation of SMAD3. Effect of Shh on cell motility was not observed after treatment of cells with anti-TGF-B blocking antibody or TGF-Bl siRNA [199]. Hepatocellular carcinoma Reduced TBRII expression was observed in approximately 25% of hepatocellular carcinoma (HCC) patients; this event is associated with aggressive phenotype of HCC and intrahepatic metastasis. TBRII down-regulation also correlated with an early recurrence time and higher grade of tumor suggesting that TBRII down-regulation is a late event in HCC development. In addition, TGF-B is a tumor suppressor in the majority of HCCs expressing TBRII [200]. Mutations in intracellular signaling components have been observed: SMAD2 mutations occur in 5% of HCC, while loss of SMAD4 expression was found in 10% of HCC [201,202]. Several studies of HCC indicated that over-expression of SMAD3 promotes TGF-B-induced apoptosis [203,204]. Pro-apoptotic activity of SMAD3 requires both input from TGF-B signaling and activation of p38 MAPK, which occurs selectively in liver tumor cells. SMAD3 represses transcription of an important apoptotic inhibitor, BCL-2, by directly binding to its promoter [203]. Therapeutic options for patients with HCC are still limited; however, it was recently described that blocking the TGF-B signaling pathway with LY2109761, a kinase inhibitor of TBRI, is associated with inhibition of molecular pathways involved in neo-angiogenesis and tumor growth. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to significant reduction of HCC growth and dissemination. Currently, LY2109761 is being tested in clinical trial phase II [205-207]. Colorectal cancer In colorectal cancer (CRC), TGF-Bl inhibits proliferation of less aggressive tumor cells but stimulates growth of tumor cells at later stages by autocrine manner. High level of TGF-Bl correlates with tumor progression [208]. In colorectal cell lines, TGF-B induces proliferation by Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 12 of 24 transcription control of expression Figure 5 The E2F1/miR-106b-25/p21 pathway. In gastric cancer, miR-106b-25 cluster is activated by E2F1 in parallel with its host gene, Mcm7. In turn, miR-106b and miR-93 regulate E2F1 expression, establishing a miRNA negative feedback loop. Over-expressed miR-106b, miR-93, and miR-25 inhibit the synthesis of p21clpl and Bim (TGF-(3 downstream effectors) and therefore prevent cell cycle inhibition and apoptosis. RAS-independent manner [209]. In a recent study, TGF-|3, T|3RI, TBRII, SMAD4, pSMAD2/3 and E-cadherin were found to be closely related to TNM stage of CRC. Therefore, TGF-B, TBRII, SMAD4, pSMAD2/3 and E-cadherin come into view as valuable independent bio-markers of prognosis in CRC patients [140]. Inactivating mutations in SMAD2 and SMAD4 are frequent especially in pancreatic and colorectal carcinomas, although they do not stand for the most frequent tumor changes. Most of SMAD2 mutations have been found in the MH2 protein domain, thereby preventing complex formation with SMAD3 and SMAD4. Alterations of SMAD2 are present in about 6% of colorectal carcinoma cases [210]. SMAD3 mutation is a very rare event in human solid tumors; however, a missense mutation in SMAD3 gene (leading to reduced activity of SMAD3 protein) was found in human colorectal cell lines [211]. Inactivation of SMAD4 is a genetically late event in gastrointestinal carcinogenesis. It was identified with less frequency in advanced colon cancers and in 16% of colon carcinomas [212,213]. Nevertheless, recent studies revealed that some of the TGF-B induced pathways are SMAD4 independent [214]. Proteomic screen of SMAD4 wt and SMAD4 deficient cell lines detected different protein levels in cell lines pointing to SMAD4 dependent and independent TGF-B responses in colon carcinoma cells [215]. Another study indicated that novel genetic variant -4 T(10) in the SMAD4 gene promoter affects its activity. Obtained preliminary results indicate that SMAD4 gene promoter haplotype -462 T(14)/-4 T(10) represents a potentially relevant genetic marker for pancreatic and colorectal cancer [216]. This downstream inactivation of TGF-B signaling components promotes colon adenoma to carcinoma progression. Mutations of TBRII are frequent alterations of the TGF-B signaling pathway (reviewed in [217]). They are present in approximately 30% of CRC cases and were reported in cancer cell lines, sporadic colon cancers and patients with hereditary non-polyposis colorectal cancer with microsatellite instability and in a smaller percentage in microsatellite stable cancers [123,218,219]. TBRII mutations occur in >90% of microsatellite unstable (MSI) colon cancers and most principally affect a polya-denine tract in exon 3 of TBRII, the BAT-RII; however, non-BAT point mutations in TBRII were found with less frequency also in microsatellite stable cancers [164,219]. Interestingly, it has been recently published that restoration of TBRII in cancer cell lines with microsatellite instability (MSI), bearing mutated TBRII, promoted cell survival and motility. Therefore, it is plausible that such mutations contribute to favorable outcome in MSI patients [220]. In contrast to TBRII, mutations in TBRI are less common. They are rare in colon as well as pancreatic cancer. Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 13 of 24 Decreased T|3RI allele expression is associated with higher risk of colon cancer development [221]. Recently, it has been described that TpRIII mRNA expression is not significantly altered in human colorectal cell lines; however, protein levels of TpRIII are frequently increased, suggesting a distinct role for TpRIII in colon cancer. Thus, enhanced expression of TpRIII is possibly involved in cancer progression [222]. Other mechanisms, such as crosstalk between TGF-|3 and Wnt/|3-catenin pathways, are involved in colon cancer progression [214]. It has been shown that SMAD4 restoration is associated with suppression of Wnt/|3-catenin signaling activity, decrease of |3-catenin/Tcf target genes expression and with induction of functional E-cadherin expression [223]. Recently, the role of microRNA in colon cancer has been established. Elevated levels of miR-21 and miR-31 promote motility and invasiveness of colon cancer cell line and enhance the effect of TGF-|3. It seems that miR-21 and miR-31 act as downstream effectors of TGF-|3 [224]. Pancreatic cancer Pancreatic cancer has the poorest prognosis among GI cancers due to aggressiveness, frequent metastases and resistance to treatment. SMAD4, also called DPC4 (deleted in pancreatic carcinomas), suggests close relationship between loss of this gene and pancreatic cancer. Mutation or deletion of SMAD4 is a well-characterized disruption in the TGF-|3 pathway - it occurs late in neoplastic progression, at the stage of histologically recognizable carcinoma. In pancreatic cancers, SMAD4 is homozygously deleted in approximately 30% of cases, inactivated in 20%, while allelic loss of the whole 18q region was found in almost 90% of cases [225]. These mutations are present mostly in the MH2 domain; however, missense, nonsense or frame-shift mutations are present within the MH1 domain as well [226,227]. Dual role of SMAD4 was established in a mouse model. Smad4 or TjiRII deletion in pancreatic epithelium did not affect pancreatic development or physiology. However, when activated K-Ras was present in cells, loss of Smad4 or TjiRII or Smad4 haploinsuffi-ciency led to progression to high-grade tumors. Thus, it is possible that Smad4 mediates the tumor inhibitory action of TGF-|3 signaling, mainly in the progressive stage of tumorigenesis [115]. In concordance with colorectal cancer, mutations in T|3RII were found in cancers with microsatellite instability; however, mutations in T|3RII and also in T|3RI are less common [217]. Frequency of mutations in TjiRII is about 4% and even less for TjiRI [228]. Interestingly, polymorphism within the TjiRI gene, which is less effective in mediating anti-proliferative signals than wild type, was described [229]. High level of TGF-|3 was found in serum of patients with pancreatic adenocarcinoma suggesting that TGF-|3 could possibly become a marker for monitoring disease activity [230]. As previously mentioned in HCC, targeting T|3RI/II kinase activity in pancreatic cancer with the novel inhibitor LY2109761 also suppressed pancreatic cancer metastatic processes. LY2109761 suppressed both basal and TGF-|3l-induced cell migration and invasion and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival and reduced spontaneous abdominal metastases [231]. Lung cancer In non-small cell lung carcinoma (NSCLC), elevated expression of TGF-|3 correlates with disease progression [232]. Furthermore, significantly higher serum concentrations of TGF-|3l cytokine were found in lung cancer patients. Presumably, elevated expression and higher levels of serum TGF-|3 represent an important prognostic factor that could serve as a complementary diagnostic test in lung cancer detection [233]. Defective expression of T|3RII was observed in primary NSCLC, where T|3RII acts as a tumor suppressor. Down-regulation of T|3RII on transcriptional level could be explained by aberrant methylation of the TjiRII promoter [234]. Moreover, reduced expression of TpRIII has been found in NSCLC cells compared to normal human bronchial epithelial cells [235]. Downstream components of TGF-|3 signaling pathways are important in NSCLC development. Jeon et al. observed a correlation between better tumor-related survival and absence of SMAD6. Moreover, SMAD6 contributes to lung cancer progression by limiting TGF-|3-mediated growth inhibition of cell lines, which was proven by knockdown of SMAD6 that resulted in increased apoptosis in lung cancer cell line [236]. TGF-|3 signaling is also required for lung adenocarcinoma (LAC) progression. In a study on LAC cell line A549, knockdown of T|3RII resulted in suppression of cell proliferation, invasion and metastasis and induced cell apoptosis [237]. TGF-P in hematological malignancies Leukemia Myeloid leukemia TGF-|3 is a potent inhibitor of human myeloid leukemia cells [238]. In acute myeloid leukemia (AML), t(8;21) translocation results in the formation of a chimeric transcription factor AML1/ETO. Jakubowiak et al. used transient transfection assays and a reporter gene Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 14 of 24 construct that contained SMAD and AML1 consensus binding sequences and demonstrated that AML1/ETO represses basal promoter activity function and blocks response to TGF-|3l. AML1/ETO possibly binds to SMAD3, instead of activating TGF-|3l signaling pathway. It represses TGF-|3l-induced transcriptional activity and blocks TGF-|3l signaling, thus contributing to leukemia genesis [239]. In addition, in AML, dominant negative mutations in SMAD4 were found. They are characterized by a mis-sense mutation in the MH1 domain and a frameshift mutation in the MH2 domain of SMAD4. Mutated SMAD4 lacks transcriptional activity [240]. The t(3;21) translocation fusion product AML1/EVI-1 likely interacts with SMAD3 through the first zinc finger domain, represses SMAD3 activity by preventing SMAD3 from interacting with DNA, thereby repressing TGF-|3-mediated growth suppression in hematopoietic cells. This way, AML1/EVI-1 contributes to leukemogenesis [241]. In acute promyeloytic leukemia (APL), t(15;17) translocation in which the retinoic acid receptor a (RARa) gene on 17ql2 fuses with a nuclear regulatory factor PML on 15q22 results in the fusion protein PML-RARa [242]. PML is normally found in 2 isoforms, a nuclear isoform and a cytoplasmic isoform. Cytoplasmic isoform is required for association of SMAD2/3 with SARA and for the accumulation of SARA and TGF-|3 receptors, resulting in SMAD phosphorylation (Figure 6). The PML-RARa oncoprotein antagonizes with cytoplasmic PML function by withdrawing cytoplasmic PML from the SMAD/SARA/T|3RI/T|3RII complex resulting in defects in TGF-|3 signaling [243]. In chronic myeloid leukemia (CML), t(9;22) (the so-called Philadelphia chromosome) results in the formation of BCR-ABL fusion gene [244]. The fusion protein is an active tyrosine kinase which enhances resistance of malignant cells to TGF-|3-induced growth inhibition and apoptosis. BCR-ABL protein targets AKT and transcription factor FOX03 and thus impairs the cytostatic effect of TGF-|3l [245]. In addition, by improving protea-somal degradation, BCR-ABL blocks TGF-|3l-induced expression of p27 . Thus, BCR-ABL kinase promotes activation of cyclin-dependent kinase and cell cycle progression [246]. In CML, expression of EVI-1, a proto-oncogene that is expressed at very low levels in normal hematopoietic cells, is increased. [247]. EVI-1 binds to the MH2 domain of SMAD3 repressing its DNA-binding ability and transcriptional activity and this way attenuates TGF-|3 signaling [248]. Moller et al. showed that BCR-ABL up-regulates TGF-|3 signaling when expressed in Cos-1 cells. In Cos-1 cells, the expression of BCR-ABL up-regulates TGF-|3-mediated transcriptional activity by interaction between T|3RI and kinase domain of BCR-ABL, which leads to increased activity of SMAD3 promoter and increased SMAD2 and SMAD3 protein expression level [249]. Lymphoid leukemia In children T-cell acute lymphoblastic leukemia (ALL), SMAD3 protein is absent or significantly decreased, however SMAD3 mRNA is present in T-cell ALL and normal T-cells at similar level. The level of SMAD3 is decisive for the T-cell response to TGF-|3. A reduction in SMAD3 interplays with other oncogenic events, such as alterations in the retinoblastoma pathway, to precede T-cell leukemogenesis. It was proven that the loss of Smad3 can work in tandem with a loss of p27KIP1, which is also frequently altered in human T-cell ALL, to promote T-cell leukemogenesis in mice [250]. The t(12;21) translocation found in ALL generates the TEL-AML1 chimeric protein. Loss of sensitivity to TGF- PML-RARa Figure 6 TGF-fi signaling in APL. Cytoplasmic isoform of PML (cPML) protein interacts with SMAD2/3 and SARA and is required for accumulation of SARA-SMAD2/3 and TGF-p" receptors in early endosome. However, the PML-RARa oncoprotein physically interacts with cPML and thus leads to impaired TGF-p" signaling Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 15 of 24 |3 could be an important component of the function of TEL-AML1; it was shown that TEL-AML1 blocks the ability of TGF-|3 to suppress proliferation via activation of p27 . The exact mechanism needs to be elucidated; however, a possible alternative is that TEL-AML1, in addition to binding SMAD3, binds co-repressors NcoR and SIN3A and this complex is able to transcriptionally activate the key cell cycle negative regulators, including p27KIP1[251]. Scott et al. showed that mRNA of downstream components of TGF-|3 pathway, such as p21CIP1 and pl5 , are absent in ALL cell lines with high frequency, while p27 mRNA levels are not reduced. These findings suggest epigenetic silencing of TGF-|3 signaling in molecular pathogenesis of ALL and possibly pl5 and p21 are inactivated by this mechanism. In ALL, pl5' mRNA absence is often connected with promoter methylation, whereas reduced p21CIP1 expression happens independently of promoter methylation, indicating that within the same malignancy, epigenetic silencing of TGF-|3 signaling is methylation-dependent or independent [252]. In adult acute T-cell leukemia, TGF-|3 signaling is inactivated through the activity of viral oncoprotein Tax. This oncoprotein compromises trans-activation of TGF-|3 responsive promoters by inhibiting the ability of SMAD proteins to mediate TGF-|3-induced transcriptional activation by interfering with transcriptional factor CBP/p300 [253]. Another model of its function is that Tax interacts with the MH2 domains of SMADs 2, 3 and 4 in order to inhibit formation of the SMAD3/4 complex, disturb the interplay of the SMAD proteins with transcriptional factor CBP/ p300, prevent binding of the SMAD complex to its target DNA sequence and thus inhibit TGF-|3 signaling [254]. The Tax repressor effect is mediated by activating JNK leading to increased phosphorylation of c-Jun, which is followed by formation of SMAD3/c-Jun complex that inhibits the ability of SMAD3 to bind DNA [255]. In hairy-cell leukemia (HCL), higher levels of TGF-|3l were observed in bone marrow (BM), serum and plasma from peripheral blood. The main source of this cytokine in active and latent form is hairy cell (HC). HCs produce TGF-|3l, which is stored in BM near bone marrow fibroblasts; it activates them to synthesize collagen and re-ticulin fibers. TGF-|3l is important in fibrosis and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL [256]. Lymphoma Peripheral and cutaneous T-cell lymphoma In cutaneous T-cell lymphoma and Sezary syndrome, reduced levels of T|3RI and TpRII correlate with decrease in T|3RI and TpRII mRNA levels. This leads to the loss of TGF-|3 growth inhibitory responses [257]. Knaus et al. detected a single point mutation (Asp-404-Gly [D404G]) in the kinase domain of TpRII in advanced lymphoma. This dominant negative mutation prevents cell surface expression of normal TpRII. The ability of the mutant receptor to prevent function of normal TGF-|3 receptors is a new mechanism for loss of responsiveness to the TGF-|3 in tumorogenesis. Since T|3RI is not able to bind TGF-|3 in the absence of T|3RII, no T|3RI is detected on the surface of these cells. This mutant receptor binds to normal receptor in an intracellular compartment, likely the endoplasmic reticulum, and blocks development of the normal receptor on the cell surface [258]. In addition, a 178-bp deletion in exon 1 in the gene for T|3RI was reported to be responsible for loss of T|3RI expression on the cell surface in anaplastic large cell lymphoma cell line JK. This deletion was confirmed to be present also in patients' samples. Also, loss of T|3RI is followed by loss of its tumor suppressive properties in human T-cell lymphoma [259]. Non-Hodgkin s lymphomas (NHL) ATL, adult T-cell leukemia/lymphoma is a rare form of Non-Hodgkin's lymphoma (NHL). Zinc-finger E-box binding homeobox 1 (ZEB1) is a candidate tumor suppressor gene since mRNA of ZEB1 was found to be down-regulated in ATL. Physiologically, ZEB1 binds phosphorylated SMAD2/3 to enhance TGF-|3 signaling, and it can counteract the SMAD7-mediated inhibition of TGF-|3l function. Down-regulation of ZEB1 mRNA together with over-expression of inhibitory SMAD7 mRNA in ATL leads to loss of responsiveness to TGF-p-mediated growth arrest. Therefore, ZEB1 has an important role in regulation of TGF-|3l signaling pathway by binding to R-SMADs and also I-SMADs [260]. SMAD1 protein level is elevated and it is phosphorylated in response to TGF-|3l signaling in NHL. This suggests a role of SMAD1 in mediating the effects of TGF-|3 in NHL [261]. In B-cell lymphoma, Bakkebo et al. found that phosphorylation of SMAD 1/5 is surprisingly an important event for the TGF-|3-mediated anti-proliferative effects. T|3RI was highly expressed in these cells and likely is important for signaling through SMAD 1/5 pathway. Also, the regulation of TGF-|3-mediated proliferation is at least partly dependent on activated p38 MAPK [262]. In B-cell lymphoma, the cell line resistant to TGF-|3l did not possess functional TpRII. This led to the absence of nuclear translocation of phosphorylated SMAD3 and SMAD2, the lack of nuclear expression of p21 and the down-regulation of c-Myc. Chen et al. found that methylation of promoter (CpG methylations at -25 and -140) plays an important role in TpRII gene silencing [263]. Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 16 of 24 In diffuse large B-cell lymphoma (DLBCL), miR-155, which is over-expressed in aggressive type of B-cell lymphoma, targets SMAD5 by binding to the 3/ UTR of the SMAD5 gene. Treatment of DLBCL cell line with TGF-pi resulted in phosphorylation of SMAD2/3 but also of SMAD1/5 indicating an active non-canonical signaling. Over-expression of miR-155 in this cell line significantly limited the cytostatic effect of cytokine due to impaired TGF-|3l-mediated induction of p21 . In miR-155-overexpressing and SMAD5 knockdown DLBCLs, the disruption of p21CIP1 induction was independent of the inhibitory effects of TGF-|3l thus creating a link between miR-155, TGF-|3 pathway and lymphoma-genesis [264]. In small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL), the CLL cells are resistant to the growth-inhibitory effects of TGF-|3 in spite of T|3RII expression which is similar as in normal B cells. Therefore, the loss of responsiveness to TGF-|3 is most likely due to altered binding of TGF-|3 to the receptor complex or downstream signaling pathway [265]. Lagneaux et al. attributed the loss of responsiveness of CLL cells to TGF-|3 especially to decreased cell-surface expression of T|3RI. CLL cells resistant to TGF-|3l showed no surface T|3RI able to bind TGF-|3l, but the expression of T|3RII was normal. On the other hand, both TGF-|3l-sensitive and TGF-|3l-resistant CLL cells contained normal levels of T|3RI and T|3RII mRNAs. The absence of functional T|3RI on the surface of CLL cells, in spite of normal mRNA level, could be explained by point mutations in the TjiRI gene [266,267]. In CLL, Schiemann et al. found mutations in the signal sequence of T|3RI (Leul2Gln substitution together with an in-frame single Ala deletion) which leads to reduced gene transcription stimulated by TGF-|3 [268]. In addition, CLL cells exhibited an increased expression of the TGF-|3 co-receptor, TpRIII, which is normally not expressed entirely in hematopoietic cells [269]. On the other hand, Lotz et al. found over-expression of TGF-|3 in CLL cells; all primary cells in this study were sensitive to the growth-inhibitory effects of this cytokine [270]. MM cells proliferation osteoblasts differentiation bone marrow stromal cells T cells disruption of proliferation and activation Figure 7 TGF-P signaling in the bone marrow microenvironment of multiple myeloma. Myeloma cells are able to produce TGF-p" cytokine which influence cells of bone marrow microenvironment such as osteoblast progenitors, bone marrow stromal cells. Moreover, it disrupts T eel oroliferation and activation Kubiczkova et al. Journal of Translational Medicine 2012, 10:183 httpy/www.translational-medicine.com/content/10/1/183 Page 17 of 24 In Burkitt's lymphoma, TGF-|3-mediated growth arrest is associated with transcriptional repression of the E2F-1 gene. On the other hand, over-expression of the E2F-1 gene overcomes the TGF-|3-mediated Gl arrest. So, the transcriptional repression of the E2F-1 gene is required for growth arrest suggesting that TGF-|3 can effectively exert tumor suppression also in cells without c-Myc, pl5iNK4B and p21ciPi regUiation [27i]. Inman and Allday reported that in Burkitt's lymphoma, cells express normal levels of T|3RI RNA and protein, but decreased levels of T|3RII RNA, leading to lack of responsiveness to TGF-|31 [272]. Multiple myeloma In multiple myeloma (MM), higher levels of TGF-|3 are secreted by myeloma cells as well as bone marrow stromal cells (BMSC). TGF-|3 secretion escalates with the stage of B cell differentiation (Figure 7). Increased production of TGF-|3 is followed by increased interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion by BMSC, related to tumor cell proliferation. TGF-|3 is the major inducer of IL-6 and VEGF, two important cytokines of MM. On the other hand, TGF-|3 inhibits proliferation and Ig secretion of normal B cells [273]. After treatment with T|3RI kinase inhibitor (SD-208), decreased production of IL-6 and VEGF and also attenuated tumor cell growth was observed. Mechanism of action of SD-208 is blocking nuclear accumulation of SMAD2/3 and related production of IL-6. This leads to inhibition of MM cell growth, survival, drug resistance and migration [274]. In MM, no mutations in T/3RI or TjiRII genes were described; MM cells contain T|3RI and T|3RII proteins in the cytoplasm. Resistance to the growth-inhibitory functions of TGF-|3 signaling develops, possibly due to defective trafficking of T|3RI and T|3RII to the cell surface in these cells [275,276]. Possibly, the loss of T|3RII expression on the cell surface is the result of gene silencing by hypermehylation correlating to poor survival [277]. TpRIII expression is diminished on mRNA and protein level in MM, enhancing cell growth, proliferation, mobility, heterotrophic cell-cell adhesion and contributing to disease progression [278]. Serum level of TGF-|3 is an important prognostic factor in MM. Higher levels of this cytokine mean lower levels of normal Ig resulting in immune impairment [279]. TGF-|3 secreted from MM cells disrupts proliferation, activation and IL-2 responsiveness in T cells. TGF-|3 is important in this immune-suppression, and its intensity of suppression is tumor burden dependent [280]. In MM patients, TGF-|3 represses bone formation in bone lesions. Initially, TGF-|3 enhances proliferation of osteoblast progenitors and promotes mineralization of bone matrix. Then, TGF-|3 inhibits subsequent phases of differentiation of osteoblasts and represses mineralization of matrix. This effect can be abrogated by inhibitors of T|3RI kinase domain (reviewed in [281]). Conclusion TGF-|3 signaling is complex and finely regulated fundamental pathway, which has an important role during human development and adult life. It is broadly intertwined with other signaling pathways. Moreover, it is involved in cancerogenesis of solid tumors as well as hematological malignancies. Paradoxically, TGF-|3 is both a tumor suppressor and tumor promoter. The tumor suppressor activities are widely described as anti-proliferative and apoptotic effects. During cancer progression, tumor frequently avoids tumor suppressive activities of TGF-|3 either by acquiring mutations of signaling components or by inhibiting its anti-proliferative response. This switch' helps the tumor to use TGF-|3 as an oncogenic factor inducing tumor motility, invasion, metastasis and epithelial-to-mesenchymal transition. Advances in the study of molecular mechanisms that elucidate oncogenic activities of TGF-|3 lead to a strong desire to target TGF-|3 signaling in cancer therapy. However, the exact mechanisms involved in the malignant transformation of TGF-|3 needs to be clarified. Only then, it will be possible to develop successful therapeutic strategies as well as provide new therapeutic targets to restore the normal TGF-|3 function. Competing interests The authors declare that they have no competing interests. Authors' contributions _.K. wrote the original manuscript, L.S. and R.H. cooperated on revising the manuscript. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit o Central Mechanism of immunomodulatory drugs in multiple myeloma Sedlarikova L, Kubiczkova L, Sevcikova S, Hajek R. LeukRes. 2012 Oct;36(10):1218-24. PMID: 22727252 IF vroce 2012: 2,764 Leukemia Research 36 (2012) 1218-1224 Contents lists available at SciVerse ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Leukemia Research Invited review Mechanism of immunomodulatory drugs in multiple myeloma Lenka Sedlarikova, Lenka Kubiczkova, Sabina Sevcikova*, Roman Hajek Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic ARTICLE INFO Article history: Received 18 March 2012 Received in revised form 18 March 2012 Accepted 25 May 2012 Available online 21 June 2012 Keywords: Multiple myeloma Treatment Immunomodulatory drugs Thalidomide Lenalidomide Pomalidomide ABSTRACT Multiple myeloma is the second most common hematological cancer in the world. It is characterized by accumulation of malignant plasma cells in the bone marrow, osteolytic lesions and monoclonal immunoglobulins in blood/urine. With the introduction of immunomodulatory drugs into the treatment protocol, the outcome of multiple myeloma patients has dramatically improved with more than 30% of patients surviving for 10 years thus shifting multiple myeloma to a treatable condition. © 2012 Elsevier Ltd. All rights reserved. Contents 1. History of multiple myeloma treatment.............................................................................................................1218 2. Immunomodulatory drugs...........................................................................................................................1219 2.1. Thalidomide..................................................................................................................................1219 2.2. Lenalidomide.................................................................................................................................1219 2.3. Pomalidomide................................................................................................................................1220 3. Mechanisms of immunomodulatory drugs in multiple myeloma..................................................................................1220 3.1. Direct antitumor effects......................................................................................................................1220 3.2. Immunomodulatory effects of IMiDs........................................................................................................1220 3.2.1. Co-stimulation of T cells............................................................................................................1220 3.2.2. Enhancement of NKT and NK cells.................................................................................................1220 3.2.3. Inhibition of regulatory T cells.....................................................................................................1221 3.3. Anti-angiogenic activity of IMiDs............................................................................................................1221 3.4. Anti-inflammatory properties of IMiDs......................................................................................................1221 3.5. Effects of IMiDs in the bone marrow of multiple myeloma.................................................................................1223 4. Conclusion............................................................................................................................................1223 Conflict of interest.....................................................................................................................................1223 Acknowledgments...................................................................................................................................1223 References...........................................................................................................................................1223 1. History of multiple myeloma treatment Multiple myeloma (MM) is a plasma cell malignancy. The World Health Organization ranks MM among immunosecretory * Corresponding author at: Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic. Tel.: +420 5 4949 3380; fax: +420 5 4949 8480. E-mail address: sevcik@med.muni.cz (S. Sevcikova). 0145-2126/$ - see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.Org/10.1016/j.leukres.2012.05.010 peripheral neoplasms of B lymphocytes [1]. MM has complex pathophysiology characterized by accumulation of clonal malignant plasma cells in the bone marrow accompanied by production of monoclonal immunoglobulins or light or heavy chains, resulting in clinical manifestation of the disease. Clinically, MM manifests by osteolysis, impaired immune system, hypercalcemia, peripheral neuropathy and renal insufficiency [2,3]. For MM, first 'treatment attempts' included rhubarb pill and infusion of orange peel which were given to the first documented patient by Dr. Solly in 1844 [4]. Six years later, William Macintyre L. Sedlarikova et al. / Leukemia Research 36 (2012) 1218-1224 O Fig. 1. Structure of IMiDs. (A) Structure of thalidomide, (B) structure of lenalidomide and (C) structure of pomalidomide. described phlebotomy as 'maintenance therapy' for MM [5]. In the middle of the 20th century, Loge and Rundles used urethane to decrease the number of myeloma cells and pain in the bones [6], The first truly successful treatment strategy was melphalan in combination with prednisone (MP) which has been used since the 60softhe 20th century [7]. Next, polychemotherapy regimens were introduced and achieved good treatment responses - for example, combination of vincristine, adriamycin and dexamethasone (VAD) treatment used as induction chemotherapy before autologous transplantation [8]. Unfortunately, these regimens were not curative. In the first decade of the 21st century, a major effort was put into creating novel therapies, such as proteasome inhibitors and immunomodulatory drugs (IMiDs) that successfully and dramatically altered the therapeutic landscape for MM treatment. Moreover, several new highly efficient drugs (pomalidomide, carfil-zomib, bendamustin), which will increase treatment options very soon, will play a key role in overcoming resistance to previous treatment or increase survival of patients [3], In this review, we will focus on the role and molecular mechanism of IMiDs in the treatment of multiple myeloma. 2. Immunomodulatory drugs Immunomodulatory drugs (IMiDs) are a group of new therapeutic agents, thalidomide-derivatives lenalidomide and pomalidomide (Fig. 1). The development of thalidomide, lenalidomide and pomalidomide represents a paradigm shift in the treatment of MM. These drugs possess pleiotropic properties against MM, including the ability to modulate host immune responses, impact cytokine 1219 secretion, angiogenesis, inflammation and they also have a direct effect on MM cells via induction of apoptosis (reviewed in [9]). Thalidomide and lenalidomide have been approved by the FDA for treatment of MM. While pomalidomide has not been approved yet, it is widely expected that the approval will be granted in 2012. 2.1. Thalidomide Thalidomide, 2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (C13H10N2O4), is a synthetic derivative of glutamic acid. It contains one chiral centre and is a racemic mixture of two optically active enantiomers, S and R, with the ability of rapid chiral inter-conversion at physiological pH. The S enantiomer is responsible for the teratogenic and anti-tumor properties of thalidomide, while R enantiomer has sedative effects (reviewed in [10]). Despite its infamous history as a human teratogen, thalidomide is an effective anti-inflammatory and anti-tumor agent [10]. Singhal and colleagues were the first to describe its effect in the treatment of MM, where it induces apoptosis of myeloma cells, down-regulates expression of adhesion molecules thereby disrupting mutual interactions between myeloma cells and bone marrow stromal cells; thalidomide also possesses anti-angiogenic and anti-inflammatory properties and stimulates host immune cells [11-14]. Thalidomide was first synthesized by Chemie Griinenthal in 1954 and was broadly used as a sedative and hypnotic agent for morning sickness during pregnancy. In 1961, its teratogenic effect on children whose mothers took thalidomide was first described. Thalidomide caused limb hypoplasia (phocomelia), absence of ears, deafness, malformations of gastrointestinal system and heart, facial and palatal defects. Thalidomide was taken off the market immediately, but affected development of nearly 10,000 children all around the world (reviewed in [10]). A lucky chance led to the discovery of its anti-inflammatory properties in the treatment of erythema nodosum leprosum, a cutaneous complication of leprosy [15]. Thus, thalidomide was introduced back into the market, but its use has been limited by obligatory program STEPS (System for Thalidomide Education and Prescribing Safety) in order to minimize its teratogenic potential [16]. The use of thalidomide for the treatment of graft versus host disease after allogeneic transplantation of the bone marrow revealed more of its effects [17]. In addition, D'Amato showed that malformations caused by thalidomide are the consequences of its anti-angiogenic properties; such a mechanism of inhibition of growth of tumoral vessels might be utilized even in treatment of cancer [12]. Later, its effect on relapsed and refractory MM was examined, leading to the discovery of its strong anti-tumor activity [11]. 2.2. Lenalidomide Lenalidomide, 3-(4-amino-l-oxo-l,3-dihydro-2H-isoindol-2-yl) piperidine-2,6-dione (C^H^IS^C^), has preserved chiral centre and occurs as a racemate, in a mixture of two optically active enantiomers, S and R. Lenalidomide is generated by adding an amino group to position 4 of the phthaloyl ring of thalidomide and removing a carbonyl group from the 4-amino substituted phthaloyl ring (reviewed in [18]. Clinical experience with thalidomide led to initiation of research of its analogs with more favorable toxic profile and increased effectiveness (reviewed in [9]). Lenalidomide (Revlimid, formerly CC-5013, Celgene) is ranked among these derivatives. Unfortunately, its teratogenic potential is preserved; for that reason, its use comes under the safety program as well [9]. L. Sedlarikova et al. / Leukemia Research 36 (2012) 1218-1224 1220 Similarly to thalidomide, lenalidomide inhibits angiogenesis and adhesion of MM cells to bone marrow cells, reduces secretion of growth factors, induces apoptosis of MM cells, inhibits production of inflammatory cytokine TNF-a and supports cytotoxic activity of NK cells and T cells [19-21]. However, this IMiD mediates activation of Wnt/P-catenin signaling which is a mechanism of inducible chemoresistance to lenalidomide at the transcriptional and post-transcriptional levels [22]. 2.3. Pomalidomide Chemical formula of pomalidomide is 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-l,3-dione (C13H11N3O4). This compound is derived from thalidomide by adding an amino group to position 4 of the phthaloyl ring and exists in two forms, enantiomers S and R(reviewed in [18]). Another analog of thalidomide is pomalidomide (Actimid, Cel-gene), which has a pleiotropic effect on myeloma cells (reviewed in [9]). It induces apoptosis of MM cells, inhibits angiogenesis, has strong immunomodulatory abilities and is most effective in TNF-a mRNA degradation when compared to thalidomide and lenalidomide [19,20,23]. 3. Mechanisms of immunomodulatory drugs in multiple myeloma There are several mechanisms of IMiDs in multiple myeloma. They have direct antitumor, immunoregulatory and anti-angiogenic activity. We shall also discuss anti-inflammatory properties and effects these drugs have on the bone marrow microenvironment in multiple myeloma. 3.1. Direct antitumor effects The direct antitumor effect of IMiDs on myeloma cells was discovered in the study of Hideshima et al. In that study, myeloma cell lines were treated with IMiDs, and this treatment led to CDK inhibition and cell cycle arrest in phase [23]. The IMiDs-induced cell cycle arrest of clonal plasma cells in Gi phase is mediated via increased levels of p21WAF_1 protein through epigenetic modifications. Reduction of histone methylation in the promoter of p21WAF_1 gene and increased histone acetylation make transcriptional factors (such as Spl, Sp3, Egrl and Egr2) accessible to DNA [24]. Increased p21WAF_1 expression subsequently induces p2\wAF-i ancj 2,4 or 6 complex formation, leading to inhibition of their kinase activity [25]. Afterwards, pRb hypophosphorylation occurs preventing the cell from Gi /S phase transition. In the study of Gandhi et al., lenalidomide induced pi 5 and p27 tumor-suppressor gene expression in myeloma cells [26]. Myeloma cells are protected from apoptosis by anti-apoptotic proteins that are regulated via NF-kB transcriptional factor [27]. In these cells, IMiDs prevent NF-kB from activation which leads to decreased expression of anti-apoptotic proteins, for example cellular inhibitor of apoptosis 2 (cIAP2), FLICE inhibitory protein (FLIP), X-linked inhibitor of apoptosis protein (XIAP) which prevents cas-pase 8 activation, TRAIL/Apo2L and Fas sensitivity. In addition, IMiDs directly induce activation of caspase 8 and subsequently activation of caspase 3 [28,29]. IMiDs block IGF-1 production by decreased NF-kB transcriptional activity. IGF-1 induces phosphorylation of transcriptional factor FKHRL-1 thereby disrupting its pro-apoptotic activity and also increasing levels of apoptotic inhibitors cIAP2, FLIP and XIAP. IMiDs reduce IGF-1 effects which lead to increase in sensitivity to TRAIL/Apo2L in myeloma cells [30]. Another study showed that lenalidomide downregulates levels of interferon regulatory factor 4 (IRF4) in MM. It was associated with decreased MYC levels, as well as Gi phase cell cycle arrest, decreased cell proliferation and cell death. So, lenalidomide-induced IRF4 inhibition partially mediates anti-proliferative and pro-apoptotic effects of the compound [31 ]. The direct target of IMiDs is still unknown but the requirement of cereblon (CRBN) expression has been shown to be important for anti-myeloma activity of these agents. IMiDs are able to bind CRBN which leads to its cytotoxic activity. In the case of CRBN absence, cells show IMiDs resistance (reviewed in [32]). In addition, dys-regulation of Wnt/P-catenin pathway might be a possible cause of lenalidomide resistance and phosphorylated P-catenin as a possible substrate of CRBN. In the presence of IMiDs, CRBN is not able to form ubiquitin ligase which results in accumulation of P-catenin. In conclusion, CRBN inhibition may have a key role in the treatment of MM [22]. 3.2. Immunomodulatory effects of IMiDs 3.2.1. Co-stimulation of T cells Activation of T cells is mediated via the T cell receptor (TCR), but also requires a secondary co-stimulation signal mostly mediated by antigen presenting cells (APCs). IMiDs are able to co-stimulate partially activated CD3+ T cells [13,33]. This stimulation is equal for both CD4+ and CD8+ cells (Fig. 2). IMiDs improve their proliferation and augment production of Thl type cytokines, IL-2 and interferon ~y (IFN-7). Subsequently, secretion of IL-2 and IFN-7 increases number of natural killer cells (NK cells), improves their function and mediates lysis of myeloma cells. One of the mechanisms of augmenting IL-2 production via IMiDs is mediated by increase of activation protein-1 (AP-1) transcriptional activity. AP-1 is a key factor of IL-2 production [34]. IL-2 and IFN-7 production is also mediated via JAK/STAT signaling pathway, where activated STAT proteins induce expression of target genes. On the other hand, these genes can be negatively regulated by the suppressor of cytokine signaling (SOCS). SOCS1 is a negative regulator of IL-2 and IFN-7. In immune cells, the SOCS1 expression is significantly inhibited by IMiDs [35]. Activation of T cells can be abrogated by various factors. Lenalidomide overcomes blockage of cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), disrupting cell proliferation and cytokine secretion [36]. So, lenalidomide triggers tyrosine phosphorylation of CD28 on T cells (B7-CD28 pathway) followed by activation of NF-kB and facilitation of to S phase transition. In addition, phosphoinositide 3 kinase (PI3K) is activated during CD28 phosphorylation and leads to PI3K/Akt signaling pathway activation and facilitation of nuclear factor of activated T cells 2 (NFAT2) translocation, resulting in IL-2 secretion [37]. Pomalidomide does not provide co-stimulative signal by itself, but is able to increase signal from another cell or molecule. Pomalidomide also augments promotor of IL-2 gene activity and so IL-2 production [34]. 3.2.2. Enhancement ofNKT and NK cells Natural killer T cells (NKT) are T cells with NK cell surface markers. They have direct cytotoxic anti-tumor properties. Dendritic cells (DC) with NKT ligand, a-galactosylceramide, are responsible for activation and expansion of NKT cells [38]. When these cells are exposed to lenalidomide, increased NKT expansion via DC occurs. IFN-7 secretion by NKT cells leading to partial activation of NK cells and proliferation is associated with this exposure as well [39]. NK cells are irreplaceable elements of innate immunity. They protect the organism by killing tumor cells and virus-infected cells. IMiDs enhance NK cell proliferation in the presence of IL-2, which facilitates killing of myeloma cells [13]. In addition, lenalidomide improves antibody-dependent cell-mediated cytotoxicity (ADCC) and thus increases granzyme B and Fas ligand (FasL) expression APC I. Sedlarikova et al./Leukemia Research 36 (2012) 1218-1224 APC 1221 APC Fig. 2. Co-stimulatory activity of IMiDs. APCs activate T cells by binding peptides of major histocompatibility complex (MHC) to the TCR. The B7-CD28 secondary co-stimulatory pathway is also required for effective T cell activation. IMiDs are able to enhance T cell stimulation in the absence of the secondary signals. in NK cells leading to tumor cells apoptosis. Increased monocyte chemotactic protein-1 (MCP-1) expression is associated with this process as well. MCP-1 attracts T cells in order to migrate to the tumor. Granulocyte macrophage colony-stimulating factor (GM-CSF) expression is also increased during this process, improving anti-tumor response [21]. 3.2.3. Inhibition of regulatory T cells Regulatory T cells (Tregs) play an active role in establishing and maintaining immunological unresponsiveness to self antigens and negative control of various immune responses to non-self antigens. Regulatory function for Tregs is provided by a master molecule FoxP3. At present, several studies showed that Tregs were expanded both in hematological malignancies and solid tumors suppressing the function of naive T cells [40]. The study of Galus-tian and colleagues showed that lenalidomide and pomalidomide inhibit proliferation of Tregs via decreased FoxP3 mRNA expression. Another possible mechanism was prevention of CD134 expression on the surface of Treg cells. This process abrogates T cells activation (Fig. 3). Unlike its analogs, thalidomide had no effect on Treg cells in this study [41]. On contrary, several other studies presented different results. Muthu Raja et al. found that patients responding to lenalidomide and dexamethasone combination had increased number of Tregs and concluded that this treatment strategy was not able to enhance the immune anti-tumor response [42]. Similarly, Gupta and colleagues discovered a decrease in the frequency of Tregs with reduced expression of FoxP3 in previously untreated MM patients. However, the immunosuppressive potential of Tregs was preserved proposing normal Tregs function. After treatment with thalidomide, an increase in the number of Tregs was observed in this study [43]. These conflicting results might be caused by different identification approaches of Treg cells. At this point, data about Tregs in MM are contradictory, so no clear conclusions can be presented [44]. 3.3. Anti-angiogenic activity of IMiDs IMiDs have been shown to have anti-angiogenic effects which are independent of their immunomodulatory effects (Fig. 4). While anti-angiogenic properties of thalidomide prevail, lenalidomide and pomalidomide have an immunomodulatory potential [12,20]. IMiDs modulate factors affecting endothelial cell migration, especially TNF-a, VEGF and bFGF which are secreted by bone marrow stromal cells. According to Dredge and colleagues, IMiDs reduce Akt phosphorylation and thus interfere in the PI3K/Akt signaling pathway affecting the expression of these factors and restraining angiogenesis [45]. VEGF expression induces VE-cadherin tyrosine phosphorylation via Src kinase. It disrupts contact of endothelial cells leading to necessary migration during angiogenesis [46]. Lenalidomide blocks Src kinase activity and disrupts subsequent VE-cadherin tyrosine phosphorylation during angiogenesis [47]. In addition, myeloma cells produce VEGF after IL-6 stimulation. Endothelial cells and bone marrow stromal cells respond to VEGF production via IL-6 secretion thereby closing the paracrine loop [48]. Gupta et al. observed that thalidomide and lenalidomide decreased expression of these factors and thus inhibit growth of new vessels and myeloma cells nutrition [49]. Lu et al. found an inhibitory effect of lenalidomide on expression of endothelial cells hypoxia-inducible factor la (HIF-la) which has a key role in hypoxia-mediated effects, together with angiogenesis and metastasis promoting aggressive tumor phenotype. The inhibition of HIF-la expression is at least partially mediated via PI3K/Akt signaling pathway suppression. In this study, pomalidomide seemed to have less impressive inhibitory effect on HIF-la expression and thalidomide had almost none [50]. 3.4. Anti-inflammatory properties of IMiDs Cyclooxygenase 2 (C0X-2) catalyzes conversion of arachidonic acid into several inflammatory prostaglandins (PG). For example, PG-E2 contributes to inflammation, tumor-induced angiogenesis and production of IL-6 [51]. IMiDs have been shown to inhibit the expression of C0X-2 via reducing the half-life of C0X-2 mRNA and thereby reducing levels of PG-E2 [52]. In addition, lenalidomide increases IL-10 production which also plays a partial role in C0X-2 mRNA degradation. Thalidomide has an inhibitory effect on TNF-a, inflammatory cytokine produced by monocytes and macrophages [53]. TNF-a inhibition is mediated via its mRNA degradation [54]. Unlike thalidomide, lenalidomide inhibits TNF-a production more effectively, but the most effective inhibitor of TNF-a is pomalidomide [19]. 1222 L. Sedlarikova et al. / Leukemia Research 36 (2012) 1218-1224 Fig. 3. Overview of the immunomodulatory effects of IMiDs. These effects include co-stimulation of T cells, inhibition of regulatory T cells with suppressor effects on the host immune system and enhancement of NK and NKT cell function and proliferation. Anti-inflammatory and anti-angiogenic properties of thalidomide are partially controlled by NF-kB transcriptional factor. In the cytoplasm, NF-kB is bound to IkB and thereby inhibited. Stimulation by IL-ip or TNF-a leads to IkB dissociation, and then, the expression of inflammatory genes is activated. Thalidomide abrogates IkB phosphorylation, dissociation and resultant inflammatory cytokine secretion [55]. However, IMiDs inhibit not only TNF-a and IL-ip production, but also IL-6, IL-12 and TGF-P production. These cytokines enhance growth and survival of myeloma cells, drug resistance, cell migration and adhesive molecule expression. On the other hand, IMiDs increase anti-inflammatory cytokine IL-10 production [56]. IL-6 production is partially mediated via JAK/STAT signaling pathway and modulated by SOCS1 expression. In 75% of MM stromal cells Fig. 4. Overview of non-immunomodulatory effects of IMiDs. These effects consist of anti-angiogenic activity, inhibition of cell growth and enhancement of MM cells apoptosis, inhibition of osteoclasts activity and reduction of myeloma cells/stromal cells interactions. L. Sedlarikova et al. / Leukemia Research 36 (2012) 1218-1224 1223 cases, promoter of SOCS1 gene is silenced by CpG dinucleotide hypermethylation leading to uncontrolled IL-6 production, myeloma cells growth and suppression of host immune system. Pomalidomide particularly demethylates promoter of SOCS1 gene in MM cells, induces SOCS1 transcription and thus inhibits IL-6 production [35]. 3.5. Effects oflMiDs in the bone marrow of multiple myeloma Osteolytic lesions are the most striking symptom of MM. IMiDs prevent osteoclast maturation and thus inhibit bone structure disruption [57]. Mechanism of inhibition lies in reduced expression of the cysteine protease cathepsin K, a protease associated with matrix degradation and bone resorption, and in reduced levels of aV(33-integrin, a marker associated with osteoclast differentiation mediating interactions between cells and the extracellular matrix [58]. These mechanisms are consequences of downregu-lated levels of transcriptional factor PU.l, which is a key mediator of osteoclastogenesis - it regulates the differentiation of myeloid cells to osteoclast precursor cells [59]. In addition, Breitkreutz et al. observed that lenalidomide decreases MlP-la secretion, which is one of the most important factors for growth and survival of osteoclasts. This agent also inhibits RANKL, a key mediator of osteoclastogenesis [57]. Adhesive molecules, induced by TNF-a, facilitate interaction between clonal plasma cells and bone marrow stromal cells [14]. It has been shown that for example very late antigen 4 (VLA-4) interactions with fibronectin are important for migration and homing of MM cells into the bone marrow milieu. This molecule is expressed on myeloma cells as well as on physiological plasma cells promoting terminal B cell differentiation and secretion of immunoglobulins (reviewed in [60]). Nevertheless, IMiDs inhibit production of TNF-a thereby decreasing VLA-4 and lymphocyte function-associated antigen 1 (LFA-1) expression. VLA-4 and LFA-1 are adhesive molecules on the surface of plasma cells. IMiDs modulate expression of adhesive molecules, such as ICAM-1, E-selectin, L-selectin and VCAM-1, on the surface of bone marrow cells [61]. This goes along with consequent inhibition of adhesion-mediated cell signaling as well as cytokine production in the bone marrow milieu [49]. Thalidomide significantly inhibits SDF-la and CXCR4 receptor expression on MM cells. Their interaction is important for myeloma cell adhesion and migration. However, this mechanism has not been fully clarified [62]. Reduced contact between myeloma cells and the bone marrow leads to decreased IL-6 and VEGF production supporting survival of MM cells [14,49]. This mechanism also overcomes cell adhesion mediated drug resistance to apoptosis of tumor cells [48]. 4. Conclusion The introduction of the novel drugs, especially IMiDs, turned multiple myeloma into a chronic disease. The possibility of actually curing the patients is within our reach and combinations of these drugs with the upcoming drugs from the pipeline may bring this in the very new future. Even though multiple myeloma is a very heterogenous disease, IMiDs have been able to attack the malignant cells by various mechanisms, ranging from direct anti-myeloma effect, immunomodulatory effects to anti-angiogenic effect and many others. Conflict of interest The authors have nothing to declare. Acknowledgments This work was supported by grant of The Czech Science Foundation GAP304/10/1395, project of The Ministry of Education, Youth and Sports: MSM0021622434, and by the IGA grants of Ministry of Health of the Czech Republic NT12130 and NT12215 and NT11154. Contributions. L.S., L.K. and S.S. cooperated on writing the manuscript. RH. approved the final document. References [1] Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, et al. 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DOI: 10.3109/10428194.2012.704030 neaitnCaľe LETTERTOTHE EDITOR Serum miR-29a as a marker of multiple myeloma •g a Sabina Sevcikova1*, Lenka Kubiczkova1*, Lenka Sedlarikova1, Ondrej Slaby2& Roman Hajek13'4 ^Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, •'-Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Brno, Czech Republic, and ^Department of internal Medicine - Hematooncology and4Laboratory of Experimental Hematology and Cell Immunotherapy, Department of Clinical Hematology, University Hospital Brno, Czech Republic MicroRNAs (miRNAs) are short non-coding RNAs about 21-25 nucleotides in length; they have been reported to be involved in the initiation and progression of solid tumors as well as hematological malignancies [1]. Moreover, circulating miRNAs have been observed in serum. These circulating serum miRNAs are very stable, resistant to RNAse treatment, VI patients wil presence of serum miRNAs in MM patients with MM. Our data show that serum miR-29a is differentially expressed in MM patients with MM versus healthy donors. Serum samples from 91 MM patients with MM obtained at the time of diagnosis (prior to any treatment) were included in this study. These MM patients with MM (age range 41-88 and differ between healthy subjects and patients with col- years) were diagnosed between 2001 and 2010 at the Faculty orectal and lung cancer as well as diabetes [2]. It has further Hospital Brno, and were included in this study only after been described that elevated expression levels of some signing the informed consent form approved by the ethical serum miRNAs of patients with colorectal cancer decrease after surgery [3]. A recent paper described circulating serum miRNAs as markers of renal cell carcinoma [4]. Multiple myeloma (MM) is the second most common hematological malignancy worldwide [5]. MM is characterized by malignant proliferation of plasma cells (PCs) that accumulate in the bone marrow and displace normal hematopoiesis [6]. Currently, there are no specific markers for MM prediction. With the introduction of novel drugs and a very real possibility of targeted therapy in the near future, such markers are becoming increasingly important. If a specific marker were to be found in the peripheral blood, it would be easily accessible and could be obtained at various time points of treatment and even at the time of remission for frequent monitoring. For our study, we chose four miRNAs based on their possible relationship to MM pathogenesis. miR-410 is encoded by the 14q32.31 locus, which is frequently involved in MM translocations, and it has previously been described as a prognostic marker in neuroblastoma [7]. Aberrant expression of miR-660 has also been linked to MM [8]. miR-142-5p has been found to be aberrantly expressed in MM and monoclonal gammopathy of unknown significance (MGUS) [9,10], andmiR-29a to be up-regulated in MM PCs compared with normal PCs [9]. We decided to investigate whether these miRNAs (miR-29a, miR-142-5p, miR-410 and miR-660) are present in the serum of MM patients with MM. To the best of our knowledge, we are the first to report the committee. Serum was separated after clotting and cen-trifugation of whole blood and stored at — 80 °C. As controls, blood donors with no tumor diagnosis were included (age range 45-64 years). Clinical parameters of the MM patients with MM and healthy donors are described in Table I. F Total RNA enriched for small RNAs was isolated from 200 uL of serum by miRNeasy Mini Kit (Qiagen, Germany) according to modified manufacturer's protocol. Each sample was mixed with 800 uL of QIAzol solution and 1.25 uL of 0.8 |ug/|U,L MS2 RNA carrier (Roche, Switzerland). Extracted RNA was eluted in 30 uL of RNase free water. Quantification and purity measurements of RNA/miRNA were performed by Nanodrop ND-1000 spectrophotometer (Thermo Scientific, USA); samples with absorbance A260/280>1.8 were stored at — 80 °C for further processing. Reverse transcription (RT) was performed using the Taq-Man MicroRNA Reverse Transcription Kit (Applied Biosys-tems, USA) and small RNA-specific RT primers for hsa-miR-16, hsa-miR-29a, hsa-miR-142-5p, hsa-miR-410 and hsa-miR-660 (ID: 000391, 002112, 002248, 001274 and 001515; all Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the TaqMan Small RNA Assay on a 7500 Real Time PCR System (Applied Biosystems) using 1.4 uL of RT product according to the manufacturer's instructions. All experiments were run in duplicate. Average threshold cycle and standard deviation (SD) values were calculated. *These authors contributed equally. Correspondence: Sabina Sevcikova, PhD, Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic. Tel: 420-5-4949-3380. Fax: 420-5-4949-8480. E-mail: sevcik@med.muni.cz Received 17 April 2012; revised 11 June 2012; accepted 13 June 2012 RIGHTS L I M K4y 2 S. Sevcikova et al. Table I. Basic characteristics of patients and healthy donors. Healthy donors Patients («) (n) Total 91 30 Sex Male 49 14 Female 42 16 ISS Stage 1 28 — Stage 2 32 — Stage 3 26 — Not classified 5 — Durie-Salmon I 10 — II 14 — III 58 — Not classified 9 — Type of monoclonal Ig IgD 2 — IgA 22 — IgG 46 — IgM 2 — LC only 16 — Biclonal 1 — Non-secretory 2 — Age according to ISS stage (years), median/mean (range) Total 63.9/64.9 (41-88) — Stage 1 60.4/60.7 (41-81) — Stage 2 65.3/65.5 (48-82) — Stage 3 70/69 (47-83) — Not classified 55.9/63.2 (49-88) — Age according to sex (years), median/mean (range) Total — 55.5/55.4 (45-64) Male — 55.5/56.4 (51-64) Female — 55.5/54.6 (45-58) ISS, International Staging System; Ig, immunoglobulin; LC, light chain. Analysis of the qRT-PCR data was performed using SDS 2.0.1 software (Applied Biosystems) (settings: automatic baseline, threshold 0.2). Expression data were normalized to the expression of miR-16 reference miRNA. Statistical differences between miRNA levels in patients with MM and healthy donors were evaluated using the non-parametric Mann-Whitney [/-test. Sensitivity, specificity and area under the curve (AUC) for serum miRNA levels were determined using receiver operator characteristic (ROC) analysis. All calculations were performed using MedCalc software version 12.2.1.0. p-values of less than 0.05 were considered statistically significant. Results were processed using the non-parametric Mann-Whitney [/-test. Correlation was assessed using the Spearman correlation coefficient. We used qRT-PCR to test differences in serum miRNA expression between samples from an independent cohort of MM patients with MM and samples from healthy donors. In total, 91 MM patients with MM and 30 healthy donors were included in this study. Expression levels of serum miR-142-5p, miR-660, miR-410 and miR-29a were analyzed (Table II). For normalization of expression data, miR-16 was used as previously reported [10]. We chose miR-16 as a reference as it was the most stable in our preliminary experiments (data not shown). The expression of miR-142-5p, miR-660 and miR-29a in serum was significantly increased in patients with MM compared to healthy donors (p = 0.0183, p = 0.0062andp< 0.0001, respectively) [Figures 1(A)-1(C)]. As the difference in miR-410 expression level between MM and healthy donor sera did not reach statistical significance [p = 0.2918), miR-410 was excluded from further analysis. ROC curve analysis revealed that the serum level of miR-29a might serve as a useful biomarker for differentiating serum of patients with MM from that of controls, with an AUC of 0.832 (95% confidence interval [CI], 0.753-0.894). At a cutoff value of 0.0103 for the relative expression of miR-29a normalized to miR-16 levels, the sensitivity was 88% and specificity was 70% [Figure 1(D)]. Expression of miR-29a was not significantly different between patients at various Durie-Salmon (p = 0.223) or International Staging System (ISS) stages (p = 0.677). Although there were statistically significant differences between the ages of MM patients with MM and those of healthy volunteers (p >0.01), there was no correlation between age and level of miR-29a within the group of MM patients with MM (p = 0.442), as well as within the group of healthy donors (p = 0.411). Markers of diagnosis, remission and relapse of MM are obtained from the bone marrow after purification of the target cell population [6]. As bone marrow sampling is an invasive procedure, it cannot be repeated as often as needed. On the other hand, markers from peripheral blood are easily accessible and can be used for frequent monitoring. Table II. Expression levels* and basic characteristics of miRNAs. Expression level miRNA HD MM FC p-Value Putative target genes miR-29a 0.0089 0.0080-0.0119 0.0199 0.0129-0.0302 2.2371 < 0.0001 IGF1, NQ02, CDK2, AKT2, MYCN, MMP8, BCL11A, PTEN, CCNA2 miR-142-5p 0.0031 0.0024-0.0041 0.0046 0.0027-0.0075 1.4839 0.0183 CENTB2, RAD50, ELK4, IGF1, MCL1, MYCN, MAPK6, PTEN miR-410 0.0002 0.0001-0.0003 0.0002 0.0001-0.0004 1.0000 0.2918 CDC85A, CREB1, FGF2, FGF7, IGF, SMAD7 miR-660 0.0033 0.0029-0.0039 0.0040 0.0032-0.0049 1.2121 0.0062 E2F3, GDA, BCL2L11, CDK2 ^Presented as median and interquartile range. HD, healthy donors; MM, multiple myeloma; FC, fold change. RIGHTS LIN KÍ} Serum miRNA- the new kids in multiple myeloma 3 •g a (A) 0.1 r 1 i 1 3 0.O1 r 0,001 r 0,0001 (C) « 0,1 (B) to 0,1 1 36 0,01 0.001 r™R-660 P=0.0062 miR-142-5p-[vW mrR-142-5p-HD (D) 0.01 0.001 miR-29a niR-660-MM miR-29a-MM miR-660-HD mrR-29a-MM miR-29a-HD 40 60 SO 100- Specificity Figure 1. Comparison of serum miRNA expression levels and ROC analysis. (A-C) Comparison of serum miR-142-5p (A), miR-660 (B) and miR-29a (C) expression levels (loglO scale on y-axis) in patients with multiple myeloma (MM) (n = 91) and healthy donors (HD) (n = 30). Expression of each miRNA was normalized to expression of miR-16. Lines represent mean value, 25-75% quartile and min-max values. Statistically significant differences were determined using Mann-Whitney [/-test. (D) Receiver operating characteristic (ROC) curve analysis of serum miR-29a in patients with MM showed 0.832 AUC, 88% sensitivity and 70% specificity at cut-off value > 0.0103. Unfortunately, at this point, they are not perfect. Also, a good marker for prediction of early relapse is missing. Circulating serum miRNA might represent a novel putative, easily accessible and stable marker. Further investigation is needed to estimate whether these circulating miRNAs are directly associated with changes occurring in MM, as they may also reflect indirect pathological effects of the disease, such as bone lesions or renal failure in MM [11,12], Also, a possible relationship of miRNA levels found in PCs of patients with MM and corresponding sera is not clearly understood. Our results show that miR-142-5p, miR-660 and miR-29a are up-regulated in the serum of MM patients with MM. Further analytical characteristics of miR-29a (sensitivity 88%, specificity 70%) proved that it is potent in discriminating MM serum from healthy donor serum. To our knowledge, this is the first report concerning circulating serum miR-29a differentially expressed in MM patients with MM. Although our observations are promising, further large-scale studies and validations are needed to establish miR-29a as a marker of the disease. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal. References [I] Hunter MP, Ismail N, Zhang X, et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS One 2008;3:e3694. [2] Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum. Cell Res 2008;18:997-1006. [3] Ng EK, Chong WW, Jin H, et al. Differential expression of microRNAs in plasma of patients with colorectal cancer. Gut 2009;58:1375-1381. [4] RedovaM., PoprachA., Nekvindova J, et al. CirculatingmiR-378 and miR-451 in serum are potential biomarkers for renal cell carcinoma. J Transl Med 2012; 10:55. [5] Hajek R, Krejci M, Pour L, et al. Multiple myeloma. Klin Onkol 2011;24(Suppl.): S10-S13. [6] Anderson KC, Carrasco RD. Pathology of myeloma. Annu Rev Pathol Mech Dis 2011;6:249-274. [7] Gattolliat CH, Thomas L, Ciafre SA, et al. Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma. Br J Cancer 2011;105:1352-1361. [8] Chi J, Ballabio E, Chen XH, et al. MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival. Biol Direct 2011;6:23. [9] Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis. Proc Natl Acad Sci USA 2008;105:12885-12890. [10] Zhao H, Shen J, Medico L, et al. A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer. PLoS One 2010;5:el3735. [II] Hu Z, Chen X, Zhao Y, et al. Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer. J Clin Oncol 2010;28:1721-1726. [12] Reid G, Kirschner MB, van Zandwijk N. Circulating microRNAs: association with disease and potential use as biomarkers. Crit Rev Oncol Hematol 2011:80:193-208. RIGHTS LIN Kt} Soft-tissue extramedullary multiple myeloma prognosis is significantly worse in comparison to bone-related extramedullary relapse Pour L, Sevcikova S, Greslikova H, Kupska R, Majkova P, Zahradova L, Sandecka V, Adam Z, Krejci M, Kuglik P, Hajek R. Haematologica. 2013 Sep 13. PMID: 24038024 IF vroce 2013: 5,935 Articles Multiple Myeloma Soft-tissue extramedullar multiple myeloma prognosis is significantly worse in comparison to bone-related extramedullar relapse Luděk Pour,1 Sabina Sevcikova,23 Henri eta Greslikova,2 Renata Kup ska,2 Petra Majkova,2 Lenka Za hradová,1 Viera Sandecka,1 Zdenek Adam,1 Marta Krejci,1 Petr Kuglik,2 Roman Hajek234 'Department of Internal Medicine - Hematooncology, University Hospital, Brno; 2Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno; ^Department of Clinical Hematology, University Hospital, Brno; and 'Department of Hematooncology, Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic ABSTRACT Even in the era of new drugs, multiple myeloma patients with extra medullary relapse have a poor prognosis. Our goal was to analyze the frequency and outcome of extramedullary relapse occurring in relapsed multiple myeloma patients. In total, we analyzed the prognosis of 226 relapsed multiple myeloma patients treated between 2005 and 2008 and evaluated them for presence of extramedullary relapse. We found evidence of extramedullary relapse in 24% (55 of 226) of relapsed multiple myeloma patients. In 14% (32 of 226) of patients, the lesions were not adjacent to the bone, while extrameduEary relapse adjacent to the bone was documented in 10% (23 of 226) of cases. Patients without extramedullary relapse had significandy longer overall survival than patients with extramedullary relapse (109 vs. 38 months; P<0.001). Moreover, patients with soft tissue-related extramedullary relapse had significantly poorer overall survival compared to bone-related extramedullary relapse patients (30 vs. 45 months; F=0.022). Also, overaE survival from diagnosis was as low as five months for soft tissue-related extrameduEary relapse patients when compared to 12 months overaE survival for bone-related extrameduEary relapse. This is the first study that shows a significant difference in prognosis for different types of extrameduEary relapse. If the extrameduEary myeloma infiltration was not bone-related, overaE survival after relapse was extremely short (5 months). Introduction Multiple myeloma (MM) is characterized by malignant proliferation of clonal plasma cefls that usually produce a unique monoclonal immunoglobuEn. MM comprises approximately 1% of all cancers and is the second most common hematologic malignancy.1 Unfortunately, the etiology of MM is stiE unknown/ Current treatment strategies combine novel agents, such as thalidomide, bortezomib, lenalidomide, with conventional chemotherapy, corticosteroids and autologous stem cell transplantation and significantly prolong long-term patient outcome. More than 30% of patients undergoing intensive treatment Eve for more than ten years, and there is a clear but Emited possibility for some patients with low-risk disease to be cured.3 However, relapse is still a frequent event; whenever it occurs, there is no chance for a cure with current treatment options. Even in relapsed disease, remissions can be obtained in the majority of patients"1'5 although these tend to be shorter than first treatment responses.6,7 Extramedullary myeloma is not frequent but is always associated with a significantly shorter overaE survival, even in the era of novel agents.""0 Extramedullary myeloma (EM) is a type of MM defined by the presence of extraskeletal (i.e. soft tissue or visceral) clonal plasma ceEs infiltrates.12 EM can be present either at the time of initial diagnosis (primary EM) or at the time of relapse (secondary EM).3 Clinically, three types of extrameduEary lesions can be described: a) tumor mass adjacent to bone and extending into soft tissues; b) soft tissue or visceral tumor that is not connected to the bone; or c) diffuse infEtration of organs by plasma ceEs without any obvious focal lesion. However, the majority of studies do not discriminate between these three types of EM lesions. There are very few large studies focusing on incidence of EM, Primary EM is found in approximately 4-16% of MM patients at the time of diagnosis. Secondary EM is found in 6-20% during further MM disease course." Unfortunately, the prognosis of EM patients is generaEy poor, and there is no effective treatment for EM.15 The primary objective of this study was to analyze the frequency and outcome of secondary EM occurring in relapsed MM patients. In addition, we identified the clinical difference and outcome of bone-related and bone-unrelated subtypes of EM. Methods All consecutive relapsed MM patients (in total 226 patients) treated in our department at the University Hospital Brno, Czech Republic, between 2005 and 2008 were included in this analysis and prospectively evaluated for the presence of EM. They were included in this study only after signing the informed consent form approved by the Ethical Committee of die hospital. The median age of patients was 60.8 years (range 27.9-83.5), and the median follow up was 3.7 years {range 0.1-22) after diagnosis. Other base-line patients' characteristics are shown in Table 1. Out of 226 MM patients, we excluded 6.2% (15 of 226) of patients with pri- ©2013 Ferrate Storti Foundation. This is an open-access paper. Haematologica 2013;98. doi:10.3324/haematol.2013.094409 Manuscript received on July 8.2013. Manuscript accepted on September 11,2013. Correspondence: mm. najek@seznam. cz 360 haematologlca | 2014; 99(2) Poor outcome for soft-tissue extramedullar/ relapse mary EM, i.e. evidence of extra me dullaiy disease at the time of MM diagnosis. EM was diagnosed using imaging methods, such as ultrasound, computed tomography (CT) or magnetic resonance (MR). Biopsies were carried out if the lesion was accessible to confirm the presence of clonal plasma cells on cytological or histological examination. EM was defined as the presence of a pathological soft tissue mass by imaging in patients with other findings compatible with MM progression/relapse or a finding of clonal plasma cells in biopsy or aspirate from the extramedullary lesion. Patients with clinical suspicion of extramedullary progression underwent further examinations. All patients with new neurological symptoms had MRI of spine or brain if there were no contraindications. All patients with feptomeningeal infiltration had lumbar punction with flow-cytometric evaluation of cerebrospinal fluid. If there were some contraindications, CT was performed. Ultrasound was performed in patients with hepatic lesion and also if soft tissue infiltration was suspected. Patients with EM were divided into two groups: 1) soft tissue-related EM (EM-S), i.e. the presence of soft tissue or visceral masses not linked to skeletal involvement by MM, or diffuse organ infiltration by malignant plasma cells; 2) bone-related EM (EM-B), i.e. a plasma cell mass adjacent to a bone lesion. Overall survival (OS), time to EM, and previous treatments were analyzed for both groups of patients. The treatment was rather heterogeneous but all patients had received either thalidomide or bortezomib prior to relapse. EM therapy consisted of a novel agent that had not been used previously, in combination widi corticosteroids and chemotherapy. Patients with a good performance status and available stem cell autograft received high-dose chemotherapy with melphalan followed by stem cell rescue. Regimens used for EM treatment included thalidomide in 33 % of patients, bortezomib in 38% and lenalidomide in 5%. Forty-two percent of patients were treated with high-dose melphalan and stem cell transplantation. Chromosomal abnormalities were evaluated using FISH as reported previously.15"18 Statistical analysis Kaplan-Meier estimates were used for survival analysis. Differences between survival times in patient subgroups were tested using the log rank test. Differences in categorical variables were analyzed using the M-L yj test. The level of statistical significance was 5% for all tests. Results We Eound evidence of EM in 24% (55 of 226) of evalu-able relapsed MM patients. In 14% (32 of 226) of these patients, the lesions were not adjacent to bone and thus were classified as EM-S, while EM-B was documented in 10% (23 of 226) of cases. In the EM-S group, the most common site of EM was skin and subcutaneous tissue (69%), while extramedullary masses extending from vertebrae (78%) were most common in the EM-B group. Histological evaluation was performed in 66% of proven EM cases. In other cases, MRI (22%), CT (4%) and ultrasound (2%) were used for proven EM cases (Table 2). EM occurred early in the course of the disease: for 53% of patients (29 of 55 patients) at first relapse, 33% (18 of 55) at second relapse, 14% (8 of 55) at third and higher relapse. In both groups, more than half of patients were diagnosed with EM during the first relapse (Table 3). Time from diagnosis to EM relapse was similar for both EM-S and EM-B groups and the difference was not statistically significant (21 vs. 23 months). Conventional chemotherapy was used in 20% (11 of 55) of patients prior to EM relapse. Thalidomide-containing regimens, bortezomib-containing regimens and high-dose chemotherapy with autologous stem cell transplantation had been given to 38% (21 of 55), 29% (16 of 55) and 53% (29 of 55) of patients, respectively. Differences in the treatment regimens of EM-B and EM-S groups prior to relapse were not statistically significant (Table 4), Median OS for all the 226 MM patients followed was 89 months with median follow up of 44.4 months (range 6.5-264). There was no difference in incidence of EM in gender (P=0.54) or median age at the time of EM diagnosis (^=0.132). Overall survival was significantly longer for patients without EM than for patients with EM (109 vs. 38 months; P<0.001) (Figure 1A). However, there were no differences Table 1. Patients' characteristics. Characteristics H. Gender M/F 115/111 Median age in years (range) 60.8 (27.9-8Í.5) Durie-Salmon stage 1/11/IU 35/41/145 Durie-Salmon stage A/B [87/37 Isotope: IgG/lgA/LCyotber 135/S0/25/16 Light chain: kappa/lambda 142/74 ESS stage l/lllll 57/K5/37 Median follow-up in years after diagnosis (range) 3.7 (0.1-22) EM-S 32 KM B_& ISS: International Staging System, in total. 226 MM patients were analyzed. EM-S: extramedullary relapse - soft tissue, EM-R: extramedullary relapse - bone related. Table 2. Organ involvement of 55 patients with extramedullary relapse of multiple myeloma Involved organ N. [%) Biopsy (%) I Bone related - in total (EM-B) 23 (41.8) 8(14.6) Bone related - spine 18 (32.7) 1(73) Bone related - other 5 (9.1) Soft-tissue related - in total (EM-S) 32 (58.2) 4 (73) 28(51*) Skin 22 (49) 22(«) Central nervous system 2 (IS) Retroperitoneal tumor mass 4 (7.3) 2(3.6) Lung; Í (&*} 1 Lymph nodes 1 (1.8) 1 (1.8) liver 1 (1.8) 1 (1.8) Table 3. Occurrence of extramedullary lesions in relapse of MM. EM-B, N. (%) EMS, N. (%) la relapse 12 (52.2) 17 (53.1) 2"1 relapse 7 (30.4) 11 (34.4) 3"1 relapse 2 (8.7) 3 (9.4) 4» relapse 1(4.3) 1(3.1) 6» relapse 1(4.3) 0 (0.0) haematofogica | 2014; 99(2) 361 L. Pour etat. in overall response rate (ORR) or complete response rate (CR) in first-line treatment between these two groups (P=0.201). Also, TTP after first-line treatment was similar in patients with and without EM (20 months vs. 16 months; ir>=0.112). Interestingly, we did not find any difference between TTP after first-line treatment between the EM-B and EM-S groups of patients (14 months vs. 18 months; ^=0.128). Overall response rate after EM relapse was as low as 24% (13 of 55) in EM patients, with 5% (3 of 55) and 19% (10 of 55) of patients achieving complete and partial responses, respectively. Time to next progression (TTP) was only 5.4 months in EM patients. When we analyzed the two groups of EM patients, we found that the EM-S group of patients had significantly poorer survival compared to EM-B patients (30 vs. 45 months; P=0.022) (Figure IB). Analysis of OS from diagnosis of EM confirmed the poorest outcome for patients with EM-S when compared to EM-B (median OS 5 vs. 12 months; P=0.006) (Figure 2). Results of cytogenetic analysis using FISH at the time of diagnosis were available for 22% (38 of 171) of patients without EM and 24% (13 of 55) of EM patients. The following chromosomal abnormalities with presumed impact on the prognosis of MM1417 were studied: RBI deletion, p53 deletion, IgH gene disruption, translocation t(4;14), amplification lq21 and hyperploidy. No differences were shown in the incidence of any of these chromosomal abnormalities between EM patients and those without EM (Table 5). Discussion Even in the era of new drugs, extramedullary relapse remains incurable. EM has been mostly studied in MM Table 4. Treatment of patients before diagnosis of extramedullary relapse. 1 Treatment EM-B, n.(%) EM-S, n. (%) Conventional regimens 1 (26.1) 5 (15.6) Thalidomide 9 (39.1) 12 (375) Bortezomib S(21.7) 11(34.4) Autologous transplantation 13(56.5) Iii (SM) Interferon alia 11 (47 S) 11(34.4) Table 5. Cytogenetic aberrations of MM patients. We found cytogenetic aberrations in 51 patients at the time of diagnosis, in 38 patients without and 13 patients with extramedullary relapse. There was no statistical difference in incidence of aberrations between both groups. EM relapse N n. (K) o EM relapse P I n. (%) level Deletion RBI positive 13 (54) 37(62) 0.744 Deletion p53 positive 6(33) 25 (8) * IgH gene disruption positive 10 (80) 18 (61) 0.417 Translocation t(4;14) positive 12(33) 26 (31) 1.00 Gain lq21 positive 9(56) 28 (50) 1.00 Hyperdiploidy positive 4(0) 14 (36) * N: number of patients evaluated; %: percent ofpatients with positive aberration out of n patients; * analysis not performed due to tow number of patients. patients treated with high-dose chemotherapy, and most published studies evaluated patients at first relapse.*!521 The incidence of EM has been estimated to be 10-15% of all MM relapses.B,5,!3 Recently, Usmani et at. analyzed 1965 MM patients for presence of EM at the time of MM diagnosis and at the time of relapse. The presence of extramedullary disease at the time of diagnosis was reported between 2.41% and 4.5% depending on the type of therapy. At the time of relapse/disease progression, extramedullary involvement was noted in 3.43-7.24% of patients.3 In our cohort of 226 relapsed MM patients, the incidence of EM, after excluding all patients with known extramedullary disease at the time of diagnosis, was 24%, including 14% of EM-S and 10% of EM-B, respectively. However, the absence of an exact EM definition and consequently different format of EM calculation and reports make correct comparison difficult, even between trials. In our study, we systematically analyzed all consecutive 0 50 100 150 200 250 300 Time (months) B 0.0 0 20 40 60 80 100 120 140 Time (months) Figure i. Overall survival and survival from diagnosis of EM patients. (A) Overall survival from diagnosis in patients with extramedullary relapse (EM) is significantly shorter than in other MM patients (38 vs. 109 months P>0.001). (B) Comparison of survival from diagnosis in patients with bone related extramedullary relapse (EM-B) is significantly longer than in patients with soft-tissue related extramedullary relapse (EM-S) (30 vs. 45 months; P-0.022) 362 haematotogica | 2014; 99(2) Poor outcome for soft-tissue extra medullary relapse 0 10 20 30 40 Time (months) Figure 2. Overall survival from diagnosis of patients with bone related extramedullar/ relapse (EM-B) is significantly longer than in patients with soft-tissue related extra medullary relapse (EM-S) (4 vs. 12 months; P=0.006). patients who relapsed during a 3-year period, from 2005 to 2008; up to 53% of EM occurred in the first relapse. We were not able to detect any association between EM relapse and any novel agent (thalidomide or bortezomib). In this single center experience, EM-B was observed only in 2% (4 of 113) of patients who underwent initial therapy with classical treatment protocol without any novel agents in the period 1996-2002 (4 x VAD and melphalan 200 mg/m3).11 The most important finding in our analysis is the significant difference in prognosis for the two different types of EM. In accordance with the results of most research groups, we noted survival of approximately 12 months in our EM patients if the extramedullary mass was adjacent to the bone. However, if the extramedullary myeloma infiltration was not bone-related, the overall survival was extremely short and not longer than four months. Based on these results, it is possible to divide patients with EM relapse into two different prognostic groups: 1) 'bone-related' extramedullary myeloma (EM-B), i.e. myeloma mass is adjacent to a distinct bone. The patients with this type of EM have an OS that is less than 50% shorter than patients who relapse without the EM component; 2) 'bone-unrelated' extramedullary myeloma (EM-S), i.e. myeloma mass of soft tissue with no relation to the bone. Such patients have the worst prognosis with a limited OS of less than 4-6 months. This would suggest that the biological behavior of these two types of EM is probably different. Clear identification of one type of EM from another is now easily available with widespread use of PET/CT and/or whole body MRI and should be standardized in every future published cohort of EM patients. Our analy- sis covers only the relapsed setting, and the prognostic significance of the subtype of EM remains to be validated in primary EM. Thus, we believe that all studies analyzing EM should include details about primary or secondary EM as well as division of patients into EM-S and EM-B groups. We completely excluded patients with primary EM from our analysis. We strongly believe that it is important to do so as we found that OS and TTP are similar after first-line treatment in all MM patients. However, survival rapidly decreases if EM develops during the course of the disease. Similarly, the presence of EM at the time of diagnosis was a very strong negative prognostic factor in the Arkansas analysis; it was the only parameter that was significantly important in all groups of patients. Also, patients with primary EM had the worse prognosis.3 According to these data and our results, it is clear that the prognosis of MM patients is similar until the development of EM. But after this event, survival is very poor for patients with EM regardless of whether it is primary or secondary EM. Our cohort of relapsed EM patients is one of the largest ever published. While no patient with skin and subcutaneous plasmocellular masses was seen in our center prior to 2005, as many as 22 cases were diagnosed between 2005 and 2008. It is likely that the recent changes in treatment strategies are associated with the increased incidence of EM, although the trend is unlikely to be associated with any particular drug. Our results suggest that the incidence of soft tissue EM is increasing in the era of novel drugs. These data need to be confirmed in prospective studies and comparative studies with historical controls. Many analyses, including ours, show that survival of MM patients has improved significantly after the implementation of novel therapeutic regimens.2,11,13,32,23 However, patients with EM generally do not benefit from these agents and their survival remains extremely poor. Thus, EM remains one of the major ongoing issues in the care of MM patients. Acknowledgmen ts The authors would like to thank all patients and their caregivers without whom this study would not have been possible, as well as nurses and data managers who helped with this study. We would like to thank John B. Smith for proofreading the manuscript. Funding This study was supported by grants of the Ministry of Health NT1Z150 and NT4H54, grant of The Ministry of Education, Youth and Sports MSM0OZ16224M and grant MUNI/11/InGAi 7/201Z. Authorship and Disclosures Information on authorship, contributions, and financial <$2 other disclosures was provided by the authors and is available with the online version of this article at www.haematoiogica.org. References 1. Hajek R, Krqci M, Pour L, Adam Z. Multiple myeloma. Kin Onkol. 2011;(24 SuppI):S10-3. 2. Harousseau JL, Shaughnessy J, Richardson P. Multiple myeloma. Hematology Am Soc Hematol Educ Program. 2004:237-56. 3. Usmani SZ, Heuck C, Mitchell A, Szymonifka J, Nair B, Hoering A, et al. Extramedullary disease portends poor prognosis in multiple myeloma and is over-repre- sented in high-risk disease even in the era of novel agents. Haematologica. 2012;97(11): 1761-7. 4. Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, et al. A phase 2 study of bortezomib in relapsed, haematologica | 2014; 99(2) 363 L. Pour etal. refractory myeloma. N Engl J Med. 2003] 348(26):2r509-17. 5. Dimopoulos M, Spencer A, Attal M, Prince HM, Harousseau JL, Dmoszynska A, et al. Lenalidomide plus dexamethasone for relapsed or refractory multiple myeloma. N Engl J Med. 2007,357(21)=2123-32. 6". Krivanova A, Hajek R, Krejci M, Scudla V, Indrak K, Bacovsky J, et al. Second autologous transplantation for multiple myeloma patients relapsing after the first autograft — a pilot study for the evaluation of experimental maintenance therapies. Report of the prospective non-randomized pilot study of the Czech Myeloma Group. Onkologie. 2004,27(3):275-9. 7. Barlogie Bp Anaissie E, van Rhee E, Shaughnessy JD, Szymonifka J, Hoering A, et al. Reiterative survival analyses of total therapy 2 for multiple myeloma elucidate follow-up time dependency of prognostic variables and treatment arms. J Clin Oncol. 2010;28(18):3O23-7. 8. Terpos Er Rezvani Kh Basu S, Milne AE, Rosc-PE, Scott GL, et aL Plasmacytoma relapses in the absence of systemic progression post-high-dose therapy for multiple myeloma. Eur J Haematol. 2005;75(5):376^3. 9. Alegre A, Granda A Martinez-Chamorro Ch Diaz-Mediavilla J, Martinez R, Garcia-Laraha Jr et al. Different patterns of relapse after autologous peripheral blood stem cell transplantation in multiple myeloma clinical results of 280 cases from the Spanish Registry. Haematologica. 2002;87(6):609-14. 10. Srikanth M, Davies^FE, Wu P, Jenner MW, Ethell ME, Potter MNh et al. Survival and outcome of blastoid variant myeloma following treatment with the novel thalidomide containing regime DT-PACE. Eur J Haematol. 2008,31(6) :432-6". 11. Laura R^ Cibeira MT, UriburuC, Yantorno S, Salamero O, Blade J, et al. Bortezomib: an effective agent in extramedullary disease in multiple myeloma. Eur J Haematol. 2006^76 (5):405-8. 12. Blade J, Fernandez de Larrea C, Rosin ol L, Cibeira MT, Jimenez R, Powles R, Soft-tissue plasmacytomas in multiple myeloma: incidence, mechanisms of extramedullary spread, and treatment approach. J Clin Oncol. 2011,29(28):3805-12. 13. Weber DM. Solitary bone and extramedullary plasmacytoma. Hematolo Am Soc Hematol Ecuc Program. 2005:373-6. 14. Oriol A Multiple myeloma with extramedullary disease. Adv Trier. 2011^28 (Suppl 7):l-6. 15. Varettoni M, Corso A, Pica G, Mangiacavalli S, Pascutto C, Lazzarino M. Incidence, presenting features and outcome of extramedullary disease in multiple myeloma: a longitudinal study on 1003 consecutive patients. Ann OncoL 2010;21(2):325-30. 16. Nemec F, Zemanova Z, Greslikova H, Michalova K, Eilkova H, Tajtlova J, et al. Gain of lq21 is an unfavorable genetic prognostic factor for multiple myeloma patients treated with high-dose chemotherapy. Biol Blood Marrow Transplant 2010flo~(4):548-54. 17. Greslikova Hp Zaoralova R, Eilkova Hp Nemec P, Oltova A, Kupska R, et al. Negative prognostic significance of two or more cytogenetic abnormalities in multiple myeloma patients treated with autologous stem cell transplantation. Neoplasma. 2010,57(2): 111-7. 18. Fon&eca R, Bergsagel PL, Drach J, Shaughnessy ], Gutierrez N, Stewart AK, et al. Lntemational Myeloma Working Group molecular classification of multiple myeloma: spotlight review. Leukemia. 2009^23 (12):2210-21. 19. Zeiser R, Deschler B, Bertz HP Einke J, EngelhardtM. Extramedullary vs medullary relapse after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) in multiple myeloma (MM) and its correlation to clinical outcome. Bone Marrow Transplant 2004;34(12):1057-65. 20. Perez-Simon JA, Sureda A, Femandez-Aviles F, Sampol A, Cabrera JR, Caballero D, et al. Reduced-intensity conditioning allogeneic transplantation is associated with a high incidence of extramedullary relapses in multiple myeloma patients. Leukemia. 2006;20 (3):542-5. 21. Zamagni E, Nanni C, Patriarca EP Englaro E, Castellucci PP Geatti O, et al. A prospective comparison of 18F-fluorodeoxyglucose positron emission tomography-computed tomography, magnetic resonance imaging and whole-body planar radiographs in the assessment of bone disease in newly diagnosed multiple myeloma. Haematologica. 2007;92(l):50-5. 22. Krejci M, Scudla V, Tothova Ep Schutzova M, Koza Y Adam Z, et al. Long-term outcomes of autologous transplantation in multiple myeloma: significant survival benefit of novel drugs in post-transplantation relapse. Clin Lymphoma Myeloma. 2009;9(6):A36A2. 23. Kumar S, Fiinn I, Richardson PG, Hari Pp Callander Np Noga SJ, et al. Randomized, multicenter, phase 2 study (EVOLUTION) of combinations of bortezomib, dexamethasone, cyclophosphamide, and lenalidomide in previously untreated multiple myeloma. Blood. 2012;119(19):4375-82. 364 haematologica | 2014; 99(2) Inhibitory proteazomu v léčbě mnohočetného myelomu Kubiczková L., Matějíková J., Sedlaříková L., Kryukov F., Hájek R., Ševčíková S. Klinická onkologie. 2013; 26(l):ll-8 PMID: 23528167 IF v roce 2013: - PŘEHLED Inhibitory proteazomu v léčbě mnohočetného myelomu Proteasome Inhibitors in Treatment of Multiple Myeloma Kubiczková L., Matějíková J., Sedlaříková L., Kryukov R, Hájek R., Ševčíková S. Babákova myelomová skupina, Ústav patologické fyziologie, LF MU Brno Souhrn Mnohočetný myelom, maligní onemocnění plazmatických buněk, zůstává stále velmi obtížně léčitel ným h ema too nko logic kým onemocněním, pro které je nutné hledat nové možnosti terapie ovlivňující jak plazmocyty samotné, tak i mikroprostředí kostní dřené. Klonálníplazmocyty se vyznačují zvýšenou regulací ubikvitin-proteazomové kaskády, což zvyšuje jejich citlivost k působení inhibitorů proteazomu. Léčebné přístupy využívající inhibitory proteazomu patří do éry nových léků a v posledních letech se ukázaly být velice důležitou součástí léčby pacientů s mnohočetným myelomem. Prvním inhibitorem proteazomu schváleným pro léčbu mnohočetného myelomu se stal bortezomib, který vykazoval silné antimyelomové účinky. Bohužel, navzdory jeho vysoké účinnosti se u velkého procenta pacientů s mnohočetným myelomem pacientů po čase objevuje rezistence k jeho působení. Ve snaze překonat rezistenci k borte-zomibu a vyvinout inhibitor proteazomu s lepším toxickým profilem byty vyvinuty inhibitory proteazomu druhé generace - carfilzomib, marizomib a MLN9708, které by mohly nadějně změnit průběh mnohočetného myelomu v onemocnění chronické. Klíčová slova mnohočetný myelom - inhibitory proteazomu - bortezomib - carfilzomib - marizomib -MLN9708 Summary Multiple myeloma, a plasma cell malignancy, still remains a hard-to-treat hematological disease that desperately needs new therapy targeting plasmocytes but also the bone marrow micro-environment Clonal plasmocytes are characterized by increased regulation of ubiquitin-pro-teasome pathway which augments their sensitivity to proteasome inhibitors. Treatment strategies based on proteasome inhibitors belong to the era of new drugs, and they have become increasingly important for treatment of multiple myeloma in recent years. Bortezomib became the first proteasome inhibitor approved for the treatment of multiple myeloma and showed remarkable anti-myeloma activity. However, despite its high efficiency, a large proportion of patients have became bortezomib resistant. The second generation of proteasome inhibitors -carfilzomib, marizomib and MLN9708 - were developed in an effort to overcome bortezomib--resistance and find proteasome inhibitors with a better toxic profile. These drugs brought a chance that multiple myeloma would become a chronic disease. Key words multiple myeloma - proteasome inhibitors - bortezomib-carfilzomib-marizomib-MLN9708 Práce byla podpořena výzkumnými projekty: Ministerstva školství, mládeže a télo-výchovy MSM0021622434, Grantové agentury ČR GAP304/1071395 a Interní grantové agentury Ministerstva zdravotnictví NT13190 a NT)) 154. This study was supported by scientific program of the Czech Ministry of Education, Youth and Sports No 1622434, by grant of C.'.-ch Science Foundation Mo. GAP304/10/1395 and by grants of Internal Grant Agency of the Czech Ministry of Health No. NT13I90 and NT) 1154. Autoři deklaruji, že v souvislosti s předmětem studie nemají žádné komerční zájmy. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. Redakční rada potvrzuje, že rukopis práce splnit ICMJE kritéria pro publikace zasílané do biomedíctnských časopisů. The Editorial Board declares that the manuscript met the ÍCMJE 'uniform requirements" for biomedical papers. R N Dr. Sabina Ševčíková, Ph.D. Babákova myeíorrtová skupina Ústav patologické fyziologie LF MU Kamenice 5, A3 625 00 Brno e-mail: sevcik@med.muni.cz Obdrženo/Submitted: 2.10.2012 Prijato/Accepted: 15. Í0.2Q12 KlinOnkol 2013; 26(1): 11-18 1 1 INHIBITORY PROTEAZOMU V LÉČBĚ MNOHOČETNÉHO MYELOMU Úvod - inhibitory proteazomu Proteazom jako nová buněčná struktura byl poprvé identifikován na počátku 70. let skupinou doktora Harrise [1]. Mnoho dalších objevů bylo učiněno později, koncem let 70. a počátkem let 80. v laboratoři prof. Avrama Hershka, a bylo zjištěno, že jeho funkce spočívá v ATP-dependentní degradaci intracelu-lárních proteinů a jeho specifita je dána interakcí pouze s takovými proteiny, které jsou označeny polyubikvitinovým řetězcem anebo obsahují specifickou sekvenci aminokyselin [2,3]- Za tento významný objev získali prof. Ciechanover, Hershko a Rose v roce 2004 Nobelovu cenu za chemii. Proteazom je útvar cylindrického tvaru. Skládá se ze čtyř homolog-ních prstenců tvořených sedmi pod-jednotkami a nebo B, které jsou nad sebou uspořádány v pořadí a-B-B-a [4]. V této struktuře lze rozeznat tři různé typy proteolytických podjednotek -BI, B2 a B5. Každá z nich obsahuje aktivní místo na svém N-konci, kde se nachází zbytek aminokyseliny threoninu (Thrl) [51. Ačkoli proteazom obsahuje více katalytických míst, k inhibici jeho funkce postačuje pouze zablokování podjednotky 85, která vykazuje chy-motrypsinovou aktivitu [6]. V hemato-poietických buňkách je hlavním typem proteazomu jeho inducibilní izoforma - imunoproteazom, jehož výskyt koreluje s hladinou cytokinů. Je charakterizován nahrazením proteolyticky aktivních podjednotek p1, p2 a 65 jejich Obr. 1. Struktura bortezomibu {The Pub-Chem Project). ekvivalenty Bii (LMP2), B2i (MELO) ap5i(LMP7) 17]. Nejčastějšími proteiny určenými k degradaci jsou špatně sbalené proteiny a proteiny s krátkým biologickým poločasem, které mají většinou regulační funkci [8]. Produkty proteolytické reakce jsou pak oligopeptidové řetězce s průměrnou délkou 8-12 aminokyselin [9]. Použití inhibitorů proteazomu (IP) patří v současné době mezi jedny z nej-úspěšnéjších strategií pro léčbu mnoho-četného myelomu (MM), nádorového onemocnění způsobeného maligní transformací B lymfocytů s charakteristickou klonální proliferací a akumulací plazmatických buněk v kostní dřeni [10]. IP jsou zpravidla krátké peptidy, ke kterým je kovalentně připojen farmako-for - skupina atomů, která se váže do katalytických míst proteazomu, a tím zabraňuje jeho správné funkci. Konečným důsledkem inhibice proteazomu v mye-lomových buňkách je indukce apopto-tických drah, překonání rezistence ke konvenční chemoterapii a senzitizace vůči dalším terapeutikům. V tomto přehledovém článku se zaměříme na mechanizmus účinku a roli IP v léčbě mnohočetného myelomu. První generace inhibitorů proteazomu Bortezomib Bortezomib, známý také pod označením PS-341 a komerčním názvem Vel-cade (Millenium Pharmaceuticals), je prvním a jediným IP, který by! doposud oficiálně schválen pro klinickou praxi. Po chemické stránce se jedná o dipeptidy-lový derivát kyseliny borité se sumárním vzorcem C19H25BN404 (obr. 1). Poprvé byl bortezomib syntetizován v polovině 90. let minulého století společností Myogenics [11]. O jeho vysoké specifitě, účinnosti a oxidační stabilitě vypověděly výsledky studií in vitro u 60 rakovinných linií [12]. První klinická studie s Velcade pro léčbu hematologic-kých malignit byla zahájena v listopadu 1999. Tým pod vedením dr. Orlowského ve studii s nízkými dávkami léku, sloužícími pouze k ověření jeho bezpečnosti, zaznamenal úplné vymizení příznaků (CR) MM u 47leté pacientky. U ostatních osmi pacientů z ceikového počtu jede- náct došlo alespoň k minimální léčebné odpovědi (MR) nebo stabilizaci onemocnění [13]. Výsledek to byl natolik převratný, že po ověření v dalších fázích klinických studií vedl k urychlenému schválení bortezomibu pro léčbu rela-bujícího a refrakterního myelomu v roce 2003 v USA a o rok později také v České republice [14,15]. Mechanizmus účinku Mechanizmus inhibice proteazomu bor-tezomibem spočívá v jeho kovalentní vazbě na B5 podjednotku, případně LMP7 podjednotku imunoproteazomu. S nižší afinitou se bortezomib váže také na podjednotky BI a p2 [16]. Rozdílnost v afinitě je dána odlišnými interakcemi postranních řetězců inhibitoru s jednotlivými podjednotkami [17]. Vazebná konformace je zaujímána v podobě antiparalelního p skládaného listu, který je stabilizován vodíkovými můstky mezi atomy hlavního řetězce agens a konzervovanými zbytky katalytických míst (Gíy47N,Thr21N,Thr210 a Ala490). Samotná inhibice je zprostředkována farmokoforovou skupinou, v tomto případě zbytkem kyseliny borité. Atom boru zde kovalentně in-teraguje s nukleofilem, kterým je volný elektronový pár kyslíku ThrlO. Vzniklá elektronová mezera je stabilizována vazbou Gly47N na hydroxyl boru. Tetrahed-rická struktura proteolyticky inaktivního produktu je dále zpevněna vazbou druhého hydroxylu na aminoskupinu v katalytickém místě [17]. Vzniklý adukt je charakterizován nízkým stupněm diso-ciace, a proto, i když se jedná o rever-zibilní reakci, zůstává po několik hodin prakticky vysoce stabilní. Jelikož je proteazom zapojen do obratu intracelulárních proteinů, patří mezi primární důsledky jeho inhibice hromadění nefunkčních proteinů a chyby v signálních drahách, které vyúsťují v narušení adheze myelomových buněk, potlačení novotvorby cév, zastavení buněčného cyklu, omezení odpovědi na poškození DNA a indukci apoptózy [18]. Původní hypotézou hlavního biologického účinku bortezomibu na myelo-mové buňky byla inhibice transkripčního faktoru NF-kB, a tím zabránění tran- 12 KlinOnkol 201 3; 26(1): 11-18 INHIBITORY PROTEAZOMU V LÉČBĚ MNOHOČETNÉHO MYELOM U škripce genů, které blokují apoptózu. Hlavní zástupce NF-kB se nachází v cyto-plazmě v podobě inaktivního komplexu s vlastním inhibitorem I-kB, jehož degradace proteazomem je klíčem k aktivaci samotného transkripčního faktoru. Model proteazomové inhibice počítal se zastavením degradace I-kB, a tedy konstitutivní inhibicí NF-kB [19]. Ačkoli se tento předpoklad nepotvrdil, existuje ještě alternativní nekanonická dráha aktivace NF-kB, která by eventuálně mohla být bortezomibem blokována [20]. Klíčovou událostí v navození apoptózy myelomových buněk bortezomibem zůstává aktivace iniciačních kaspáz 8 a 9, které předávají apoptotický signál efekto-rovým kaspázám štěpícím obsah buňky zevnitř. Iniciační kaspázy mohou být aktivovány nejrůznějšími způsoby. U bortezomibem navozené inhibice protea-zomu byla pozorována zvýšená aktivita c-Jun N-terminální kinázy (JNK), která souvisí s apoptózou odehrávající se přes Fas receptor. Fas patří do rodiny TN F receptoru, které po navázání ligandu spouštějí proapoptotický signál, který, pokud není interferován, vyústiv buněčnou smrt. Zablokováním přirozeného obratu intra-celulárních proteinů dochází také k disproporci onaci apoptotických proteinů rodiny B c 1-2 ve prospěch jejích proapoptotických členů, což vyústí v permeabilizaci vnější mitochondriální membrány a taktéž v aktivaci efektorových kaspáz. Další cestou je aktivace proteinu p53 s výraznými proapoptotický m i účinky a jeho následná stabilizace štěpením příslušného ubikvitín-li-gačn ího enzymu MDM2 [21,22]. Dále bortezomib zabraňuje opravám poškozené DNA, indukuje stres endoplaz-matického retikula (ER) v buňkách MM, snižuje adhezi nádorových buněk k buňkám kostní dřeně inhibicí signalizační dráhy MAPK, zabraňuje nádorové angio-genezi a podílí se na apoptóze osteoklastů a diferenciaci osteoblastů [23,24] (obr. 2). Klinické studie Klinické studie fáze II, SUMMIT a CREST, potvrdily antimyelomový účinek borte-zomibu, který byl pozorován ve studiích pře klinických fází a v první fázi klinického výzkumu [25,26]. Do studie SUMMIT bylo zařazeno 202 pacientů, z nichž 193 bylo následně hodnoceno. Léčebné odpovědi dosáhlo 35 % z nich, přičemž kompletní remise byla pozorována v sedmi případech, a to ve velmi krátké době (medián odpovědi 1,3 měsíce). U dalších 24 % pacientů byla pozorována stabilizace nemoci. Navíc, 74 pacientů s nedostatečnou odpovědí na monoterapii bortezomibem dostalo v kombinaci dexametazon a v 13 případech bylo dosaženo následného zlepšení léčebné odpovědi [25]. Mezinárodní randomizovaná klinická studie fáze III APEX srovnávala účinnost bortezomibu v porovnání s dexameta-zonem. Do studie bylo zapojeno 669 pacientů s relabujícím MM, z nichž bylo nakonec vyhodnoceno 627. Již při prvních výsledcích bylo zřejmé, že bortezomib je signifikantně mnohem účinnější než dexametazon v léčebné odpovědi (38 % vs 18 %), kompletní remisi (6 % vs < 1 %) i v jednoročním přežití (80 % vs66%) [18]. Klinická studie fáze III VISTA byla podkladem pro schválení bortezomibu pro narušení mechanizmu opravy DNA indukce stresu ER u MM buněk aktivace kaspáz 8 a 9 inhibice PÍ3/AKT, MAPK signálni dráhy snížení a ntia poptotických proteinů rodiny BCL-2, stabilizace proapoptotických členů rodiny BCL-2 degradace IkB stromální buňky kostní dřcnč rusta přežíváni MM buněk aktivace NF-kB dráhy a transkripce MM buněčných cytokinů J Obr. 2. Mechanizmus působení bortezomibu. Bortezomib blokuje proces degradace IkB, která je klíčem k aktivací NFkB. Dále bortezomib snižuje adhezi plazmatických buněk ke stromálnim buňkám kostní dřeně a navozuje apoptózu aktivaci kaspáz 8 a 9. Bortezomib v buňce narušuje mechanizmus opravy DNA, indukuje apoptózu narušením membrány endoplazmatického retikula, inhibuje MAPK signální dráhy, snižuje hladiny a ntia poptotických proteinů a stabilizuje hladiny proapoptotických proteinů. Klin Onkol 2013; 26(1): 11-18 13 INHIBITORY PROTEAZOMU V LÉČBĚ MŇOHOČETNÉHO MYELOMU primoléčbu. Cílem její pozdější analýzy bylo potvrdit, že kombinace bortezo-mibu s melfalanem a prednisonem zvyšuje celkové přežití nemocných [27]. Minulý rok vyšla studie francouzské skupiny, která porovnávala použití bortezomibu subkutánně (s.c.) proti intravenóznímu podávání (i.v.) s překvapivými výsledky. Ve studii bylo hodnoceno celkem 222 nemocných rando-mizovaných v poměru 2 : 1 (s.c. : i.v.). Z výsledků studie vyplývá, že všechny sledované parametry související s léčebnou účinností léků byly velmi podobné a nezávislé na použití aplikační cesty. Ale četnost nežádoucích účinků při podkožním podání byla pro určité typy toxicit nejméně o 10 % nižší (gast-rointestinální, dýchací potíže a potíže související s mediastinem a hrudníkem, průjem), především však byla nižší četnost výskytu periferní polyneuropatie, která při intravenózním podání bortezomibu znamená závažný problém. Periferní neuropatie stupně 2 a výše byla pozorována u 38 % při podkožním podání oproti 53 % u nemocných s intravenózním podáním. Pro polyneuropatie stupně 3-4 byly poměry rovněž ve prospěch podkožního podání (6 % vs 16%). Při podkožním podání nelze konstatovat, že by poškození nervů přestalo být problémem, ale jde o významnou redukci četnosti nežádoucího účinku výhradně změnou cesty podání [28]. V EU se přechází na podání bortezomibu jednou týdně, lze tedy očekávat - v kombinaci s podkožním podáním - další redukci četnosti polyneuropatie. Evropská komise EMA již odsouhlasila na základě těchto výsledků možnost podkožního podání bortezomibu v běžné praxi. Toxicita, rezistence a limity Původní hypotéza, že biologické účinky inhibice proteazomu bortezomibem jsou nezávislé na typu buňky s fatálním dopadem pro lidský organizmus, byla vyvrácena. Faktem však zůstává, že působení bortezomibu na ostatní buňky organizmu se odráží v podobě relativně vysoké míry nežádoucích účinků. Mezi ty hematologické patří trombocytope-nie, kdy s každým podáním bortezomibu dochází k poklesu počtu trombo-cytů. Dále anémie, dyspeptické poruchy a zejména periferní neuropatie, které bývají hlavním důvodem pro ukončení léčby [13,15,29]. Periferní neuropatie se vyskytuje s incidencí 35-55 % po podání bortezomibu a je pravděpodobně způsobena účinkem bortezomibu na prote-ázu HtrA2/Omi - stresem indukovanou proteázu důležitou pro přežití nervových buněk a formaci neuritů [30]. Navzdory vysoké účinnosti bortezomibu disponuje až 60 % pacientů primární rezistencí nebo se u nich v průběhu léčby vyvine rezistence sekundární [25]. Doposud bylo identifikováno několik málo molekulárních mechanizmů, pomocí kterých tyto rezistence mohou vznikat. Jedním z nich jsou mutace v propeptidovém lo-kusu pro B5 podjednotku proteazomu a její nadměrná syntéza [31]. Se zvýšenou rezistencí nádorových buněk také koreluje zvýšená hladina anti-apoptotických proteinů rodiny Bcl-2 a proteinů tepelného šoku Hsp 27, 70 a 90 [32,33]. 5tudie Zhang et al (2011) odhalila, že u buněčných linií rezistentních k bortezomibu zůstala enzymatická aktivita proteazomu inhibovaná i po další léčbě bortezomibem stejně jako u buněčné linie citlivé k tomuto léku. Tyto výsledky naznačují, že mechanizmus rezistence se objevuje až později v signalizační kaskádě [34]. V poslední době se navíc ukázalo, že přírodní produkty obsahující vicinální dioly jsou schopné inhibovat účinek bortezomibu vazbou na zbytek kyseliny borité. Mezi tyto produkty patří zelený čaj obsahující epigalokatechin-3-galát a vitamin C [35,36]. Druhá generace inhibitorů proteazomu Úspěch bortezomibu vzbudil zájem vědecké obce o proteazomové inhibitory. Optimalizace dávek a kombinace bortezomibu s jinými proti nádorovým i te-rapeutiky sice omezily jeho vedlejší účinky a částečně potlačily rezistenci, bylo však jasné, že druhá generace inhibitorů proteazomu může přinést daleko lepší výsledky. Carfilzomib, Marizomib a MLN9708 reprezentují druhou generaci IP a nabízejí řadu výhod v podobě zvýšené účinnosti, bezpečnosti lékového profilu a překonání rezistence k bortezomibu díky své odlišné chemické struktuře, biologickým vlastnostem, mechanizmu účinku, i/reverzibilité inhibice proteazomu a způsobu užívání [37]. Carfilzomib Carfilzomib (známý též jako PR-171, Kyprolis, Onyx Pharmaceuticals) je tetra-peptidový epoxyketon se sumárním vzorcem C40H57N5O7 (obr. 3). V pre-klinických výzkumech byl identifikován jako vysoce potentní IP. Bylo prokázáno, že dokáže navodit apoptózu u bortezo-mib-naivních i u bortezomibem před-léčených myelomových buněk bez zvýšené toxicity a je schopen překonat primární i sekundární rezistenci [38,39]. Ačkoli mechanizmus překonání rezistence nebyl doposud zcela objasněn, jedním z možných vysvětlení může být odlišný způsob a typ vazby, který dovoluje obejít důsledky mutací v genech pro proteazomové podjednotky. Jiným vysvětlením může být jeho vyšší selektivita vůči katalytickým podjednotkám imu-noproteazomu, jehož význam doposud nebyl dostatečně reflektován [39-41]. V červenci 2012 schválila FDA carfilzomib pro léčbu pacientů s MM, kteří už mají za sebou více než dvé linie léčby včetně bortezomibu a nějakého imu-nomodulačního léku a u kterých došlo k progresi onemocnění do 60 dnů od ukončení předchozí léčby (www.onyx. com). Předpokládá se, že nejpozději do dvou let od schválení v USA bude lék schválen i pro Českou republiku. Mechanizmus působení Inhibice chymotrypsinové podjednotky proteazomu carfilzomibem je z mechanického hlediska ireverzibilní reakcí snižující aktivitu proteazomu na méně než 20 %. Znovunastolení proteazomové aktivity v buňce je možné pouze na-syntetizováním jednotlivých podjed-notek a jejich sestavením do nových proteazomu [38]. Carfilzomib se primárně navazuje na B5 katalytickou podjednotku proteazomu a LMP7 podjednotku imunopro-teazomu s vyšší selektivitou než borte-zomib [7]. Navázáním epoxybutanového farmakoforu, který vykazuje vysokou specifitu vůči hydroxylové a především 14 KlinOnkol 201 3; 26(1): 11-18 INHIBITORY PROTEAZOMU V LÉČBĚ MNOHOČETNÉHO MYELOMU aminové skupině N-terminálního Thrl, vzniká šestičlenný morfolinový kruh. Tato intermolekulámí cyklizace probíhá dvoustupňovým mechanizmem, kdy tedy kyslík hydroxylové skupiny Thrl nukleofilně atakuje uhlík epoxyketonu za vzniku hemiacetalu. Druhým krokem je nukleon lni atak a-amino dusíku Thrl na C2 uhlík epoxidového kruhu, což má za následek vytvoření morfolinového aduktu [42,43], V buňkách MM vystavených působení carfilzomibu byla pozorována indukce vnější i vnitřní apoptotické kaskády s výrazným zvýšením hladiny kaspázy 3, 7, 8 a 9. Programovaná buněčná smrt byla asociována s aktivací JNK, depola-rizací mitochondriální membrány, vylitím cytochromu c, počátečním poklesem fosforylovaného elF2 v souvislosti se stresem endoplazmatického retikula navozeným akumulací nefunkčních proteinů a zvýšením hladiny proapoptotic-kého proteinu Noxa, který je členem rodiny proteinů Bcl-2 [7,38]. Klinické studie První klinická studie s carfilzomibem pro léčbu hematologických malignit byla zahájena v září 2005. Studie 29 pacientů prokázala snášenlivost a klinickou aktivitu terapeutika. Objektivní léčebné odpovědi bylo dosaženo u dvou z deseti pacientů s MM [44]. Pokračováním fáze I bylo vyhodnocení bezpečnosti a účinnosti carfilzomibu v kombinaci s lenalidomidem a dexametazonem, standardními léky pro pacienty s re-labujícím myelomem. Ačkoli se jedná teprve o první zhodnocení studie, Nie-svizky et al (2009) zaznamenali klinický přínos v 78 % případů [45]. U šesti pacientů došlo ke kompletní remisi a nebyly pozorovány závažné vedlejší účinky. Výsledky by měly být potvrzeny v rámci fáze III začínající klinické studie ASPIRE. Nedávno byly publikovány výsledky otevřené multicentrické studie fáze II s carfilzomibem v monoterapii pro relabující/ /refrakterní myelom. Do studie bylo zařazeno 129 pacientů, z nichž 47,6 % dosáhlo léčebné odpovědi. Ve třech případech došlo ke kompletní remisi [46]. V klinické studii fáze l/ll u nově diagnostikovaných pacientů s MM léčených kombinací carfilzomibu, lenalidomidu a nízko dávkovaného dexametazonu dosáhlo 62 % pacientů téměř kompletní remise [47]. Odpovědna léčbu se zlepšovala v průběhu času a tento léčebný režim byl vyhodnocen jako vysoce účinný a dobře tolerovatelný. Toxický profil carfilzomibu není zdaleka tak bohatý jako u bortezomibu. Periferní neuropstie byly pozorovány u relativně nízkého procenta pacientů a nejčastějšími vedlejšími účinky byly únava, anémie, trombocytopenie a nauzea [46]. Marizomib Marizomib, známý také jako NPI-0052 nebo Salinosporamid A, je prvním přírodním proteazomovým inhibitorem, který byl zařazen do klinického výzkumu MM. Jedná se o produkt obligátní mořské bakterie, aktinomycéty Salinispora tropica [48]. Po chemické stránce je marizomib bicyklo v-laktam-B-lakton se sumárním vzorcem C15H20CINO4 (obr.4). Na rozdíl od předešlých IP ve své struktuře neobsahuje pepttdový řetězec. V preklinických výzkumech s buněčnými liniemi MM bylo prokázáno překonání rezistence na konveční terapie a léčbu bortezomibem spolu s vyšší účinností. Kombinace bortezomibu s marizomi-bem by mohla umožnit používat takovou koncentraci jednotlivých léků, která nepůsobí na pacienty toxicky a zlepšuje společný antimyelomový účinek jednotlivých léků [49]. Mechanizmus působení Marizomib se přednostně navazuje do B5 a B1 katalytického místa proteazomu a s nižší afinitou také na P2 podjednotku. Za ireverzibilnost vazby zodpovídá chloretylová skupina substituující B-lak-ton. Skupina se navazuje do S2 vazebné kapsy aktivního místa a chlor se chová jako odstupující atom, čímž umožní vytvořit stabilní komplex konečného produktu po acylaci Thrl O p-laktonem inhibitoru [49]. Na rozdíl od bortezomibu, který aktivuje kaspázu 8 i 9, je apoptotický účinek marizomibu zprostředkován především kaspázou 8, v menší míře pak kaspázou 9, ale s odlišným mechanizmem aktivace než u bortezomibu, čímž překonává rezistenci myelomových buněk s mu- Obr. 3. Struktura carfilzomibu (The Pub-Chem Project). tacemi v genech pro proteiny rodiny Bcl-2. Dochází tedy k uvolnění cytochromu c a proteinu Smac z mitochon-driídocytosolu, generaci kyslíkových radikálů a aktivaci výše zmíněných kaspáz. Studie Chauhana et al (2005) prokázala, že marizomib je schopen indukovat apoptózu u myleomových buněk dokonce i v přítomnosti myelomových růstových faktorů, IL-6 a IGF-1 [32]. Navíc se podílí na zablokování sekrece IL-6 stromálními buňkami kostní dřeně, aniž by došlo k ovlivnění jejich životaschopnosti. Marizomib významně blokuje migraci buněk MM indukovanou VEGF, a potvrzuje tak i své antiangiogenní účinky. U marizomibu byla pozorována jako u jednoho z mála IP i inhibice kanonické NF-kB dráhy a následné sekrece cytokinů [33]. Klinické studie V současné době se marizomib nachází v doposud nevyhodnocené I. fázi klinické studie [50]. Další klinické studie I právě nabírají pacienty. Jedná se o studii pacientů s relabujícím nebo refrak-terním myelomem léčených marizomi-bem (NPI-0052-101) a studii pacientů trpících některou z pokročilých malignit (NPI-0052-102). Mezi zatím nejčastěji pozorované nepříznivé účinky tohoto léku patří únava, nespavost, ne- Klin Onkol 2013; 26(1): 11-18 15 INHIBITORY PROTEAZOMU V LÉČBĚ MNOHOČETNÉHO MYELOMU Cl v_:_j Obr. 4. Struktura marizomíbu (The Pub-Chem Project). volnost, průjem, zácpa, bolesti hlavy, halucinace, změny v kognitivních funkcích, ztráta rovnováhy nebo horečky. Významný výskyt periferní neuropatie však nebyl pozorován. Tato data naznačuji, že marizomib by mohl být bezpečným lékem bez zkřížené rezistence s ostat- Obr. 5. Struktura MLN9708/MLN2238 (The PubChem Project). nimi IP a aktivním u pacientů refrakter-ních k bortezomibu [51,52]. MLN9708 MLN9708 je analog kyseliny borité a první orálně podávaný IP druhé generace, který v preklinických studiích prokázal větší potenciál účinku proti buňkám MM in vivo než bortezomib [53]. Jde o reverzibilní typ IP, který ve vodných roztocích či plazmě okamžitě hyd-rolyzuje na MLN2238, jeho biologicky aktivní formu (obr. 5) [54]. Je tedy schopen rozsáhlejší distribuce do krve ve stabilní formě a má vyšší farmakodyna-mtcký účinek vtkaních [51]. Mechanizmus působení MLN9708 (MLN2238), stejně jako bortezomib, inhibuje především chymotrypsi-novou proteolytickou (p5) podjednotku 20S proteazomu. Navíc je schopen ve vyšších koncentracích inhibovat kaspá-zovou (BI) a trypsinovou (B2) proteolytickou podjednotku a indukovat a kumulaci ubikvitinovaných proteinů [53,55]. K rozpadu 20S podjednotky proteazomu po léčbě MLN9708 dochází šestkrát rychleji než po léčbě bortezomibem. MLN9708 je zodpovědný za kaspá-zově dependentní indukci apoptózy myelomových buněk. Po podání dochází k aktivaci kaspáz 8,9 a 3; dále k navýšení hladiny proteinů p53, p21, NOXA, PUMA, E2F a naopak ke snížení hladiny cyklinu Dl a CDK6. Léčba pomocí MLN9708 vedla také k indukci exprese Bip a CHOP, proteinů stresové odpovědi ER a k účinné inhibici kanonické i nekanonické dráhy NF-xB ovlivňujíce tak sekreci cytokinů důležitých pro růst a přežívání myelomových buněk stromálními buňkami kostní dřeně. Takto jsou narušeny cytoprotektivní účinky mikropro-středí kostní dřeně. Chauhan et al (2011) dále pozorovali snížení počtu buněk nesoucích VEGFR2 a PECAM, což naznačuje inhibici nádorem indukované angiogeneze [53]. Lee etal {2011) ve své studii na myších modelech prokázali, že na rozdíl od bortezomibu, MLN9708 pravděpodobně také zmírňuje osteolýzu kostí, nejčas-tější příznak MM [54], Profilování miRNA v buňkách MM léčených MLN9708 prokázalo zvýšenou expresi miR-33b. Zvý- šená exprese této miRNA je asociovaná se sníženou schopností migrace a životaschopností buněk MM stejně jako se zvýšenou apoptózou a citlivostí myelomových buněk k léčbě MLN9708. Navíc zvýšená exprese miR-33b vede k negativní regulaci onkogenu PIM-1. Ve studii Tiana et al (2012) bylo tedy naznačeno, že miR-33b funguje jako nádorový supresor, který se podílí na apoptóze myelomových buněk vyvolané léčbou MLN9708, což vede k inhibici růstu nádoru a prodloužení přežití lidských myelomových xenoimplantátových modelů [56], Klinické studie V současné době hodnotí několik klinických studií fáze I bezpečnost MLN9708 u různých populací pacientů léčených různými dávkami tohoto léku. Dvě probíhající studie (Cl6004 a Cl 6003) hodnotí účinek MLN9708 v monoterapii u pacientů s relabujícím či refrakterním mye-lomem, kteří byli dříve léčeni některým z IP. Mezi nejčastější nepříznivé účinky léčby MLN9708 patří únava, trombo-cytopente, nevolnost, průjmy, zvracení a méně často neutropenie. Je však důležité, že po léčbě MLN9708 trpí pouze 10 % pacientů periferní neuropatií. Dáíe probíhají studie účinnosti tohoto léku v různých kombinacích u nově diagnostikovaných pacientů. Jde např. o studii účinku MLN9708 v kombinaci s melfalanem a prednísonem (Cl6006) a v kombinaci s lenalidomidem a nízkými dávkami dexametazonu (C16005 a Cl 6008) [46]. Bylo rovněž zjištěno, že MLN9708 v kombinaci s lenalidomidem vykazuje synergistickou antimyelomo-vou aktivitu a tato kombinace léků má potenciál pro klinické studie, neboť jde o vysoce účinný perorální léčebný režim bez známek periferní neuropatie [53], Budoucnost inhibitorů proteazomu Doposud byly identifikovány čtyři významné mediatory přímé antimyelo-mové aktivity IP, a to transkripční faktor NF-kB, pro- a antiapoptické faktory, protein p53 a proteiny stresové odpovědi ER. Účinek IP by však měl být chápán jako komplexní děj zahrnující na mnoha místech všechny tyto hlavní mechanizmy, 16 KlinOnkol 2013; 26(1): 11-18 INHIBITORY PROTEAZOMUVLÉČBĚ MNOHODETNÉHO MYELOMU neboť ani samotná inhibice NF-KBani samotná mutace zajišťující ínaktivitu proteinu p53 nezastaví apoptózu myelomo-vých buněk indukovanou iR Identifikace podrobných mechanizmů působení IP má velký potenciál pro odhalení možných molekulárních cílů pro budoucí léčiva- Příkladem mohou být dnes známé specifické deubikvitinační enzymy, které mohou zastavit degradaci určitých molekul bez nutné inhibice proteazomu. Na otázku, jak zajistit vyšší selektivitu, účinnost a výrazně omezit vedlejší účinky dnešních IR mohou nabídnout odpovědi specifické inhibitory imunoproteazomu. S ohledem na heterogenní podstatu MM lze do budoucna počítat se zavedením genetického screeningu jednotlivých genových sad kódujících klíčové molekuly, na jehož základě by byla zahájena optimální léčba s predikcí stupně účinnosti IP u jednotlivých nemocných. Závěr Důležitou a vysoce efektivní strategií při hledání léčebných přístupů k MM je poznání důležité role, kterou v léčbě tohoto heterogenního onemocnění hraje inhibice proteazomu bortezomibem.Toto poznání vedlo k vývoji IP druhé generace, které se liší ve svých chemických strukturách (bo-ronáty, epoxyketony, salinosporamidy), účinnosti a toxických profilech, a poskytují tak nové možností pacientům, kteří se stali rezistentními k bortezomibu. Hlavní cesta dalšího výzkumu ve vývoji nových léčiv by se tedy měla ubírat směrem lepšího porozumění mechanizmům účinku IP a především mechanizmu, kterým buňky získávají rezistenci ktěmto lékům- Literatura 1. Harris JR. The proteins released From intact erythrocyte ghosts' at tow ionic strength. Biochern J 1971; 122(5): 2Xiehanover A, Hod Y, Hershko A. A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochern BJophys ResCommun 1978; 81(4): 1100-1 IDS. 3. Ciechanover A. The ubiq u fti n-proteasome pathway: on protein death and cell life. EMBG J 1998; 17(24}: 7151-716Ú, 4. Linno M, Mizushima T, Morimoto Y et at The structure of the mammalian 20S proteasome at 2.75 A resolution. Structure 2002; 10(5): 609-618. 5. Jäger 5, Grcll M, Huber R et al. Proteasome beta-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function. J Mol Biol T999;291(4)i 997-1013. 6. Arendt CS, Hochstrasser M. Identification of the yeast 205 proteasome catalytic centers and subunit interactions required foractive-siteformation. Proc Natl Acad Sci USA1997; 94(14): 7156-7161. 7r Parlati F, Lee S, Aujay M et al. Carfilzomib can induce tumor cell death through selective inhibition of the chy-motrypsfn-like activity of the proteasome. BEood 2009; 114(16): 3439-3447. 8. Carvalho Pr Goder V. RapoporE TA. Distinct Ubiquitin-Li-gase Complexes Define Gonvergent Pathways for the De-grad ation of Eft Proteins. Gell 2006; 126(2}: 361-373. 9. Ludani F, Kesrnir C, Mishto M et al. A mathematical model of protein degradation by the proteasome. Bio-physJ 2005; 88(4): 2422-3432. 10. Adam Z, Sčudla V, Neubauer J. Mnohocetny mye-lom. In: Adam Z, Vorlíček J, Adamová Z et at. Hematofo-gte II: Přehled maligních hernatobgickych nernocF. Praha: Grada 2001-461^98. 11. Goldberg AL. Introduction to the proteasome and its inhibitors, In: Adams J. Proteasome inhibtors in cancer therapy. New Jersey: Humana Press 2004: 17-39. 12. Adams J, Palombella VJ, Sausville EA et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999; 59(11): 2615-2622. 13. Olowski RZ, Stinchcombe Tt, Mitchell BS et al. Phase 1 trial of the proteasome inhibitor PS-341 in patier-ts with refractory hematologic malignancies. J Glin Oncol 2002; 20(22): 4420-4427. 14. Kane RC, Bross PF, Farrelf ATet al. Velcade: US. FDA ap proval for the- treatment of multiple myeloma progressing on prior therapy. Oncologist 2003; 3{6): 508-513. 15. Špička I, Kleibl Z, Hájek R. Bortezomibum. Remedia 2005; 15(3): 193-203. 16. BerkersCR, Verdoes M, Lichtman E et at. Activity probe for in vivo profiling of the specificity of proteasome inhibitor bo rtezo mi b. Nek Methods 2005; 2(5): 357-362 17. Groll M. Berkers CR Ploegh HL et aL Crystal structure of the boronic acid-based proteasome inhibitor bortezo-mib in complex with the yeast 20S proteasome. Structure 2006; 14{3): 451-456. 18. Richardson PG, Sonnevefd R Schuster MW et al. As-sessment of Proteasome Inhibition for Extending Remis-sions- (APEX) Investigators. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 2005; 352(24): 2487-2498. 19. Hideshima TH Chauhan D,. Richardson PetalNf-kappa B as a therapeutic target in multiple myeloma. J BioE Chem 2002; 277(19): 16639-16647. 20. Hideshima Tr fkeda Hr Chauhan D et al. Bortezo mib induces canonical nuclear factor-kappaB activation in multiple myeloma cells. Blood 2009; 114(5); 1046-1052. 21. Mitsiades N, Mitsiades CS, Poulaki V et al. Molech tar sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci U S A 2002; 99(22): 14374-14379. 22. Hideshima T. Mitsiades Cr Akiyama M et aL Mole-cular mechanisms mediating 3ntimyeloma activity of proteasome inhibitor P5-341. Blood 2003; 101(4): 1530-1534. 23. Hideshima T, Richardson R Chauhan D et aLThe proteasome inhibitor PS-341 inhibits growth, induces apop' tosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res 2Q01;61(7); 3071-3076. 24. Mukhenee Sr Raje N, Schoonmaker JA et al. Pharma^ cologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice. J Glin Invest 2008; 118(2): 491-504. 25. Richardson PG, Barlogie B. Berenson J et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003; 348(26}: 2609-2617. 26. Jagannath 5, Barlogie B, Berenson J et al. A phase 2 study of two doses of bortezomib in relapsed or refractory myeloma. Br J Haematol 2004; 127(2): 165-172. 27. Mateos MV,. Richardson PG, Schlag R et al. Bortezomib plus meEphalan and prednisone compared with melpha-Ian and prednisone in previously untreated multiple myeloma: updated follow-up and impact of subsequent therapy in the phase III VISTA trial. J OinOncol 2010; 28(13): 2259-2266. 28. Moreau R Pylypenko H, Grosicki S et al. Subcutaneous versus intravenous administration of bortezomib in patients with relapsed multiple myeloma: a randomised, phase 3, non-inferiority study. Lancet Oncol 20M; 12(5): 431-140. 29. Hájek R, Adam Z, Mařsnar V et al. Diagnostika a léčba mnohočetného myelomu. Transfuze Hematol dnes 2012; 18(Suppl 1): 1-80. 30. Arastu-Kapur S, Anderl JL, Kraus M et al. Nonproteaso-mal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events. Glin Cancer Res 2011; 17(9): 2734-2743, 31. Oerlemans R, Franke NE, Assaraf YG et al. Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein.Blood2008j ll2(6}:2489-2499. 32. Smith AJ, Dai Hr Correia C et al. Noxa/Bcf-2 protein \n-teractions contribute to bortezomib resistance in human lymphoid cells. J Biol Chem 2011; 286(20): 17682-17692. 33. Chauhan D, Catley Lr Li G et al. A novel orally active proteasome inhibitor induces apoptosis in multiple mye-lama cells with mechanisms distinct from Bortezomib. Cancer Gell 2O05;8(5):4O7^19. 34. Zhang L, Littfejohn JE, Gui Y et al. Characterization of bortezomib-adapted 1-45 mesothelioma cells. Mof Cancer 2010; 9: 110. 35. Zou W, Yue R Lin N et al. Vitamin C inactivates the proteasome inhibitor PS-341 in human cancer cells. Clin Cancer Res 20O6;12(l): 273-280. 36. Golden EB, Lam PYj Kardosh A et ai. Green xea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors. Blood 2009; 113(23):5927-5937. 37. Lonial Sr Boise LH. Current Advances in Novel Proteasome Inhibitor-Based Approaches to the Treatment of Relapsed/Refractory Multiple Myeloma. Oncology 20 T1; 25(2). 38. Kuhn DJr Chen Q, Voorhees PM et aL Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-- proteasome pathway, against preclinical models of multiple myeloma. Blood 2007; 110(9}: 3281-3290. 39. DemoSD, KirkCJ, Aujay MA et al. Antitumor activity of PR-17)l a novel irreversible inhibitor of the proteasome. Cancer Res 2007; 67( 13): 6383-5391. 40. Suzuki E, Demo 5, Arastu-Kapur 5 etal. Bortezomib resistant cel I lines have increased proteasome levels but remain sensitive to carfilzomib. Blood 2009; í 14: Abstr 2852. 41. Wang L, Kumar S, Fridley B et al. Proteasome beta subunit pharmacogenomics: gene resequencing and functional genomics. Glin Cancer Res 2008; 14(tl): 3503-3513. 42. KÍ5selev AF, Goldberg AL. Proteasome inhibitors: from research tools to drug candidates. Chem Biol 2001; 8(8): 739-758. 43. Ruschak AM, Sfassi Mr Kay LE et al. Novel proteasome inhibitors to overcome bortezomib resistance. J Natl Cancer Inst 2011; 103(13}: 1007-1017. 44. O'Connor OAr Stewart AK, Val lone M et a I. A phase 1 dose escalation study of the safety and pharmacokinetics of the novel proteasome inhibitor carfilzomib (PR-171) in patients with hematologic malignancies. Clin Cancer Res 2009; 15(22): 7085-7091. 45. Niesvizky R, Bensinger W, Vallone M et at. PX-171-006: Phase lb multicenterdose escalation study of carfilzomib (CFZ) plus lenalidomide (LEN) and low-dose dexametha-sone (loDex) in relapsed and refractory multiple myeloma (MM): Preliminary results (Abstract). J Clin Oncol 2009; 27 (15Suppf);AbstrS541. Klin Onkol 2013; 26(1): 11-18 17 INHIBITORY PROTEAZOMU V LECBE MNOHOCETNEHO MYELOMU 46. Vlj R, Wang M, Kaufman JL et al. An open-label, single-arm, phase 2 (PX-171-004) study of single-agent carfil-zomib in bortezomib-naive patients with relapsed and/ /or refractory multiple myeloma. Blood 2012; 119(24): 5661 -5670. 47. Jakubowiak AJr DytFeid D, Griffith KAet ti A phase 1/2 study of carfilzomib in combination with lenalidomide and tow-dose dexarnethasoneas a frontline treatment for multiple myeloma. Blood 2012j 120(9); 1801-1089. 48. Fenical W, Jensen PR. Developing a new resource for drug discovery: marine actinomycete bacteria, Nat Chem Biol 2006; 2[12}: 666-673. 49. Miller CP, ManTon CA. Hale R et al. Specific and pro-longed proteasome inhibition dictates apoptcsis induc- tion by marizomib and its anatogs. Chern Biol Int 2011; 194{1): 58-68. 50. Hofmeister CC, Richardson R Zimmerman T et al. Clinical trial of the novel structure proteasome inhibitor NPl-0052 in patients with relapsed and relapsed/refrac-lory mufriple myeloma (r/r MM). J Clin Oncol 2009; 27 (15Supp[}:Abstr8505. 51. Odo EM, Mateos MV, San-Miguel JF. Novel agents derived from the currently approved treatments for MM: novel proteasome inhibitors and novel IM(Ds. Expert Opin Investig Drugs2012; 21(8): TQ75-1037. 52. Moreau R The future of thera py ft? r relapsed/re^ra et o ry multiple myeloma: emerging agents and novel treatment strategies. Semin Hematol 2012; 49 (Suppl 1}:533-S46 53. Chaühan D, Han Z, Zhou B et al. In vitro and in vivo selective antitumor activity of a novel orally bioa^ailable proteasome inhibitor MLN970S against multiple mve-kam cells. Clin Cancer Res 2011; 17(16): 5311 -5331. 54. Lee EC, Fitzgerald Mr ßannerman B et al Antitumor activity of the investigational proteasome inhibitor MLN97D8 in mouse models of B-cell and plasma cell malignances. Clin Cancer Res 2011; 17(23)^ 7313-7323. 55. Kupperman E, Lee EC, Cao Y et al. Evaluation of the proteasome inhibitor MLN97Q8 in preclinical models of human cancer. Cancer Res 2010; 70(5): 1970-1980. 56. Tian Z, Zhao JJ, Tai YT et al. investigational agent MLN970Ä/2238 targets tumor suppressor microRNA-33b in MM cells. Blood 2012; 120{ 19): 3958-3967. 18 KlinOnkol 2013; 26(1): 11-18 High-Risk Multiple Myeloma: Different Definitions, Different Outcomes? Paszekova H, Kryukov F, Kubiczkova L, Hajek R, Sevcikova S. Clin Lymphoma Myeloma Leuk. 2013 Sep 28. pii: S2152-2650(13)00425-4. doi: 10.1016/j.clml.2013.09.004. PMID: 24225331 IF vroce 2013: 1,667 Review High-Risk Multiple Myeloma: Different Definitions, Different Outcomes? Helena Paszekova,1 Fedor Krynkov,1 Lenka Kubiczkova,1'2 Roman Hajek,1'2 Sabina Sevcikova1'2 Abstract Multiple myeloma (MM) is a clonal plasma cell malignancy. Although MM is still not completely curable, it can be maintained at the level of a long-term chronic condition. Irrespective of the treatment strategy, relapse is still a major problem for most patients. Approximately 10% to 15% of all MM patients relapse early and have poor prognosis and outcome. Currently, there are many ways of identifying these high-risk patients using cytogenetics or molecular biology. Despite these various approaches to definition of high risk patients, a clear definition of high-risk MM has not been widely accepted. In this review, we discuss and compare various approaches, and their strengths and weaknesses in early identification of high-risk MM patients. Clinical Lymphoma, Myeloma & Leukemia, Vol. 14, No. 1, 24-30 © 2014 Elsevier Inc. All rights reserved. Keywords: Cytogenetics, GEP, High-risk disease, MGUS, Multiple myeloma, Prognosis Introduction Multiple myeloma (MM) is a malignant B-lymphoproliferative disease characterized by infiltration of clonal plasmocytes in the bone marrow, osteolytic lesions of the skeleton, and presence of monoclonal immunoglobulin (M-protein) in serum and/or urine.1 MM accounts for 10% of all hematologic malignancies.2 It is the second most common hematologic cancer and represents 1 % of all cancer diagnoses and 2% of all cancer deaths.3 Despite new advances in the treatment of MM, it remains mostly an incurable disease. MM progresses from a premalignant stage called monoclonal gammopathy of undetermined significance (MGUS).4 MGUS is a plasma cell proliferative disorder characterized by plasma cell content of less than 10% in the bone marrow, M-protein in serum < 30 g/L, no end organ damage including bone lesions, and no evidence of other B-cell proliferative disorder.5 Smoldering myeloma (SM), also called asymptomatic myeloma, is an intermediary between MGUS and MM. SM has M-protein in serum > 30 g/L and/or bone marrow plasma cells > 10%, and no related organ or tissue impairment or symptoms. Symptomatic MM is a disease 'Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Department of Clinical Hematology, University Hospital Brno, Czech Republic Submitted: May 3, 2013; Revised: Sep 11, 2013; Accepted: Sep 24, 2013; Epub: Sep 28, 2013 Address for correspondence: Sabina Sevcikova, PhD, Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic E-mail contact: sevcik@med.muni.cz characterized by neoplastic proliferation of a single clone of plasma cells producing M-protein, inducing end organ damage, including bone lesions, anemia, renal insufficiency, and hypercalcemia (CRAB symptoms).5 The comparison of the stages is shown in Table l.5 Extramedullary MM arises outside the bone marrow when the clonal plasma cells are capable of leaving the bone marrow niche and infiltrate virtually any organ. Extramedullary disease can accompany newly diagnosed disease or relapse and has dismal outcome for patients.6 It is considered a poor prognostic marker in newly diagnosed and in relapsed patients and is more prevalent in genomically defined high-risk MM. Generally, MM can be divided into 2 subgroups that are approximately equally distributed.7 Hyperdiploid MM is characterized mostly by numerical gains (eg, multiple trisomies) and few structural changes, and nonhyperdiploid tumors are characterized by many chromosomal rearrangements (eg, translocations involving region I4q32) and sometimes chromosome loss. MM is a heterogeneous disease at the genetic level and in terms of clinical outcome.8 The etiology is still unclear and pathogenesis is a complex multifactorial process.1 It is known that there are some changes in the microenvironment of the bone marrow that allow the tumor to grow while the function of the immune system is decreased. The outcome for patients with MM is highly variable.9 Although the median overall survival time is 3 to 4 years, the range is from less than 6 months to more than 10 years. Many reports have described a huge number of prognostic factors in MM.10 In this list, there are many factors that have been confirmed by several studies: the most important parameters are probably f^-microglobulin, proliferation index, and genetic abnormalities (Table 2).10 24 Clinical Lymphoma, Myeloma & Leukemia February 2014 2152-2650/$ - see frontmatter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/! 0.1016/j.clml.2013.09.004 Table 1 Stages of MM MGUS • M-protein in serum <30 g/L • Bone marrow clonal plasma cells <10% • No related organ or tissue impairment (no end organ damage, including bone lesions) • No evidence of other B-cell proliferative disorders Asymptomatic (smoldering) myeloma • M-protein in serum >30 g/L and/or • Bone marrow clonal plasma cells >10% • No related organ or tissue impairment (no end organ damage, including bone lesions) or symptoms Symptomatic MM • M-protein in serum and/or urine • Bone marrow clonal plasma cells • Related organ or tissue impairment (end organ damage, including bone lesions) Extramedullar MM • No M-protein in serum and/or urine • Extramedullar tumor or clonal plasma cells • Normal bone marrow • Normal skeletal survey • No related organ or tissue impairment (end organ damage, including bone lesions) Abbreviations: MGUS = monoclonal gammopathy of undetermined significance; MM = multiple myeloma; M-protein = monoclonal immunoglobulin. Adapted from International Myeloma Working Group 2003. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003; 121:749-57. Clinically, relapse is defined as > 25% increase in the serum or urine protein > 0.5 mg/dL; however, the presence of 'biochemical relapse' alone is not an indication for additional systemic therapy. Patients should also have some form of symptomatic relapse before initiation of therapy. The overall objective of creating a strong staging system for distinguishing patients with different risk is the identification of risk groups that could in particular have improved outcome because they would be considered for different treatment decisions. Novel therapies might benefit patients for whom other therapies fail.12 In high-risk patients, preliminary reports show high response rates with use of novel drugs, such as bortezomib, lenalidomide, and thalidomide, suggesting that the effect of adverse prognostic factors might be overcome when using this type of therapy. The use of genetic information for risk stratification and treatment selection continues to be investigated in clinical trials and will probably have greater significance for clinical research and patient care in the near future. Discussion MM Stratification Systems First, Durie and Salmon introduced a staging system of 3 different stages, each presented by different levels of selected clinical features that were significantly correlated with measured myeloma cell mass—extent of bone lesions, hemoglobin, and level of serum and/or urine M-protein, serum calcium, and serum creatinine.13 Creatinine level further defined lower risk (with relatively normal renal function; serum creatinine value < 2.0 mg per 100 mL) and higher risk patients (with abnormal renal function; serum creatinine Table 2 Summary of the Most Common Parameters That Accompany Poor Prognosis Parameter Poor-Prognosis Values Plasma Cell Leukemia 17p Deletion Present International Staging System Stage 3 ß2-Microglobulin >5.5 mg/L Gene Expression Profiling University of Arkansas 70-gene model or Intergroupe Francophone du Myelome 15-gene model Adapted from Avet-Loiseau. Ultra high-risk myeloma. Hematology Am Soc Educ Program 2010; 2010:489-93. value > 2.0 mg per 100 mL) in each of the 3 stages. The Durie Salmon system was created predominantly to identify some level of tumor burden at the time of diagnosis, but according to Tuchman and Lonial,14 its utility in the setting of prognosis in the era of new drugs is a bit limited. However, it is still considered a means of measuring tumor mass and should be mentioned to compare patient's outcome with previously diagnosed cases of MM.15 In an effort to ensure a more objective classification of patients, the International Myeloma Working Group (IMWG)9 described the International Staging System (ISS) based on f^-microglobulin and albumin levels (Table 3).9 These clinical parameters, chosen because of their wide availability and simplicity of their identification in blood tests, classify MM patients into 3 groups with different overall survival (62 months, 44 months, and 29 months for stages 1, 2, and 3, respectively). ISS has been validated in young and older patients, in patients treated with conventional chemotherapy, autologous stem cell transplantation, or novel agents at diagnosis and relapse, and even though it is more than a decade old, it still represents the most widely used staging system for patients with MM. ISS provides useful information regarding the baseline biological characteristics of the disease. Because of its simplicity and reproducibility, the ISS has demonstrated its value in comparing outcome of clinical trials. However, it has some important limitations, eg, ISS identifies just 3 large prognostic groups, but MM patients are described as a very heterogenic group that cannot be included in only 3 prognostic categories. Identification of the highest risk patients is achieved in only a small number of patients (from 5% to 9%) and better identification of these patients requires a more refined cytogenetic and molecular genetic classification. Another limitation is its focus on prognostication at the population level, so it is not applicable for individualized treatment decisions.10 High-Risk Definition Using Cytogenetics Because only dividing cells can be analyzed, the low proliferative activity of tumor cells early in the disease is a significant limitation of conventional cytogenetics in MM.8 This limitation has been partly overcome by the use of fluorescence in situ hybridization (FISH), multicolor FISH, comparative genomic hybridization (CGH), and spectral karyotyping. The study of Kapoor et al17 reinforced the importance of using conventional cytogenetics and interphase FISH (iFISH) for risk assessment. These methods remain independent prognostic tools despite the introduction of novel agents and are now a part of risk stratification models. Most large Clinical Lymphoma, Myeloma & Leukemia February 2014 25 High Risk Multiple Myeloma Table 3 International Staging System Stage Criteria Median Survival (Months) 1 • Serum B2-microglobulin <3.5 mg/L • Serum albumin >3.5 g/dL 62 2 • Serum B2-microglobulin <3.5 mg/L, but serum albumin <3.5 g/dL or • Serum B2-microglobulin from 3.5 to <5.5 mg/L irrespective of the serum albumin level 44 3 • Serum B2-microglobulin >5.5 mg/L 29 Adapted from Greipp et al. International staging system for multiple myeloma. J Clin Oncol 2005; 23:3412-20. series have used iFISH, although this technique has some weaknesses m MM.10 It allows analysis of only a limited number of chromosomal abnormalities and requires plasma cell identification and purification. Still, iFISH was able to detect genomic changes in almost 90% of patients at the time of diagnosis, which is approximately 3 times more frequent than with conventional cytogenetics banding methods. Chromosomal abnormalities in MM are complex, highly variable, and long chromosomes are altered numerically and structurally.8 Their complexity is reflected in a median number of 8 to 10 karyotypic changes per patient at diagnosis. Smadja et al18 were the first to describe the importance of chromosome ploidy number, and they identified the significant difference in survival between hyperdiploid and nonhyperdiploid patients. The nonhyperdiploid group is associated with poorer overall survival and with presence of structural abnormalities, typically translocations involving the immunoglobulin heavy chain locus (IgH) located at I4q32. Based on a multivariate analysis of several prognostic factors, non-hyperdiploidy was shown as the most important independent factor for overall survival. The hyperdiploid group has better overall survival and is associated with numerical aberrations (multiple trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21). There are 5 main IgH translocations involving Hql3 (CCND1 [cyclin Dl]), 4pl6 (FGFR3 [fibroblast growth factor receptor 3] and MMSET [multiple myeloma SET domain]), 16q23 (MAF [v-maf musculoaponeurotic fibrosarcoma oncogene homolog]), 20ql2 {MAFB), and 6p21 (CCND3 [cyclin D3]).19 These translocations are mostly found in the nonhyperdiploid group and are characterized by overexpression of translocated genes.20 IgH translocations are considered to be primary events and rather negative prognostic factors.21 However, the most frequent translocation t(l I;l4)(ql3;q32), which is found in 15% to 20% of patients, is usually found as neutral with regard to prognosis—in most series it seems to be associated with favorable outcome, but this effect is not strong enough to be statistically significant and there is great heterogeneity in MM patients with this translocation.22 The second most frequent translocation is t(4;l4)(pl6;q32) which occurs in 10% to 15% of patients and results in overexpression of 2 protein-coding genes located at 4p 16—FGFR3 and MMSET?3 It has been associated with poor survival and is often associated with changes of chromosome 13 24 The t(l4;16)(q32;q23) and t(l4;20)(q32;ql2) are 2 less frequent but presumably clinically important translocations that involve the MAF genes. Both of them appear to be associated with poor survival because MAF and MAFB are known oncogenes and their deregulation might play a role in MM oncogenic transformation.25 The prognostic value of t(l 4; 16) was further analyzed in a retrospective study that compared the outcome of patients with and without this translocation.26 Even though the incidence is low, the results did not confirm poor prognostic value of t(l4;16) in contrast to other prognostic parameters; its role in the distinction of high-risk MM remains unclear. Rearrangements of chromosome 1 are the most common structural aberrations, mostly involving (mainly interstitial) deletions of lp and amplifications of lq27; some cases showed more than 1 abnormality.21 Deletions of lp are associated with poor prognosis28; patients with lq21 gain or amplification detected using FISH have unfavorable prognosis and significantly shorter survival.29 An association between lq21 gain and del(13) was found, but no association with translocations t(4;l4), t(l 1;14), or del(17p), and it can be considered as another independent prognostic factor. Chromosome 13 abnormalities are found in approximately 45% to 50% of cases; most of these cases are complete monosomy 13 (90%), and the remaining 10% represent del(13).30 Initially it appeared that these abnormalities have an important effect on patients' survival—partial or complete loss of chromosome 13 seemed to be connected with aggressive clinical course and an unfavorable prognosis.31 However, subsequent analyses showed that this abnormality alone is not a negative prognostic factor and its assumed effect comes from known close association with other high-risk genetic features, such as t(4;l4),32 del(17p) or high serum level of ^-microglobulin.3 This observation further demonstrates that the presence of t(4;l4) is sufficient for shortening survival and should be considered the most relevant cytogenetic prognostic marker for MM patients. Deletion or inactivation of the TP53 gene occurring at 17pl3 is more frequent in advanced MM stages and has been identified as a clinical indicator of very poor prognosis because patients with del(17p) have more aggressive disease, higher prevalence of extramedullary disease, and overall shorter survival.33 When compared with patients lacking TP53 abnormality, del(17p) is also frequently associated with mutations of the other TP53 allele.34 Because the position of these mutations might determine the disease outcome, further and larger analysis of MM is needed. High-risk cytogenetic markers were defined in several studies3'17'35'36 as the presence of any one or more of these abnormalities—hypodiplody, monosomy of chromosome 13 or deletion 13q, deletion of TP53 (locus 17pl3), IgH translocations t(4;l4)(pl6;q32) or t(l4;16)(q32;q23), and plasma cell labeling index of 3% or greater. Decaux et al,37 confirmed that the high-risk group of patients was significantly associated with deletion of 13q, deletion of 17p, gain of lq, and translocation t(4;l4). The low-risk group was significantly correlated with hyperdiploid status determined using FISH. Considering that the prognostic value of cytogenetic abnormalities depends strongly on their coexistence with each other, eg, deletion of 13q and its association with t(4;l4), performing a systematic FISH analysis on all patients with newly diagnosed MM is still of great importance. However, some chromosomal abnormalities, such as t(4;l4) or del(17p), have been shown to be major prognostic markers and very useful in refining Clinical Lymphoma, Myeloma & Leukemia February 2014 Helena Paszekova et al Table 4 Clinical and Genetic Features of TC Molecular Subgroups of MM TC Group Recurrent Translocation Gene(s) at Breakpoint Dysregulated Cyclin D Multiple Trisomies (n) 6p21 6p21 CCND3 D3 7 11q13 11q13 CCND1 D1 40 D1 None None D1 81 D1 + D2 None None D1 + D2 21 D2 None None D2 45 None None None None 6 4p16 4p16 FGFR3/MMSET D2 42 maf 16q23, 20q11 MAF MAFB D2 19 Abbreviations: D1-3 = cyclins D1-D3; maf = v-maf musculoaponeurotic fibrosarcoma oncogene homolog; MM = multiple myeloma; TC = Translocation and Cyclin D Adapted from Bergsagel et al. Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood 2005; 106:296-303. identification of high-risk patients, yet they need to be evaluated in the context of other parameters (especially ^-microglobulin level).3 Decaux et al37 claims that, although powerful, cytogenetic abnormalities target only small subsets of patients and do not account for heterogeneity of the clinical outcome. High-Risk as Defined Using Genomics Gene expression profiling (GEP) has enabled analysis of gene expression patterns that can be involved in MM pathogenesis and might contribute to survival of MM patients. The first molecular classification system was the Translocation and Cyclin D (TC) classification,38 based on GEP of mRNA spikes involving 5 IgH translocations, specific trisomies, and over-expression of cyclin D genes (CCND1-3). The patients were divided into 8 groups (Table 4)38 defined according to early, perhaps initiating oncogenic events, with differences in GEP and clinical features; this classification suggests that dysregulation of cyclin D is an early and unifying pathogenic event in myeloma. Better survival was observed in the TC llql3 group and substantially shortened survival was noted for patients in the TC 4pl6 and TC 16q23 {MAF} groups. Zhan et al20 developed a GEP-based classification that divides MM into 7 different groups (Table 5)20 based on the presence of translocations, gene expression, or hyperdiploidy. In this system, 2 groups were connected with high-risk variables and poor prognosis—the proliferation (PR) group, characterized by overexpression of cell cycle progression and cell proliferation genes, and the MMSET (MS) group, with overexpression of MMSET and FGFR3 genes involving the t(4;l4). However, the associations between classes and survival are likely to be dependent on the type of therapy used. The first validated GEP model for prognosis predication in patients was published by Shaughnessy et al39 from the University of Arkansas, in the United States. The hypothesis of this work was that expression extremes of a subset of genes that correlates with survival might be representative of the effects of DNA copy changes in myeloma disease progression. They identified a set of 70 genes with expression level changes that allowed the identification of a small cohort of 13% to 14% of patients at high-risk for early disease-related death. A remarkable feature of the high-risk signature was the significant overrepresentation of genes located on chromosome 1—nearly 50% of 19 underexpressed genes and 30% of 51 over-expressed genes are located on chromosome 1. Most upregulated genes are mapped to lq, and downregulated genes to lp. This is in accord with the previously published suggestion that progression of disease can also be associated with the percentage of cells with 1 q21 amplification.40 It also suggests that gain of DNA material on lq and loss of lp are important determinants of high-risk MM.39 Results have shown that the low-risk myeloma group had a pattern similar to MGUS and normal plasma cells, and the high-risk group revealed a pattern similar to human myeloma cell lines. The high-risk group had a strong association with known clinical prognostic variables—higher level of f^-microglobulin in serum, creatinine, C-reactive protein, and serum lactate dehydrogenase (LDH), chromosome 13 deletion, and other cytogenetic abnormalities.39 When applied to samples from relapsed patients, 76% exhibited a high risk score. This increase in the frequency of the high-risk label from 13% at diagnosis gives molecular evidence of disease evolution that influences outcome after relapse. The model Table 5 Genetic Signatures of Expression-Defined Subgroups Group Features PR Overexpression of numerous cell cycle-and proliferation-related genes (eg, CCNB2, CCNB1, MCM2, CDCA2, BUB1, CDC2, and TYMS) and cancer-testis antigen genes; higher GEP-defined proliferation index LB Low bone disease (lower expression of genes involved in bone disease and low incidence of magnetic resonance imaging-defined focal bone lesions) MS t(4;14)(p16;q32) -> overexpression of MMSET and FGFR3 genes HY Hyperdiploidy, often associated with trisomies of chromosomes 3, 5, 7, 9. 11, 15, 19, and 21 CD-1 t(11;14)(q13;q32) -> overexpression of CCND1 genes CD-2 t(6;14)(p21;q32) -> overexpression of CCND3 genes MF t(14;16)(q32;23) and t(14;20)(q32;q11) -> overexpression of MAF/MAFB genes Abbreviations: CD = cyclin; GEP = gene expression profiling; HY = hyperdiploid; LB = low Done; MF = MAF-MAFB; MS = MMSET; PR = proliferation. Adapted from Zhan et al. The molecular classification of multiple myeloma. Blood 2006; 108:2020-8. Clinical Lymphoma, Myeloma & Leukemia February 2014 High Risk Multiple Myeloma was then modified to only 17 genes to identify high-risk patients with 97.7% accuracy of the 70-gene model. Unlike other classifications, this high-risk signature model reflects not only tumor cell proliferation, but also other features that indicate shorter survival, such as drug resistance. Decaux et al37 developed a 15-gene model that predicts survival in patients with newly diagnosed MM. The Intergroupe Francophone du Myelome (IFM) suggested that their 15-gene model improves ISS prognostication and can be more discriminating than the FISH model for stratifying MM patients according to survival. In their report, they state that myeloma cells from high-risk patients overexpress genes involved in cell cycle progression and its surveillance, whereas the low-risk patient group is more heterogeneous and includes the hyperdiploid gene signature. High-risk MM patients were characterized by overexpression of genes involved in multiple phases of the cell cycle. The list of genes contains cell-cycle regulated genes that are involved in essential cell cycle processes, such as cell-cycle control, DNA replication, DNA repair, DNA packaging, mitosis, and spindle-assembly checkpoint (SAC). Their hypothesis is that the SAC activity network is perturbed in plasma cells of high-risk patients, thereby maintaining mild chromosomal instability that will facilitate tumorigenesis and drug resistance, leading to a targeted therapeutic model in which SAC inactivation might be an efficient way to provoke plasma cell death. In low-risk patients, they identified 3 significant gene sets. Two of them are related to hyperdiploidy in MM. When compared with other prognostic factors (f^-microglobulin level in serum > 5.5 mg/L and/or t(4;l4)), their results identified subsets with different survival in low- and high-risk groups. This indicates that there are different biologic features associated with survival, and the combination of these 3 could make an independent prognostic tool to identify the highest risk patients. It was also confirmed that the high-risk group is associated with poor prognosis markers (deletion of 13q and 17p, gain of lq, and translocation t(4;l4)). Dickens et al41 used high-density single-nucleotide polymorphism (SNP) arrays to identify homozygous deletions (HZD) in genes relevant to pathogenesis and outcome in MM. This loss of material can be used to find expression signatures and specific genes with prognostic significance. When combined with global gene expression data, they were able to identify key pathologically relevant features. The resulting list of genes with HZD has significantly overrepresented deletions within the 'cell death network including 15 genes important in cell cycle regulation, apoptosis, and regulation of transcription. Deletion of any of these genes means shorter survival; therefore, it is considered to be an independent marker of poor prognosis. These changes at the DNA level need to be associated with changes at the level of gene expression—this analysis generated a list of 97 genes annotated as cell death and connected with poor outcome. From this list, a more applicable 6-gene cell death signature was derived (BUB1B vs. HDAC3, CDC2 vs. FIS1, RAD21 vs. ITM2B) that was able to identify similar subset of patients with poor prognosis with 100% specificity. If any 1 of these pairs has a ratio of > 1, then the test is positive for poor prognosis at presentation and at relapse. Although the 97-gene signature is more sensitive for identification of poor prognosis, the genes selected for the 6-gene signature are highly specific so no patients would be incorrectly classified as such. In their report, Dickens et al41 also made a comparison of the 3 signatures: the 70-gene signature from University of Arkansas group,39 the 15-gene signature from the IFM,37 and their own 97-gene cell death signature. The genes in each of the signatures are different, except 1 shared gene, BIRC5 (National Center for Biotechnology Information: location 17q25, member of the inhibitor of apoptosis gene family, which encodes negative regulatory proteins that prevent apoptotic cell death; gene expression is high in most tumors). There were 37 cases identified as poor prognosis using all 3 signatures. Overall, the 97-gene cell death signature identified 89 cases with poor prognosis, the IFM 15-gene signature identified 64, and the Arkansas group 70-gene signature identified 90 of them. Thus, it seems that the sensitivity of the 97- and 70-gene signatures are almost equal. The fact that there are no shared genes highlights the complexity of the biologic behavior of the tumor. Also, use of GEP analyses will possibly require more work.15 Moreaux et al42 derived a high-risk signature from analyzing GEP and identification of 248 heterogeneity genes in 40 human myeloma cell lines (HMCLs) and divided them into 6 groups. The HMCLs used differed in their dependence on the addition of exogenous interleukin (IL)-6 to grow in vitro (24 of 40 referred to as HMCLssemm+IL6 and the remaining 16 as HMCLssemm). Each of the 6 groups is represented by genes that are known markers of MM, such as c-MAF, CCND1, FRZB, MMSFT, FGFR3, and cancer-testis antigen genes. When applied on primary MM cells of newly diagnosed patients, the resulting clusters overlapped with 7 groups of molecular classification previously described by Zhan et al (Table 6).20,42 Considering this overlapping, the same nomenclature was used to identify HMCL groups. These data suggest that HMCLs have kept the molecular heterogeneity of MM cells of newly diagnosed patients. Assuming that some heterogeneity genes could be used as predictors for patients' survival, Moreaux et al42 found 7 of the 248 HMCL heterogeneity genes with bad prognostic value (TEAD1, CLFC11A, LRP12, MMSFT, FGRF3, NUDT11, and KIAA1671). Most of them were overexpressed in the MS and PR groups, which were previously described as high-risk.20 Using these 7 genes, a simple staging was built, scoring patients from 0 to 7, resulting in creation of the HMCL score containing 3 groups with different Table 6 Overlap Between Clustering of Primary MM Cells Based on HMCL Heterogeneity Gene Signature and 7-Group Molecular Classification HMCL Heterogeneity Gene Signature 7-Group Molecular Classification Cluster 1 100% of patients of MS group Cluster 2 71 % of patients of LB group Cluster 3 100% of patients of MF group Cluster 4 46% of patients of PR group and 29% of patients of HY group Cluster 5 92% of patients of HY group Cluster 6 89% of patients of CD-1 and CD-2 groups Abbreviations: CD = cyclin; HMCL = human myeloma cell line; HY = hyperdiploid; LB = low Done; MF = MAF/MAFB; MM = multiple myeloma; MS = MMSET; PR = proliferation. Clinical Lymphoma, Myeloma & Leukemia February 2014 Helena Paszekova et al outcome. Group 1 comprised patients with no or 1 bad prognosis gene, group 2 patients expressed from 2 to 4 bad prognosis genes, and group 3 at least 5 of them. Of course, group 3 was associated with the worst prognosis. These 7 HMCL genes share no gene with the 70-gene signature from the University of Arkansas and the 15-gene signature from IFM, and the HMCL score was shown to be more potent in some of the independent patient cohorts they used. Conclusion According to Decaux et al,37 an ideal prognostic model would probably combine f^-microglobulin level that reflects tumor burden, creatinine level that reflects renal insufficiency, general patient condition (eg, age older than 50 years, presence of primary tumor or other serious disease, long-term corticosteroid use, weight loss, and chronic inflammation of lungs or kidney), a marker of plasma cell proliferation, and genetic changes. It is still recommended to determine patient's stage using a prognostic system based on clinical parameters (Durie Salmon or ISS system) because it enables comparison of outcomes with previously diagnosed cases. After the International Myeloma Workshop Consensus Panel 2, Munshi et al15 presented recommendations for current risk stratification in MM. For newly diagnosed patients, they suggest to investigate ISS stage using serum albumin and f^-microglobulin levels (ISS stage), bone marrow cytogenetic examination for t(4;l4), t(l4;16), and del(17p) using purified plasma cells in FISH analysis, serum LDH level, immunoglobulin type A, histology for plasma-blastic disease, and additional investigation of cytogenetics, GEP, labeling index, magnetic resonance imaging/positron emission tomography scan as an emerging tool for bone disease evaluation, and DNA copy number alteration using CGH/SNP array. Patients at relapse are often characterized by changes in these risk factors; their levels are usually rising and patients should be reclassified as high-risk. Additional risk stratification criteria for relapsed patients include type of response and length of response to previous therapy. FISH analysis is mandatory for baseline risk stratification, but should only be repeated at relapse/progression for patients who were not initially classified as high-risk. If a high-risk feature has been already present at diagnosis, then there is no need to test for it again at relapse, although investigation for additional changes should be performed. Unlike cell-based staging systems that have the longest history and have been successfully validated, genomic tools (either transcriptional or DNA-based) are still evolving and their prognostic significance might be treatment-specific. Overall, the presented GEP models only share a few common genes (mainly because of high clinical and biologic heterogeneity of the disease), which represents a significant complication for accurate assessment of prognosis and for categorization of patients into groups that would be mutually comparable. It is clear that the established prognostic factors do not have a universal value, especially because of their uncertain stability in the era of novel drugs. Identifying risk groups with high predictive power could notably improve patient care— patients predicted to have poor outcome might be considered for early administration of experimental therapy regimens which might ameliorate the adverse influence of these prognostic features. Because of this promising future use, further research for multifunctional prognostic markers or stabilization of existing models is needed. The general agreement is that the risk stratification should be global and not divided for old vs. new therapy. Acknowledgments This work was supported by Research project of The Ministry of Education, Youth and Sports: MSM0021622434; IGA grants of the Internal Grant Agency of the Ministry of Health: NT13190, NT 12130, NT14575 and grant MUNI/11/InGAl7/2012. The authors thank John B. Smith for proofreading the manuscript. Disclosure The authors have stated that they have no conflicts of interest. References 1. Hajek R, Krejci M, Pour L, et al. Multiple myeloma. Klin OnkollOl 1; 24:S10-3. 2. Rajkumar SV. Treatment of multiple myeloma. Nat Rev Clin Oncol 2011; 8:479-91. 3. Avet-Loiseau H, Attal M, Moreau P, et al. Genetic abnormalities and survival in multiple myeloma: the experience of the Intergroupe Francophone du Myelome. Blood 2007; 109:3489-95. 4. Kuehl WM, Bergsagel PL. Multiple myeloma: evolving genetic events and host interactions. Nat Rev Cancer 2002; 2:175-87. 5. International Myeloma Working Group. 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Improved survival of patients with multiple myeloma after the introduction of novel agents and the applicability of the International Staging System (ISS): an analysis of the Greek Myeloma Study Group (GMSG). Leukemia 2009; 23:1152-7. 17. Kapoor P, Fonseca R, Rajkumar SV, et al. Evidence for cytogenetic and fluorescence in situ hybridization risk stratification of newly diagnosed multiple myeloma in the era of novel therapy. Mayo Clin Proc 2010; 85:532-7. 18. Smadja NV, Bastard C, Brigaudeau C, et al. Croupe Francais de Cytogenetique Hematologique. Hypodiploidy is a major prognostic factor in multiple myeloma. Blood 2001; 98:2229-38. 19. Bergsagel PL, Chesi M, Nardini E, et al. Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma. Proc Natl Acad Sei USA 1996; 93:13931-6. 20. Zhan F, Huang Y, Colla S, et al. The molecular classification of multiple myeloma. Blood 2006; 108:2020-8. 21. Mohamed AN, Bentley G, Bonnett ML, et al. Chromosome aberrations in a series of 120 multiple myeloma cases with abnormal karyotypes. Am J Hematol 2007; 82: 1080-7. 22. Fonseca R, Bergsagel PL, Drach J, et al. International Myeloma Working Group molecular classification of multiple myeloma: spotlight review. Leukemia 2009; 23: 2210-21. 23. Chesi M, Bergsagel PL, Shonukan OO, et al. Frequent dysregulation of the c-maf proto-oncogene at 16q23 by translocation to an Ig locus in multiple myeloma. Blood 1998; 91:4457-63. 24. Keats JJ, Reiman T, Maxwell CA, et al. In multiple myeloma, t(4;14)(pl6;q32) is an adverse prognostic factor irrespective of FGFR3 expression. Blood 2003; 101: 1520-9. Clinical Lymphoma, Myeloma & Leukemia February 2014 High Risk Multiple Myeloma 25- Hurt EM, Wiestner A, Rosenwald A, et al. Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interaction with bone marrow stroma. Cancer Cell 2004; 5:191-9. 26. Avet-Loiseau H, Malard F, Campion L, et al. Translocation t(l4; 16) and multiple myeloma: is it really an independent prognostic factor? Blood 2011; 117:2009-11. 27- Debes-Marun CS, Dewald GW, Bryant S, et al. Chromosome abnormalities clustering and its implications for pathogenesis and prognosis in myeloma. Leukemia 2003; 17:427-36. 28. Walker BA, Leone PE, Chiecchio L, et al. A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value. Blood 2010; 116:e56-65. 29- Nemec P, Zemanova Z, Greslikova H, et al. Gain of 1 q21 is an unfavorable genetic prognostic factor for multiple myeloma patients treated with high-dose chemotherapy. Biol Blood Marrow Transplant 2010; 16:548-54. 30. Avet-Loiseau H, Daviet A, Saunier S, , et allntegroupe Francophone du Myelome. Chromosome 13 abnormalities in multiple myeloma are mostly monosomy 13- Br J Haematol 2000; 111:1116-7- 31. Perez-Simon JA, Garcia-Sanz R, Tabernero MD, et al. Prognostic value of numerical chromosome aberrations in multiple myeloma: a FISH analysis of 15 different chromosomes. Blood 1998; 91:3366-71. 32. Gutierrez NC, Castellanos MV, Martin ML, et al. Prognostic and biological implications of genetic abnormalities in multiple myeloma undergoing autologous stem cell transplantation: t(4;l4) is the most relevant adverse prognostic factor, whereas RB deletion as a unique abnormality is not associated with adverse prognosis. Leukemia 2007; 21:143-50. 33- Drach J, Ackermann J, Fritz E, et al. Presence of a p53 gene deletion in patients with multiple myeloma predicts for short survival after conventional-dose chemotherapy. Blood 1998; 92:802-9- 34. Lode L, Eveillard M, Trichet V, et al. Mutations in TP53 gene are exclusively associated with del(17p) in multiple myeloma. Haematologica 2010; 95:1973-6. 35- Fonseca R, Barlogie B, Bataille R, et al. Genetics and cytogenetics of multiple myeloma: a workshop report. Cancer Res 2004; 64:1546-58. 36. Dewald GW, Therneau T, Larson D, et al. Relationship of patient survival and chromosome anomalies detected in metaphase and/or interphase cells at diagnosis of myeloma. Blood 2005; 106:3553-8. 37- Decaux O, Lode L, Magrangeas F, et al. Prediction of survival in multiple myeloma based on gene expression profiles reveals cell cycle and chromosomal instability signatures in high-risk patients and hyperdiploid in low-risk patients: a study of the Integroupe Francophone du Myelome. / Clin Oncol 2008; 26: 4798-805. 38. Bergsagel PL, Kuelh WM, Zhan F, et al. Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood 2005; 106:296-303. 39- Shaughnessy JD Jr., Zhan F, Burington B, et al. A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1. Blood 2007; 109:2276-84. 40. Hanamura I, Stewart JP, Huang Y, et al. Frequent gain of chromosome band lq21 in plasma-cell dyscrasias detected by fluorescence in situ hybridization: incidence increases from MGUS to relapsed myeloma and is related to prognosis and disease progression following tandem stem-cell transplantation. Blood 2006; 108:1724-32. 41. Dickens NJ, Walker BA, Leone PE, et al. Homozygous deletion mapping in myeloma samples identifies genes and an expression signature relevant to pathogenesis and outcome. Clin Cancer Res 2010; 16:1856-64. 42. Moreaux J, Klein B, Bataille R, et al. A high-risk signature for patients with multiple myeloma established from the molecular classification on human myeloma cell lines. Haematologica 2011; 96:574-82. Clinical Lymphoma, Myeloma & Leukemia February 2014 Circulating serum microRNAs as novel diagnostic and prognostic biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance Kubiczkova L, Kryukov F, Slabý O, Dementyeva E, Jarkovsky J, Nekvindova J, Radova L, Greslikova H, Kuglik P, Vetešníkova E, Pour L, Adam Z, Sevcikova S, Hajek R. Haematologica. 2013 Nov 15. PMID: 24241494 IF v roce 2013: 5,935 Multiple Myeloma Articles Circulating serum microRNAs as novel diagnostic and prognostic blomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance Lenka Kubiczkova,1-* Fedor Kryukov,1 Ondřej Slaby,3 Elena Dementyeva,1 Jiri Jarkovsky,4 Jana Nekvindova,1 Lenka Radova,5 Henrieta Greslikova,1 Petr Kuglik,1 Eva Vetešníkova,6 Luděk F*our,6 Zdenek Adam,6 Sabina Sevcikova,1-5 and Roman Hajek1-" 1Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno; ^Department of Clinical Hematology, University Hospital Brno; "Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute; 'Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk University, Brno; "Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Oiomouc; department of Internal Medicine - Hematooncology, University Hospital Brno; and 'Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine University of Ostrava, Ostrava, Czech Republic ABSTRACT Multiple myeloma still remains incurable in the majority of cases prompting a further search for new and better prognostic markers. Emerging evidence has suggested that circulating microRNAs can serve as minimally invasive biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance. In this study a global analysis of serum microRNAs by TaqMan Low Density Arrays was performed, followed by quantitative real-time PCR. The analyses revealed five deregulated microRNAs: miR-744, miR-130a, miR-34a, let-7d and let-7e in monoclonal gammopathy of undetermined significance, newly diagnosed and relapsed multiple myeloma when compared to healthy donors. Multivariate logistical regression analysis showed that a combination of miR-34a and let-7e can distinguish multiple myeloma from healthy donors with a sensitivity of 80.6% and a specificity of 86.7%, and monoclonal gammopathy of undetermined significance from healthy donors with a sensitivity of 91.1% and a specificity of 96.7%. Furthermore, lower levels of miR-744 and let-7e were associated with shorter overall survival and remission of myeloma patients. One-year mortality rates for miR-744 and let-7e were 41.9% and 34.6% for the 'low' expression and 3.3% and 3.9% for the 'high' expression groups, respectively. Median time of remission for both miR-744 and let-7e was approximately 11 months for the 'low' expression and approximately 47 months for the 'high' expression groups of myeloma patients These data demonstrate that expression patterns of circulating microRNAs are altered in multiple myeloma and monoclonal gammopathy of undetermined significance and miR-744 with let-7e are associated with survival of myeloma patients. introduction Multiple myeloma (MM) accounts for more than 10% of hematologic cancers.5 In MM, malignant bone marrow plasma cells (BMPCs) undergo massive clonal expansion resulting in high levels of monoclonal immunoglobulin (mlg, M-protein) in blood and/or urine. This is often accompanied by other clinical symptoms, such as osteolytic lesions, increased calcium level, renal insufficiency and anemia.'-2 MM evolves from a pre-malignant condition called monoclonal gammopathy of undetermined significance (MGUS) which progresses to MM at a rate of 1 % per year.*1 Although there are serum markers used for diagnosis of MGUS and MM, such as levels of FLC or mlg,4-6 recently a lot of attention has been paid to circulating microRNAs that could serve as new diagnostic and/or prognostic tools.7"5 MicroRNAs (miRNAs) are a class of short, non-coding, single stranded RNAs with regulatory function.'0,11 MLRNAs play crucial roles in a variety of basic biological processes; they even contribute to tumor formation and development.12 In tumors, different miRNAs expression profiles compared to healthy tissues were described and resulting miRNAs signatures correlated with patients' survival and prognosis. Such observations highlighted miRNAs as promising biomarkers for diagnosis and even possible targets for therapies.13 So far, a number of studies, using BMPCs as the source of miRNAs, found several deregulated miRNAs in MM and MGUS, and implicated miRNAs in signaling pathways deregulated in MM pathogenesis.14"17 Some of these miRNAs have a therapeutic potential in vitro and in vivo, such as miR-34a.ls Nevertheless, obtaining a marker from the bone marrow (BM) is an invasive procedure for patients; therefore, there is still a need for a minimally invasive test that can be easily repeated. There is now a greater possibility of using miRNAs as biomarkers after the discovery that they are present in various body fluids." Moreover, they are very stable, as they are protected from degradation by association either with secreted membrane vesicles (exosomes, apoptotic vesicles) or with RNA-binding proteins (nucleophosmin, Argonaut 2 (Ago2)).""21 It was shown that the miRNAs profile of body fluids reflects physiological or pathological conditions and can be used for patient ©2014 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2013.093500 The online version of this article has a Supplementary Appendix. Manuscript received on June 18,2013. Manuscript accepted on November 12,2013. Correspondence: sevcik@med.muni.cz haematologica | 2014; 99(3) 511 L. Kubiczkova ef al. classification.22 In this study, a new serum miRNAs expression profile, potent enough to distinguish newly diagnosed MM and MGUS patients from healthy controls, was created based on TaqMan Low Density Arrays (TLDA). This profile was validated by quantitative realtime PCR (qPCR) on a larger cohort of newly diagnosed and relapsed MM as well as MGUS patients. Moreover, miRNAs levels were correlated with clinical, biochemical and cytogenetic characteristics and survival data. Methods Patients and healthy donors Peripheral blood (PB) serum samples from 103 newly diagnosed MM patients, 18 MM patients in relapse, 57 MGUS and 30 healthy donors (HD) from the Faculty Hospital Brno, Czech Republic, were obtained for this study. PB serum samples were collected as follows: centrifugation 3500 rpm/15 min/20°C. Samples were frozen as 0.5 mL aliquots, stored at -80"C and thawed only once. For 70 MM and 36 MGUS samples, BMPCs were obtained for routine interphase fluorescence in situ hybridization analysis (I-F1SH), as described previously."3 Patients' and donors' characteristics are described in Table 1 and in the Online Supplementary Table Si. For 6 newly diagnosed MM patients, BMPCs and exosomal and non-exosomal fraction from PB serum were collected. Ail patients signed an informed consent form approved by the hospital ethical committee before enrollment into this study. MiRNA extraction Total RNA enriched for miRNAs was extracted from all serum samples using miRNeasy Kit (Qiagen) modified for circulating miRNAs according to the manufacturer's instructions, MiRNA/RNA quantity was assessed on a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) as measurement of each sample 2 times with mean SD=0.292 ng/ul. All samples fit into the Nanodrop ND-1000 validated measuring range. Exosomes precipitation Exosomes were collected using Exo Quick Exosome Precipitation Solution (System Biosciences). Serum samples were centrifuged for 3500 rpm/10 min/4°C, 250 uL of serum was combined with 63 ul of ExoQuick, incubated for 30 minATC and centrifuged for 2 min/13000 rpm. Exosomal and non-exosomal fraction was used for miRNA/RNA extraction, as described above. TaqMan Low Density Arrays Megaplex profiling using human TaqMan Low Density miRNA Arrays A+B, vo.O (TLDA) (Life Technologies) was performed to evaluate the expression of 667 miRNA (see Online Supplementary Methods). QPCR was performed on the ABI7900HT system; raw data were analyzed using SDS software v.2.3, RQ Manager v 1.2.1 (Life Technologies). Candidate miRNA confirmation by qPCR and quantification of miRNA Individual TaqMan miRNA assays for 6 miRNA (hsa-miR-222-002276", hsa-miR-744-002324, hsa-miR-130a-000454, hsa-miR-34a-000426, hsa-let-7e-002406, hsa-let-7d-002283, Life Technologies) were used for qPCR on a 7500 Real-Time PCR System. QPCR and reverse transcription was performed following the manufacturer's recommendations (see Online Supplementary Medians). Absolute quantification to determine the copy number of each miRNA per 1 rig of total miRNA/RNA was performed, as described previously24 (Online Supplementary Appendix and Online Supplementary Figure Si). For determination of assay precision see the Online Supplemmtary Methods. interphase fluorescence in situ hybridization analysis Interphase fluorescence in situ hybridization analysis (I-FISPÍ) was performed as a part of routine diagnostic procedure on CD138' BMPCs, as previously described2^ (Online Supplementary Methods). Statistical analysis TaqMan Low Density Arrays data were analyzed according to the manufacturer's recommendations; multiple testing correction was applied using Benjamini-Höchberg correction for assessment of adjusted P values. For determination of the relative expression levels of target miRNAs see the Online Supplementary Appendix. Normalized expression data from the screening phase of the study were statistically evaluated using the R statistical computing language using the Bioconductor package and LIMMA model combined with hierarchical clustering (HCL).25"25 Other statistical methods used are described in the Online Supplementary Afetkods. P<0.05 was considered statistically significant. Data were statist!-caEy analyzed with IBM SPSS Statistics, v.20 and R v.2.15.3 with survival ROC package. Results Low density arrays study Screening of 667 miRNAs using TXDA was perfonned on 4 newly diagnosed MM patients, 4 HD and 5 MGUS samples to identify differentially expressed circulating miRNAs that could serve as putative biomarkers. Fourteen miRNAs were significantly deregulated (all P<0.003, adjusted P<0.05) between MM patients and HD: 7 miRNAs were up-re gula ted (miR-222, miR-218, mi R-3 4a, miR-1274A, miR-138, miR-lOb*, miR-1243), 7 miRNAs were down-regulated (miR-191, miR-130a, let-7d, miR-103, let-7e, miR-744, miR-151-5p) in MM patients (Figure 1). Out of these, miR-222, miR-744, miR-34a, miR-130a, let-7d and let-7e were further validated, as their position at the top of the list, fold change and favorable expression levels (Ct<30) were taken into account (Online Supplementary Appendix and Online Supplementary Table SZ). However, no significant change in miRNAs expression was observed between MM and MGUS samples (data not shown). Therefore, we used the same 6 miRNAs to look for the difference between MGUS samples and healthy donors. Validation of candidate miRNAs using qPCR Since qPCR is more sensitive and more quantitative over a wider dynamic range than TXDA, we employed miRNA specific assays (miR-222, miR-744, miR-130a, miR-34a, let-7d and Iet-7e) on a larger cohort of 103 newly diagnosed MM patients and 30 HD to confirm the pattern of candidate miRNAs expression between MM/HD samples and also on 57 MGUS and 18 relapsed MM samples. To accurately determine expression differences between groups, miRNAs were normalized as amount of miRNA copy numbers per 1 ng of total RNA/miRNA using absolute quantification approach. Standard curves for all 6 validated miRNAs were obtained (Online Supplementary Appendix and Online Supplementary Figure S1), and individual assays imprecision was also assessed (Online Supplementary Appendix and Online Supplementary Figure 512 haematologica | 2014; 99(3) Serum microRNAs as markers of MM and MGUS Table 1. Patients' and healthy donors' base-line characteristics used for RT-PCR. MM MGUS HD N, of patients/donors 103 57 30 Cender: males-females 49.5%50.5% 66.7%-33.3% 46.794-53.3% Age median (min-max) [years) 66(47-83) 67 (54-80) 55 (45-64) 1SS stage: Ill-Ill 34%-2894-38% ND ND Durie-Salmon stage: l-ll-lll 10.9%-17.894-71.3% ND ND Durie-Salmon substage:A-B 79.6%-20.4% ND ND lg isotype: IgG-lgA-lgM-lgD-LC only- 52.49^27.2«-IMHÄ-1Ü.7W 81.8%-3.6%-12.7%-0%-1.8% ND NonSecr.-Biclonal -3.99t-1.0% 0%-0% Light chains: kappa-lambda 59,2%36.9% 53.794-46.3% ND N. of previous treatment lines None (first-tine treatment! 103 (100%) 57 (100%) ND First-line based treatment: thalidomtde- 7694-18%«% ND ND Bortezomib -lenahdomide Biochemical parameters median (min-max) Hemoglobin (g/L) 108 (62.7-157) 138 (104-166) ND Thrombocytes (count xlO") 215 (37.6-561) 233 (112-483) ND Calcium (mmoI/L) 2.41 (1.85-4.94) 2.34 (104-2.67) ND Albumin (g/L) 39.0 (22.1-50.4) 43.8 (30.6-53.3) ND Creatinine (umol/L) 92.0 (48.0-884.Ö) 86.0 (50.0-779.0) ND ^-microglobulin (mg/L) 3.82 (1.10-42.6) 2.11 (1.21-35.0) ND Lactate dehydrogenase (ukat/L) 3.16(1.15-18.69) 3.43 (1.92-7.88) ND C-reacttve protein (mt/l) 4.0(0-174.3) 3.1 (0-280.6) ND Monoclonal Ig (g/L) 26.65 (0-88.5) 8,7 (0-26.6) ND Plasma cell infiltration of bone marrow (94) 27D (10.0-94.0) 2.0 (0-8.4) ND C hromo som al abnor mai i ty I3ql4 deletion 30 (44.1%) 9 (23.7%) ND I7pl3 deletion 9 (13.2%) 1 (2.6%) ND Translocation tf 4:14'j 9 (18.4%) 3(12.5%) ND lq2l gain 24 (37.5%) 3 (9.4%) ND Hipirfiliplüidy 29 (45i%) 5115.2%") ND ND: not determined. 52) . As the difference in miR-222 expression between MM and HD was not significant (P=0.3022) it was excluded from further studies. A significant decrease was observed in expression of miR-744, miR-130a, let-7d and let-7e (all P<0.001) in the MM group. However, miR-34a was significantly increased (P<0.0001) when compared to the HD group (Online Supplementary Appendix and Online Supplementary Table 53) . These data confirm the results of the screening phase (for the correlation between TLDA and qPCR data, see Online Supplementary Appendix and Online Supplementary Figure Si). Similarly, expression of miR-744, miR-130a, let-7d and Iet-7e was decreased in MGUS samples (all P<0.0001) and the expression of miR-34a was increased in MGUS when compared to HD (P<0.0001) (Online Supplementary Appendix and Online Supplementary Table S3). Receiver operating characteristics (ROC) curve analysis revealed that serum levels of all validated mrRNAs can be used to distinguish MM and MGUS patients from HD (Online Supplementary Appendix and Online Supplementary Table £))■ Moreover, multivariate logistical regression analysis showed that the combination of miR-34a and let-7e could improve the stratification power characterized with area under the curve (AUC) of 0.898, sensitiviry of 80.6% and specificity of 86.7% for MM, and with AUC 0.976, sensitivity of 91.1% and specificity of 96.7% for MGUS (Figure 2). MiRNA expression pattern correlates with biochemical parameters but not with PCs infiltration To determine the correlation of miRNAs expression levels with clinical parameters, stage (ISS, Durie-Salmon (DS)) and percentage of BMPC infiltration, Spearman bivariate correlation was performed. All studied miRNAs significantly correlated with higher levels of hemoglobin: miR-744, miR-130a, l.et-7d and let-7e positively; miR-34a negatively. Moreover, levels of miR-744, miR-130a, let-7d and let-7e showed a significant positive correlation with thrombocyte count and a significant negative correlation with levels of creatinine and beta(p)2-microglobulrn. Expressions of miR-744. let-7d. let-7e showed a significant positive correlation and miR-34a significantly negatively correlated with levels of albumin, and miR-744 and let-7e a significant negative correlation with C-reactive protein (CRP) level. Only let-7e expression showed a significant negative correlation with level of monoclonal immunoglobulin (Ig). Similar data were obtained for MGUS patients, where levels of all studied mrRNAs showed a significant positive correlation with hemoglobin level. In addition, levels of miR-744, miR-130a, Iet-7d and let-7e were significantly associated with levels of albumin and inversely correlated with levels of creatinine and p2-microglobulin. Also, levels of miR-744, miR-130a and let-7d showed a significant negative correlation with CRP levels. In contrast to MM patients, none of the studied miRNAs in MGUS correlated haematologica | 2014; 99(3) 513 L. Kubiczkova et al. with thrombocyte count (Table 2). In MM, expression levels of miR-744, let-7d and let-7e were linked to advanced ISS stage; this trend was also observed formiR-130a, although it did not reach statistical significance. Also, only let-7e correlated with DS stage; levels of miR-744, miR-130a, let-7d and miR-34a were associated with DS sub-stage. However, none of the studied miRNAs in MM and MGUS correlated with percentage of PC infiltration in BM, which confirms previous observed findings28 (Online Supplementary Appendix and Online Supplementary Table S$). Level of circulating iet-7e correlates with dei(13ql4) in PCs Little is known about the origin of circulating miRNAs and their relationship withBMPCs. Therefore, the expression levels of five miRNAs were correlated with typical chromosomal MGUS and MM aberrations, such as gain of lq21; 13ql4 deletion, 17pl3 deletion, translocation t(4;14) and hyperdiploidy (HY) status (HY of chromosomes 5, 9 and 15). We found that presence of del(13ql4) in MM showed a significant correlation with lower levels of let-7e, and we also observed a trend for lower levels of miR-744 to be linked with this aberration (Online Supplementary Appendix and Online Supplementary Table S6). Derivation of evaluated miRNAs To further investigate potential derivation of all studied miRNAs, we measured their levels in exosomal and exo-some-depleted supernatant of 6 newly diagnosed MM patients. Concentration of miR-744, miR-130a, let-7d and let-7e (all P<0.05) was found to be significantly higher in the exosome pellet compared to the exosome-depleted supernatant. However, there was no significant difference between these two fractions for miR-34a (Online Supplementary Figure S4A). For the same patients, we obtained miRNAs from BMPCs, and we observed levels of miR-744, miR-34a, let-7d and ler-7e to be significantly Table 2. Correlation of serum microRNAs in MM and MGUS with biochemical parameters. For correlation of the data, Spearman coefficient was adopted; significant coefficients of correlation (P<0.05) are marked with bold , rS miR-744 Multiple myeloma miR130a miR 34a let-7d let-7e miR-744 MGUS miR-130a miR-34a let-7d let-7e Monoclonal Ig (g/L) -0.175 -0.011 0.135 -0.087 -0.199 0.059 0.062 0.116 0.000 0.051 Hemoglobin (g/L) 0.543 0283 -0.258 0.387 0.585 0.383 0.465 0270 0.290 0.424 Thrombocytes (countxlif) 0.555 0.390 -0.190 0.427 0.515 -0.007 -0.092 -0.127 0.000 0.024 Albumin (g/L) 0.355 0.093 -0.204 0.302 0.355 0.464 0341 0.221 0.401 0.309 Creatinine (umoU) -0.415 -0.354 -0.007 -0.310 -0.406 -0.369 -0.330 -0.090 -0.468 -0367 ß2-microglobulin (mg/L) -0.575 -0.236 0.170 -0.439 -0.571 -0.451 -0279 -0211 -0.484 -0277 Lactate dehydrogenase (ukat/L) -0207 -0.095 0.141 -0.202 -0.176 -0.115 -0.115 -0.180 -0.061 -0.216 C-reactrve protein (mg/L) -0221 -0.086 0.122 -0.178 -0.253 -0.331 -0.349 -0.019 -0.299 -0.224 514 haematologica | 2014; 99(3) Serum microRNAs as markers of MM and MGUS higher in BMPCs than in exosomal fraction (all P<0.05) (Online Supplementary Figure S4B}. Interestingly, levels of rniR-130a were comparable in BMPCs and exosomes (P=0.8438). However, there was no correlation found between miRNAs from BMPCs and from exosomal fraction (data not shown). Dynamics ofmiRNA levels during disease progression As deregulated miPsNAs expression in MGUS and MM patients was observed at the time of diagnosis, the next step was to check if this profile changes during disease progression. For 18 MM patients, who had not undergone PBMC transplantation, serum samples at the time of diagnosis and in relapse (after 2 lines of treatment) were collected. All of the miRNAs in MM samples differed significantly from HD at the P<0.0001 (miR-744: FC=0.270; miR-130a: FC=0.487; miR-34a: FC=10.083; let-7d: FC=0.243; let-7e: FC=0.300). Moreover, a significant increase of miR-34a (FC=3.560; P<0.0001) and decrease of let-7d (FC=0.460; P=0.0182) was found in relapsed samples compared to samples at the time of diagnosis. For miR-744 and let-7e, a trend toward lower expression was observed; however, no change in expression between diagnostic and relapsed sample was observed for miR-130a (Online Supplementary Appendix and Online Supplementary Figure S5). Analyses of overall survival and time to progression Furthermore, miRNAs expression was verified as a possible indicator of survival. Univariate Cox proportional hazards survival model with one explanatory variable showed prognostic impact for serum miR-744 (HR 0.670 [HR95%CI: 0.548; 0.819]; P<0.0001) and for let-7e (HR: 0.611 fHR95%CI: 0.450; 0.329]; P=0.002) for the MM cohort of patients. To characterize the prognostic significance of this miRNA. a multivariate Cox proportional hazards survival model was used. The variables in the multivariate model were the only variables which remained statistically significant when potential predictors were combined with miRNA expression and forced into the model. The results showed that neither miR-744 nor let-7e is inde-pendendy associated with overall survival (OS) when combined with other factors (miR-744: P=0.902; let-7e: P=0.472) (Online Supplementary Appendix and Online Supplementary Table 57). Survival cut-off points were established based on time-dependent ROC analysis (data not shown), which showed suitable AUC for a 0.5-1.5 year time period for miR-744 and a 1.5 year rime point for let-7e. To determine the prognostic impact of defined miR-744 and let-7e expression cut-off values, we compared OS between the 'low' and die ''high' expression subgroups (Figure 3A and B). For miR-744, worse 1-year OS was indicated in the low' expression subgroup of patients (43 of 103) in comparison with the 'high' expression group (60 of 103) (P<0.0001). One-year mortality rate for the 'low' miR-744 expression group was 41.9% (95%CI: 23.8%; 57.9%), and for the 'high' expression group it was 3.3% (95%CI: 0.8%; 12.7%), respectively. Similarly for let-7e, worse 1-year OS was indicated in the low' expression subgroup of patients (52 of 103) in comparison with the 'high' expression group (51 of 103) (P=0.001). One-year mortality rate for the low' let-7e expression group was 34.6% (95%CI: 23.4%; 49.2%) and for the 'high' expression group 3.9% (95%CI: 1.0%; 14.8%). In the same way, the Cox model showed prognostic impact for serum miR-744 (HR: 0.690 [HR95%CI: 0.584; 0.817]; PcO.0001) and let-7e (HR; 0.552 [HR95%CI: 0.424; 0.718]; P<0.0001) in time to progression (1 IF) for the MM patient cohort. Only MM patients who had an event after first-line of therapy were taken into account (86 of 103). We compared TTP between miR-744 low' and 'high' expression subgroups and between let-7e low' and 'high' expression subgroups using the cut-off value defined by time-dependent ROC analysis. The analysis showed suitable AUC for a 1-2 year time period for miR-744 and a 1-year time point for let-7e (Figure 3C and D). Shorter TTP was indicated in patients in the low' miR-744 expression subgroup (37 of 86) in comparison with the 'high' expression group of patients (49 of 86) (P<0.0001), and median time of remission was approximately 11.5 months (95%CI: 6.49; 16.50) for the low' expression and approximately 47.5 months (95%CI: 24.63; 70.37) for the 'high' expression groups, respectively. For let-7e, shorter TTP was indicated in the low' expression subgroup of patients (43 of 86) in comparison with the 'high' expression subgroup of patients (43 of 86) (P<0.0001), and median time of remission was approximately 11.5 months (95%CI: 7.17; 15.83) for the low' expression and approximately 47,5 months (95%CI: 31.61; 63.39) for the 'high' expression groups, respectively. 0.20 0.40 0.60 0.80 1-Sensititvity 1 00 1.00 0.60 0.40 0.20 0.20 0.40 0.60 0.30 1-Sensititvity 1.00 Figure 2. ROC curves curves for combination of serum miR-34a and let-7e yielded in (A) AUC of 0.898, sensitivity 80.6% and specificity 86.7% in discriminating MM from HD and in (B) AUC of 0.976, sensitivity 91.1% and specificity 96.7% in discriminating MGUS from HD. haematologica | 2014; 99(3) 515 L. Kubiczkova et al. Biochemical and stage characteristics (ISS, DS, DS sub-stage) of presented groups of MM patients and /-"-values are provided in the Online Supplementary Appendix iox both the 'high' and the low' miR-744 and let-7e expression groups (Online Supplementary Appendix and Online Supplementary Tables S8A and B, S9A and B). The miR-744 and let-7e low/high' expression groups were significantly different in levels of hemoglobin, thrombocytes, albumin, creatinine, p2-microglobulin and lactate dehydrogenase (P<0.05). Significant differences between groups in ISS and DS sub-stage distribution (P<0.05) were also observed. Interestingly, a significant association between group of patients with lower expression of let-7e and occurrence of del(13ql4) (P=0.031) was found. There was no difference between the 'high' and the 'low' miR-744 and let-7e expression groups in terms of the occurrence of the other analyzed cytogenetic abnormalities (data not shewn). Discussion It has been shown that miRNAs are present as circulating molecules in human body fluids and thus may serve as a new class of powerful and minimally invasive biomark-ers 7,b,29^i fjewever, the studies differ regarding deregulated miRNAs, the array platforms used and the normalization methods adopted. The origin of circulating miRNAs and their function is still unclear as circulating miRNAs may not always be directly associated with malignant cells but may also reflect indirect effects, could be secreted by non-malignant cells, or actively taken up by malignant cells.32,33 In this study, TLDAs were used to identify circulating miRNAs that are differently expressed in MM serum samples and could reflect this pathological condition. Fourteen differendy expressed miRNAs between MM and HD serum samples were identified. Out of these, five miRNAs (miR-744, miR-130a, Iet-7d, let-7e and miR-34a) were cho- A OS Survival functions =» 06- OP m re 0,4- E O 0.2- 0.0- Low levels of rniR-744 ■ High lewis of miR-744 500 1000 1500 2000 Time (days) Survival functions Low levels of miR-744 High levels of miR-744 0 500 1000 1500 2000 Time (days) Survival functions re OA E O G.2 GO Low levels of let-7e ■ High levels of 1et-7e 0 D TTP ~500 1000 ISM 2000 Time (days) Survival functions 0.0 Low levels of let-/e High levels of let-7e 500 1000 Trme (days) 1500 2000 Figure 3. Kaplan-Meier curves of miR-744 and let-7e and their association to (A) (B) OS (C) (D) TTP. The thresholds of cut-off points were determined using a time-dependent ROC analysis. For miR-744 OS, the cut-off value was derived from 0.5-1.5 years survival, for let-7e it was derived from 1.5 years survival. Similarly for TTP, the cut-off values were derived for miR-744 from 1-2 years progression, for let-7e from 1 year progression. Log2 scale of amount of copies/lng of miRNA/RNA was used for miRN A expression in this analysis. The Y axis represents survival probability and the X axis represents time of follow up in days. 516 haematologica | 2014; 99(3) Serum microRNAs as markers of MM and MGUS sen and confirmed as significantly deregulated on a bigger cohort of MM and MGUS patients using an absolute quantification approach. At this point, since no miRNA is accepted as a standard for serum samples, this is probably the most accurate method of quantification of serum miRNAs in MM. Therefore, to accurately determine the expression differences between groups, miRNA levels were normalized to amount of miRNAs copy numbers per 1 ng of total RNA/miRNA. Analytical characteristics of the five miRNAs (miR-744, m£R-130a, miR-34a, let-7d and let-7e) showed that they can all discriminate MGUS and MM from HD. However, the combination of serum m£R-34a and let-7e (the highest sensitivity of 91.1% and specificity of 96.7% for MGUS, and 80.6% sensitivity and 86.7% specificity for MM) proved to be an even more powerful discriminating tool. In the group of MM patients, most of the five miRNAs were observed to be associated with some of the clinical parameters, ISS or DS sub-stage. Particularly in the cases of miR-744, let-7d and let-7e, lower levels were associated with advanced ISS stage. As lower levels of miR-744, miR-130a and let-7d are related to the advanced DS sub-stage, they might reflect the renal impairment that often develops in MM patients. This observation is further supported by the relation of lower miRNA levels to higher creatinine and p2-microglobulin levels. Lactate dehydrogenase (LDH) level helps to assess tumor burden, and the level of p2-microglobulin reflects the tumor mass.6,34 Furthermore, anemia associated with MM is caused by inadequate erythropoietin levels consequent to renal impairment and the effect of inflammatory cytokines.35 C-reactive protein (CRP) as well as albumin levels are known to be hallmarks of tumor activity.3637 Taking all these facts into consideration, we can anticipate that serum miRNA levels are associated with tumor mass and disease activity. Interestingly, such correlation pattern with biochemical parameters was observed also for MGUS. However; as no correlation with infiltration of BMPCs in MM and MGUS was observed, which is in concordance with previously presented data from another group,25 our observations further suggest that circulating miRNAs reflect other MM pathological effects as well. To further investigate potential derivation of all studied miRNAs, we estimated their levels in exosomal and exosome-depleted fractions and in BMPCs. Four miRNAs were observed to be present primarily in exosomes, which is consistent with previous observations that exosome fraction is highly enriched in miRNAs.38Moreover, all of the studied miRNAs were found to be abundantly present in BMPCs when compared to levels in exosomes. Interestingly, levels of miR-130a were comparable in exosomal fraction and in BMPCs, suggesting their involvement in intercellular communication. However, as we did not find any linear dependence between miRNA levels in exosomal fiaction/exosome-depleted fraction and miRNA levels in BMPCs, it is not clear whether they originate from BMPCs. Different miRNAs expression was confirmed also in 18 paired MM samples taken at diagnosis and at relapse with higher levels oE miR-34a and lower levels of lct-7d, suggesting that deregulated levels of miRNAs reflect patient condition and are associated with more advanced disease. To the best of our knowledge, the possibility of a prognostic serum miRNAs marker in MM has not yet been investigated. In this study, lower levels of miR-744 and let-7e were found to be significantly associated with the worse OS and TTP of MM patients. It should be mentioned that this is related to a short-time period (1-2 years). For-miR-744, the observation could be partially explained by the fact that the gene for miR-744 lies in the 17pl2 region, close to the TP$3 gene (17pl3). Deletions at chromosome 17pl3.1-17pl2 were previously found to be associated with poor survival.38 Also, low TP^3 gene expression, which is highly correlated with loss of heterozygosity of the TP53 locus, was associated with shorter event-free survival and OS.™ However, we were not able to prove the relationship between low levels of miR-744 and deletion of 7P53, and thus we cannot say that absence of the 17pl3.1-17pl2 region can fully explain the lower levels of miR-744. As patients were not equally distributed across ISS stage, we assume that miR-744 and let-7e impact on OS and TTP could be explained by ISS heterogeneity. However, no differences in DS stage between groups with low/high expression of miR-744 were observed, but they were observed between groups with 'low/high'' expression of let-7e. Interestingly, the miR-744 'low1 expression group of patients was associated with presence of lq21 amplification or t(4;14), which have been previously described as unfavorable prognostic factors for MM.41,42 The 'low/high' miR-744 and let-7e groups of MM patients were also observed to be clinically heterogeneous, which was demonstrated by different levels of albumin, creatinine, p2 -microglobulin, LDH, hemoglobin and thrombocyte count between groups. As mentioned above, all listed parameters are known to be markers of tumor mass and disease activity.34'35'43 Although our initial findings concerning clinical data, such as OS and TTP, show that these miRNAs are not an independent factor, but rather a hallmark of a complex padiological process that accompanies MM, they both reflect disease status and thus can serve as new auxiliary peripheral blood prognostic markers for MM. In conclusion, we have identified for the first time a profile of five serum miRNAs which are deregulated in MV1 and MGUS sera. Levels of miR-744, miR-130a, let-7d and let-7e were significandy decreased whereas miR-34a was increased in MM and MGUS. Deregulated levels of miRNAs were observed in advanced MM suggesting that they are stable markers of MM. Moreover, levels of miR-744 and let-7e might be useful as a marker of patients' survival. Even though additional larger-scale studies are needed to address other biological characteristics of these miRNAs, it is obvious that circulating serum miRNAs have diagnostic and prognostic implications for MGUS and MM patients. Funding This work was supported by a gram of The Ministry of Education, Youth and Sports: MSM0021622434 and IGA grants of The Ministry of Health: NT12130, NTtiWO, I\JT-t4575, grant of the Ministry of Health, Czech Republic -conceptual development of research organization (FNBr, 6J269705), grant MUNI/i 1/InGAl7/20U and by the European Union Seventh Framework Programme (FP//Z007-20-13) under grant agreement n. 278570. Ackttowledgmett ts The authors would like to thank all the patients and their caregivers for participating in this work as well as lab technicians from the Faculty Hospital Brno. haematologica | 2014; 99(3) 517 L. Kubiczkova et al. Authorship and Disclosures disclosures was provided by the authors and is available with the Information on authorship, contributions, and financial d£ other online version of this article at www.haematologica.org. Referenes 1. Kyle RA, Rajkumar SV. Multiple myeloma. Blood. 2008;1 ll(6):2962-72. 2. Hajefc R, Krejci M, Pour L, Adam Z. 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Limma: linear models for microarray data. In:'Bioinformatics and Computational Biology Solutions using R and Bioconductor1. Gentleman R, Carey V, S. Dudoit S, Irizany R, Huber W (eds), Springer. New York. 2005. p. 397-420. 29. Rocci A, Hofmeister CH Omede P, Geyer SH Bringhen S, Cascione Lr et al Circulating microRNA in multiple myeloma: Differences in healthy subjects and correlation with biological parameters. 17th Congress of the European Hematology Association. 2012. 30. Yoshizawa S, Ohyashiki JH, Ohyashiki M, Umezu T, Suzuki K, Inagaki A, et al. Downregulated plasma miR-92a levels have clinical impact on multiple myeloma and related disorders. Blood. Cancer J. 2012;2(l):e53. 31. Huang JJ, Yu J, Li JY, Liu YT, Zhong RQ. Circulating microRNA expression is associated with genetic subtype and. survival of multiple myeloma. Med Oncol. 2012;29(4):2402-8. 32. Turchinovich A, Weiz L, Burwinkel B. Extracellular miBJSfAs: the mystery of their origin and function. Trends Biochem Sci. 2012;37(ll):460-5. 33. Ohshima K, Enoue K, Fujiwara A, Harakeyama K, Kanto K, Watanabe Y, et al. Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell fine. PLoS One. 2010;5(10):el3247. 34. Dimopoulos MA, Barlogie B, Smith TLh Alexanian R. High serum lactate dehydrogenase level as a marker for drug resistance and short survival in multiple myeloma. Ann Intern Med. i991;115(12):931-5. 35. Littlewood T, Mandeili F. The effects of anemia in hematologic malignancies: more than a symptom. Semin Oncol. 2002;29(3 Suppl £i):40-4. 36. Kím JE, Yoo C, Lee DH, Kim SW, Lee J5, Suh C. Serum albumin level is a significant prognostic factor reflecting disease severity in symptomatic multiple myeloma. Ann HematoL 2010;S9(4):391-7. 37. YangJ, Wezeman M_ Zhang X, Lin P, Wang M, Qian J, et aL Human C-reactive protein binds activating Fcgamma receptors and protects myeloma tumor cells from apopto-sis. Cancer Cell, 2O07;12(3):252^55. 33. Gallo Ař Tandon M., Ale viz os I, Illei G. The majority of microRNAs derectable in serum and saliva is concentrated in exosomes. PloaOne. 2012;7(3):e30679 39. Carrasco DR, Tonon G. Huang Yj Zhang Y Sinha Rj Feng Bj et al. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients Cancer Cell. 2006;9(4):313-25. 40. Xiong W Wu X, Stárneš Sh Johnson SK. Haessler J, Wang S, et al. An analysis of the clinical and biologic significance of TP53 loss and the identification of potential novel transcriptional targets of TP53 in multiple myeloma. Blood. 2008;112(10):4235-46. 41. Nemec P, Zemanova Z, Greslikova H, Michalova K, Pilková HH Tajtlova }, et al. Gain of lq21 Is an unfavorable genetic prognostic factor for multiple myeloma patients treated with high-dose chemotherapy. Biol Blood Marrow Transplant. 2010; 16"(4):543-54, 42 Keats JJr Reiman T, Maxwell CAh Taylor BJ, Larratt LMř Mant MJ, et al. In multiple myeloma, t(4:14)(p!6:q32) is an adverse prognostic factor irrespective of FGFR3 expression. Blood, 2003; 101(4): 1520-9. 43. Tichý Mj MaisnarY Falicka V, Fnedecky Br Vávrová J, Novotná H, et al. International Staging System required standardization of biochemical laboratory testing in multiple myeloma. Neoplasma. 2006;53(6):492A 518 haematologica | 2014; 99(3) Detection of tumor-specific marker for minimal residual disease in multiple myeloma patients Sedlaříková L, Bešše L, Kryukov F, Pelcová J, Adam Z, Pour L, Hájek R, Ševčíková S. Biomedical Papers, Univerzita Palackého v Olomouci, 2014. ISSN 1213-8118. doi:10.5507/bp.2014.035. PMID: 24993743 IF v roce 2014: 1,661 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. Detection of tumor-specific marker for minimal residual disease in multiple myeloma patients Lenka Sedlarikova", Lena Kubiczkova"c, Fedor Kryukov", Jana Pelcovab, Zdenek Adamb, Ludek Pourb, Roman Hajekac, Sabina Sevcikovaac Aim. Multiple myeloma (MM) is a malignant lymphoproliferative disease of terminally differentiated B lymphocytes, characterized by expansion of monoclonal plasma cells. It is the second most common hematological cancer in the world. The introduction of novel drugs is slowly turning MM into a chronic disease. The aim of treatment is hematological remission and eradication of clinical manifestation. Nevertheless, most MM patients eventually relapse. Forthis reason, research is focused on more accurate monitoring of remission and relapse by molecular biology techniques. One of these techniques is allele-specific PCR and quantitative real-time PCR based on specific detection of VDJ immunoglobulin heavy chain gene rearrangement of clonal cells. The hypervariable region of IgH rearrangement is used as a marker for detection of minimal residual disease (MRD) in MM as this sequence is used for allele-specific primers and probe design. This technique is a complementary tool for flow cytometry in MRD detection in MM. The aim of this study was to introduce detection of MRD by PCR in the Czech Republic. Results. We successfully introduced qualitative and quantitative detection of a tumor marker for MRD assessment of MM by PCR in our laboratory. Key words: multiple myeloma, minimal residual disease, tumor-specific marker, ASO PCR, RQ-PCR, IgH gene rearrangement Received: January 16, 2014; Accepted with revision: June 6, 2014; Available online: June 23, 2014 http://dx.doi.Org/10.5507/bp.2014.035 "Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University Brno, Czech Republic bDepartment of Internal Medicine - Hematooncology, University Hospital Brno department of Clinical Hematology, University Hospital Brno Corresponding author: Sabina Sevcikova, e-mail: sevcik@med.muni.cz INTRODUCTION Multiple myeloma (MM) is a plasma cell malignancy that is ranked among B lymphoproliferative neoplasias by the World Health Organization1. MM is a complex disease characterized by accumulation of clonal malignant plasma cells (PC) in the bone marrow (BM) together with production of monoclonal immunoglobulins or light/ heavy chains, resulting in clinical manifestation of the disease. Osteolysis, hypercalcemia, anemia, immune system impairment and renal insufficiency are among the most common clinical manifestations of MM (ref.2,3). With the introduction of new drugs and use of autologous stem cell transplantation, MM is slowly turning into a chronic disease. The aim of the treatment is hematological remission and eradication of clinical manifestations. Nevertheless, most MM patients eventually relapse3. This implies that not all clonogenic malignant cells had been killed and that the residue of malignant cells persisting even after treatment contributes to recurrence of the disease. For this reason, more accurate monitoring of remission and relapse by molecular biology techniques is important. One of these techniques is allele-specific (ASO) PCR and a real-time quantitative PCR (RQ-PCR) based on analysis of junctional regions of rearranged immunoglobu- lin heavy chain (IGH) gene4. The hypervariable region of IgH rearrangement is used as a tumor marker for detection of minimal residual disease in MM. Determination of such marker and its sequence analysis further allows for allele-specific (ASO) primers and probe design5. MRD detection using PCR has major advantages because of its sensitivity, accuracy, reproducibility, need of small amount of DNA and widespread and irreplaceable use in retrospective studies. On the other hand, PCR methods are more complex, expensive, take more time and allow detection of only one clone that was present at the time of diagnosis6. However, detection of tumor marker by PCR has a wide application for clinical evaluation of patients, for early relapse detection or for quantification of tumor contamination in healthy hematopoietic cells for autologous transplantation7. The use of flow cytometry (FC) for MRD detection appears to have prognostic significance as well6,7. One current approach is MRD detection using an 8-color polychromatic FC. This technique is also able to differentiate the expression of immunoglobulin light chain (IgL) k or X (ref.8,9). MRD detection via FC is applicable in approximately 90% of MM patients, which is important for routine practice6. The significance of FC use in MRD detection was shown in the large studies of Paiva et al. In these studies, patient treatment response was evaluated 1 5^56 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. via immunofixation, serum free light chain, multiparameter FC immunophenotyping and FC together with assessment of high-risk cytogenetics1011. Unfortunately, there has never been a comparative study of PCR vs. FC in a large cohort of patients treated with widely used treatment regimens. Nevertheless, according to some smaller studies - both techniques have comparable prognostic significance. Nowadays, they are considered to be complementary tools for MRD monitoring6'12. Currently, new technologies for the detection of tumor-specific marker are emerging, such as droplet digital PCR or next generation sequencing (NGS) (ref.13,14). However, the qualitative and quantitative detection of tumor-specific marker by ASO-PCR is still the golden standard technique7. Nevertheless, the technique of MM-specific marker detection using ASO-PCR has not been established in the Czech Republic so far. Therefore, the aim of this work was to introduce allele-specific qualitative and quantitative detection of MM-related marker by PCR on BM and peripheral blood samples of MM patients in our laboratory for further MRD assessment. MATERIAL AND METHODS Patients and samples Frozen genomic DNA (gDNA) derived from mononuclear cells from BM (BMMC) of 10 newly diagnosed and relapsed MM patients diagnosed between 2006 and 2007 at the Faculty Hospital Brno was included in the study (Table 1). For 6 patients, gDNA samples of mononuclear cells from peripheral blood (PBMC) at the time of relapse were available, for 2 other patients, gDNA samples of BMMC at the time of relapse were available. gDNA was isolated using phenol-chloroform extraction, and stored at -20 °C. Also, gDNA from PBMC of 10 healthy donors was included in the study. This gDNA was isolated using QIAamp DNA Mini Kit (Qiagen). All samples were included only after patients signed the informed consent approved by Ethical committee of the hospital. Amplification and sequencing of tumor-specific IgH gene rearrangement To identify tumor-related IgH rearrangements, 500 ng of gDNA was PCR-amplif ied using sets of primers for IgH variable (V), diversity (D), and joining (J) gene segments with 2mM dNTP, 20 mM MgCl2, 5x Buffer and GoTaq Flexi DNA Polymerase (Promega) (ref.1516). The reaction was carried out for initial denaturation at 94 °C for 1 min and then 33 (40) cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 30 s, with a final extension of 10 minat 72 °C (ref.17). PCR products were then run on 2% agarose gels to find clonal products. Clonal PCR products were excised and purified using MinElute Gel Extraction Kit, QIAquick PCR Purification Kit or QIAquick Gel Extraction Kit (all Qiagen) and further sequenced. Purified PCR fragments were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit on ABI3130 DNA Sequencer (Applied Biosystems). The relevant VH family or JH consensus primers were used as sequencing primers to obtain the sequence information (Table 2) (ref.151618). ASO primers design and nested PCR amplification of tumor-specific IgH gene rearrangement Patient-specific ASO primers of CDRIII region were designed using IMGT/V-QUEST (http://www.imgt.org/) and PrimerBlast (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/) and synthesized by Eurofins MWG Operon (Ebersberg, Germany) (ref.19). Nested PCR amplification using ASO primers for each IgH sequence identified earlier were performed using 2mM dNTP, 20 mM MgC12, 5x Buffer and GoTaq Flexi DNA Polymerase (Promega). PCR was performed as initial denaturation at 94 °C for 1 min, and then 33 cycles of amplification at 94 °C for 30 s 58-62 °C (dependent on specific ASO primer Tm) for 30 s, and 72 °C for 30 s, with final extension of 10 min at 72 °C. PCR products were run on 2% agarose gels17. PCR products from ASO PCR were also sequenced as described previously, to ensure the detection of the same sequence. Table 1. Patient's baseline characteristics. Total number of patients 10 Sex: male/female 5/5 Average age at diagnosis (range) [years] 64 (52-81) ISS stage: I-II-III (%) 60-30-10 Durie-Salmon stage: I-II-III (%) 40-0-60 Ig isotype: IgG-IgD-LC only (%) 70-10-20 Monoclonal Ig (g/1) 34.7 (0-79.8) Plasma cells infiltration of bone marrow (%) 31.1 (10.0-81.6) Table 2. Sequences of the consensual primers used for IgH rearrangement detection. (Symbols: R = A/ G, Y = T/C, S = G/C, K = G/T). Primer Sequence (5'-3') VH1FS CAGGTGCAGCTGGTGCARYCTG VH2FS CAGRTCACCTTGAAGGAGTCTG VH3FS GAGGTGCAGCTGGTGSAGTCYG VH4aFS CAGSTGCAGCTGCAGGAGTCSG VH4bFS CAGGTGCAGCTACARCAGTGGG VH5FS GAGGTGCAGCTGKTGCAGTCTG VH6FS CAGGTACAGCTGCAGCAGTCAG VHFR2-1 CTGGGTGCGACAGGCCCCTGGACAA VHFR2-2 TGGATC C GTC AGC C C C C AGGG AAGG VHFR2-3 GGTCCGCCAGGCTCCAGGGAA VHFR2-4 TGGATCCGCCAGCCCCCAGGGAAGG VHFR2-5 GGGTGCGCCAGATGCCCGGGAAAGG VHFR2-6 TGGATC AGGCAGTC C C CATC GAG AG VHFR2-7 TTGGGTGCGACAGGCCCCTGGACAA JHD ACCTGAGGAGACGGTGACCAGGGT 2 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. Design of probes for RQ-PCR For RQ-PCR analysis of tumor-related sequence H-chain V- region (VH) family-specific consensus reverse probes (called L-VH1 to L-VH6) derived from the germ-line sequence FR3 and designed for use in RQ-PCR in childhood ALL were used20. Because of the high rate of somatic hypermutations occurring in MM, novel probes were required. The new specific IgH probes were designed according to recommendations of Ladetto et al.17 (Eurofins MWG Operon, Ebersberg, Germany) and labeled at the 5' end with 6-carboxy-fluorescein (FAM) and 6 carboxytetramethyl rhodamine (TAMRA) at the 3' end. RQ-PCR reaction was performed in 25 uL with 500 ng of patient's gDNA using lx TaqMan Gene Expression MasterMix (Life Technologies), 10 pmol of each patient's specific ASO primer, 5 pmol specific IgH probe. Reactions were incubated in a 96-well optical plate at 50 °C for 2 min, 95 °C for 10 min, followed by 42 cycles at 95 °C for 15 s and 60 °C for 1 min. All reactions were run in triplicate on 7500 Real-Time PCR System. Standards for RQ-PCR were obtained by cloning the tumor-specific IgH region with the TOPO TA cloning Kit (Invitrogen). A variable number of white-positive colonies (colonies with correct plasmid insertion) were grown overnight in Luria-Bertani broth containing 50 mg/mL ampicillin. Plasmid DNA was purified using QIAprep Spin Miniprep Kit (Qiagen). Standard curves were prepared by tenfold serial dilutions of plasmid in gDNA obtained from healthy donors according to the European Study Group on MRD Detection in ALL (ESG-MRD-ALL criteria) (ref.21). Then, the quantitative analysis of tumor-specific sequence was related to the reference human RNase P gene (Applied Biosystems). Monoclonality of the specific sequence was verified by sequence analysis of PCR product from single colonies cloned with specific VDJ gene rearrangement of IgH. RESULTS gDNA samples often patients obtained from BMMC were used for introduction of tumor-specific marker identification by PCR. Fifty percent of the patients (5/10) were suitable both for qualitative and quantitative tumor marker detection. In 30% (3/10) of patients, the sequence was not clear and they were assessed as oligoclonal. This technique was unsuccessful in 20% (2/10) of our patients (Table 3). Tumor-specific IgH gene rearrangement amplification and sequence analysis The tumor-specific marker was established for 80% (8/10) patients by PCR with consensual primers (derived from FR1 and FR2 conservative regions). IGHV3 allele was present in 30% (3/10) of patients, IGHV2 in 10% (1/10) and IGHV4 in 10% (1/10) of patients. Sequences were analyzed using bioinformatic tool IMGT/V-QUEST for CDR2/3 hypervariable region evaluation. Only productive IgH rearranged sequences (in frame junctions, no stop codon) were used for further work. Monoclonality of the specific sequence was verified by direct sequence analysis of cloned PCR product from Table 3. A summary of individual steps of PCR detection of tumor-specific marker in MM (nd - not done). ASO PCR qPCR Patient alelle ASO primers PCR II ASO probes standard curves qPCR i IGHV2 done done done done quantifiable 2 IGHV3 done done done done quantifiable 3 oligoclonal done done nd nd nd 4 IGHV4 done done done done out of quantitative range 5 IGHV3 done done done done quantifiable 6 oligoclonal done done nd nd nd 7 oligoclonal done done nd nd nd 8 IGHV3 done done done done quantifiable 9 nd nd nd nd nd nd 10 nd nd nd nd nd nd Table 4. Results of RQ-PCR detection of tumor-specific marker in MM. Patient Status ASO probe RNase P Malignant cells./ 106 healthy cells Conclusion 1 1. relapse 14.4 43 348 662,1 positive, quantifiable i diagnosis 4 239 37 390 226 726,9 positive, quantifiable z 1. relapse 1.7 29 462 114,4 positive, quantifiable 4 1. relapse 3.9 3 331,6 2 327 positive, out of quantitative range 5 diagnosis 21 963 59 797 734 598,4 positive, quantifiable 8 1. relapse 68 3 348,1 40 606,7 positive, quantifiable 3 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. plasmid in bacteria in 5 patients. In 3 remaining oligoclo-nal patients, we identified > 3 different clones. ASO primer design, testing and qualitative PCR for tumor-specific marker detection ASO primers were designed for 5 patients with successfully obtained molecular marker and for 1 clone of 3 oligoclonal patients (Supplementary table SI). In 7 patients, ASO primers were designed according to patient's specific gDNA sequence and in 1 case, we used plasmid sequence with cloned VDJ region for primer design, because of higher quality of the sequence. Qualitative cross-reaction was performed to verify specificity of designed ASO primers to only one patient. Six out of 8 pairs of primers were specific for only one patient. Qualitative PCR with designed ASO primers was performed on gDNA diagnostic samples from 5 patients with monoclonal sequence. The original clone was present in follow-up samples of 3 patients at the time of relapse. For the other 2 patients, samples were not available. Moreover, PCR was performed on follow-up samples at the time of relapse for 3 oligoclonal patients as well, where 1 of the original clones was successfully detected in 1 oligoclonal patient relapse sample. ASO probe design and RQ-PCR for tumor-specific marker detection Quantitative detection of MRD was performed on 5 monoclonal patients. In these patients, we were able to clone monoclonal sequence into plasmids in order to obtain standard curves (Supplementary table S2, Fig. SI). These standard curves fulfilled ESG-MRD-ALL criteria21. The detection was not performed for oligoclonal patients, as the plasmids carried different inserts of the VDJ rearrangements. For RQ-PCR analysis of tumor-related marker, we started with specific consensus probes derived from the germline sequence of the FR3 region and designed for use in RQ-PCR in childhood ALL (ref.20). These consensus probes were used in 2/5 patients (LVH2 and LVH3 probes) and were fully complementary with patient's sequences. Because of high rate of somatic hypermuta-tions occurring in MM, we designed new probes according to recommendations of Ladetto et al. for 3/5 patients (Supplementary table S3) (ref.17). Quantification was based on human RNase P reference gene. The RQ-PCR reaction was successful for all 5 patients. Four samples were assessed as positive (concordant with qualitative PCR results) and quantifiable (Table 4). In one patient, MRD was assessed as positive, out of quantitative range. The RQ-PCR sensitivity was up to 106 DISCUSSION The aim of the work was to introduce MM-related marker identification for further MRD detection by PCR in our laboratory as a complementary tool for MRD assessment by FC. For the purpose of method introduction, we used retrospective patient samples from the time of diagnosis and relapse in order to confirm presence of clonogenic cells and their tumor-specific marker. MRD monitoring is important for identification of patients at increased risk of relapse and plays a key role in treatment response assessment in clinical trials7. Unlike MRD detection by FC which can be applied in approximately 90% of patients with MM thus allowing routine examination, approaches based on PCR are more complex and can be applied in approximately 75% of patients with MM because of the extensive heterogeneity of the disease and presence of several MM clones at the time of diagnosis6. Although PCR is less applicable than FC, it is a powerful technique for treatment efficacy assessment and risk stratification in MM (ref.17,22 25). In this study, with the PCR detection of clonogenic cells, we were able to obtain qualitative assessment in 80% of patients and quantitative data, important for serial monitoring, in 50% of patients (Table 3). We preferentially used FR1 or FR2 derived primers, as we need to obtain sequence of variable regions CDR2/3. Therefore, FR1/2 derived primers allowed us to obtain sequence that was shorter and more suitable for our analysis compared to using L derived primers1526. Further, we did not use FR3 derived primers because the sequences obtained after such amplification are too short which increases the risk of false positive results. For comparison, Owen et al. used FR3 derived primers although this approach allowed detection of specific IgH rearrangement only in 56% of patients27. However, all of the above mentioned approaches are possible, as described previously by van Dongen et al.16. In our case, the successfully identified VDJ rearrangements were in accordance with average frequency of VH variants16,28. Although it is possible to perform PCR reaction with several primer families, in approximately 20% cases we were not able to identify specific VDJ rearrangement sequence. This was most likely due to the presence of somatic hypermutations and subsequent loss of primer binding sites in patient-specific sequence given by extensive heterogeneity in MM clones4'715. On average, there are 8% of mutated nucleotides of VDJ rearrangement sequence in MM patients2930; however only 2% in chronic lymphocytic leukemia (CLL) and 4% in follicular lymphoma7. As the average homology of a patient's sequence with germline sequence is 92.2%, the annealing ability of primers derived from consensus regions of IgH is limited30 In our case, the primer design was successful for all patients with monoclonal sequences (5/10), and for one of the clones of all patients with oligoclonal sequence (3/10). In 1/10 case, we used sequence obtained from the plasmid with cloned hypervariable region for the primer design, as was previously described by Voena et al.15 We also verified specificity of designed primers to only one patient by qualitative cross-annealing reaction, as was shown in 6/8 designed pairs of primers. Specific ASO primers are used for tumor marker identification and its further detection during the patient follow-up. However, we cannot exclude annealing of specific ASO primers on 4 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. different MM patient. The CDR2 and CDR3 regions are relatively short; therefore, there is a high probability of similar motifs repetition. Nevertheless, the crucial thing is that designed ASO primers are not able to amplify healthy DNA; primers have to be specific for MM (ref.31). The method used for MRD quantification is RQ-PCR with ASO primers and probes. The fluorescence signal is provided by probe derived from the more conservative region of the VDJ rearrangement (FR3). First, the specific probe is chosen from previously designed probes for use in real-time PCR in childhood ALL (ref.20). Since these probes can be used for several patients, there was a significant reduction in the price of this method compared to methods using patient-specific probes17,32. Concerning the probes, we confirmed results of Ladetto et al., who analyzed the effect of mismatch between probe sequence and sequence of the patient. Probe was unsuccessful every time in the case of at least three mismatches. In contrast, probe was always successful in the case of no or one mismatch. And in the case of two mismatches, the success of probe annealing was in the type of substitution: G/C (strong interaction); A/T (weak interaction) (ref.17). In our study, the RQ-PCR was successful for all 5 monoclonal patients. Four samples were assessed as positive (concordant with qualitative PCR results) and quantifiable. In one patient, MRD was assessed as positive, but out of quantitative range. The rate of ASO RQ-PCR is considerably variable because of significant heterogeneity of MM cells but approximately ranging from 30% to over 80% (ref.22,23,32,33). In the remaining 3 oligoclonal patients, we were not able to perform the quantification because of the oligoclonal nature of the disease. In these types of samples, it is not very feasible to prepare standard curves necessary for the MRD quantification. The main advantage of MRD detection by RQ-PCR is its high sensitivity. The sensitivity is dependent on the specific ASO probe hybridisation and therefore also on the clone-specific IGHV sequence used for ASO primer and probe design17. Therefore, for this reason, we cannot reach the same sensitivity for all patients. In our work, we were able to reach RQ-PCR sensitivity of at least 104 and up to 10"6. This result is in accordance with other studies since most studies dealing with RQ-PCR detection of MRD also reach sensitivity between 104 and 106 (ref.7,21,33). Despite all the risks and complications, PCR based MRD detection has wide application, such as quantification of tumor contamination in healthy hematopoietic cells for autologous transplantation or following the dynamics of clonogenic cells and activity of the tumor load34,35. Its quantification is useful for treatment response and prognostic assessment as well as for early relapse detection17,22,23,36,37. CONCLUSION Standard techniques used for remission evaluation are able to give only superficial information about the treatment efficiency because of their limited sensitivity. There is a need for more sensitive methods to gather more detailed insight and detection of small tumor cell residues. One of these methods is PCR detection of tumor-specific marker for MRD. This approach has some limitations because of significant heterogeneity of tumor plasma cells and presence of somatic hypermutations in MM. For this reason, it is only successful in some patients and also molecular biological approaches of MM-related marker detection are provided only in a limited number of laboratories. However, we successfully managed to establish this method at both qualitative and quantitative level in MM patients for the first time in the Czech Republic. ACKNOWLEDGEMENTS We would like to thank all the patients, their caregivers and our data-managers for allowing us to do this work. We would also like to thank John B. Smith for proofreading the manuscript. This work was supported by grants of The Ministry of Education, Youth and Sports: MSM0021622434, grants of the Ministry of Health, Czech Republic - IGANT11154, NT12130, NT14575, grant of the Masaryk University MUNI/C/0963/2012 and grant of the Grant Agency of the Czech Republic GAP304/10/1395. Authorship contribution: RH, SS: mnuscript writing; LS, LK: experiment design; FK: data analysis; JP, LP, ZA: clinical data collection. 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Patient Clonality Alelle Primer Sequence (5'-3') Length (mer) T (°C) m v 7 forward CACTTATTGATTGGGATGGT 20 56 1 monoclonal IGHV2 reverse TAGTCAAAGTGCAGTCGCT 19 56 forward ATGAGTAGTGACGGGGGTA 19 58 2 monoclonal IGHV3 reverse TGTTGAAACTCTCGCACAGT 20 58 forward TAGTGGAGGTGAAACCCAAT 20 58 3 oligoclonal IGHV3 reverse TGCTTCATCTCCAGTGCCA 19 58 forward TTACACTGGGAGCACCAAC 19 58 4 monoclonal IGHV4 reverse AGATC GTAATC C GATCTC GC 20 60 forward AAATC AC C AC C C AC G GAG AT 20 60 5 monoclonal IGHV3 reverse TC C G AC ATC ATAC GC AC AGT 20 60 forward AGCTATATCACATGATGGAAGT 22 60 6 oligoclonal IGHV3 reverse TAGAACCCCCACTCCCGA 18 58 forward AC C CTAAC GTTGGTGATAC AA 21 60 7 oligoclonal IGHV1 reverse TAATCATAGTAATCTCTCGCAC 22 60 forward TACTGGTGGTGGTAGCACAT 20 60 8 monoclonal IGHV3 reverse C AATTATC ATC C GCTTTC GC 20 58 Table S2. Standard curves parameters for RQ-PCR detection of tumor-specific marker in MM. Patient Probe Slope Correlation coefficient Quantitative range Sensitivity 1 ASO probe -3.33 0.9988 1.00E-06 1.00E-05 RNaseP -3.56 0.9995 1.00E-06 1.00E-05 i ASO probe -3.18 0.9959 1.00E-06 1.00E-06 z RNaseP -3.47 0.9991 1.00E-06 1.00E-06 A ASO probe -3.33 0.9959 1.00E-05 1.00E-05 4 RNaseP -3.55 0.9985 1.00E-06 1.00E-06 ASO probe -3.07 0.9985 1.00E-04 1.00E-04 j RNaseP -3.47 0.9990 1.00E-04 1.00E-04 a ASO probe -3.35 0.9974 1.00E-06 1.00E-05 0 RNaseP -3.56 0.9995 1.00E-06 1.00E-05 Table S3. Sequences of designed probes for RQ-PCR detection of tumor-specific marker in MM. Patient Sequence (5'-3') Length (mer) 2 5 FAM: CTCTGGAGATGGTGAATCTGCC-TAMRA 22 4 5 FAM: CCGTGTTTGCGGCGGTCACA -TAMRA 20 5 5 ' FAM: GCCGTGTCCTCGACCCTCA-TAMRA 19 7 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. ASO probe RNase P y = -3.3253x +38.248 R2 = 0,9988 Log concentration ASO probe y = -3.1601x + 35.133 R2 * 0,9959 ASO probe 3.3307X + 36.26 R2 = 0.9959 Log concentration ASO probe y = -3,0726x +38.003 R2 = 0.9985 Log concentration ASO probe 3.3481x + 37.075 R2 = 0.9974 Lag concentration -3.5563X + 39.789 R2 = 0.9995 Log concentration 3.4651X + 37,434 R2 = 0,9991 C 26 RNase P 3.5513X + 37,81 R2 = 0,9985 Log concentration RNase P y = -3.688x +39.083 R2 = 0.999 Log concentration C 28 RNase P y = -3.5563X + 39.789 R2 = 0.9995 Log koncentrace Fig. SI. Standard curves for quantitative real-time PCR detection of tumor-specific marker in MM. A) Patient 1, B) Patient 2, C) Patient 4, D) Patient 5, E) Patient 8. Proteasome inhibitors - molecular basis and current perspectives in multiple myeloma Kubiczková L, Pour L, Sedlaříková L, Hájek R, Ševčíková S. Journal of Cellular and Molecular Medicine, WILEY-BLACKWELL, 2014. ISSN 1582-4934. doi:10.1111/jcmm.l2279. PMID: 24712303 IF v roce 2014: 4,75 J. Cell. Mol. Med. Vol XX, No X 2014 pp. 1-15 Proteasome inhibitors - molecular basis and current perspectives in multiple myeloma Lenka Kubiczkova a'b, Luděk Pourc, Lenka Sedlarikova a' D, Roman Hajek a-D-c, Sabina Sevcikova a, b a, b, c a, b, 3 Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic b Department of Clinical Hematology, University Hospital Brno, Brno, Czech Republic 0 Department of Hematooncology Faculty of Medicine University of Ostrava and University Hospital Ostrava, Ostrava, Czech Republic Received: December 8, 2013; Accepted: February 13, 2014 • Introduction • Proteasome inhibitors in multiple myeloma • Bortezomib - Mechanism of action - NF-kB pathway - Apoptotic pathway - Cell cycle and migration - Effect of bortezomib on MM side population - Bortezomib and microRNA - Further effects • Second-generation of proteasome inhibitors - Carfilzomib - Ixazomib - Marizomib • Novel proteasome inhibitors - Oprozomib - Delanzomib • Mechanism of resistance and cross-resistance of Pis • Induction of neuropathy • Proteasome inhibitors in other diseases • Conclusion Abstract Inhibition of proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, has emerged as a powerful strategy for treatment of multiple myeloma (MM), a plasma cell malignancy. First-in-class agent, bortezomib, has demonstrated great positive therapeutic efficacy in MM, both in pre-clinical and in clinical studies. However, despite its high efficiency, a large proportion of patients do not achieve sufficient clinical response. Therefore, the development of a second-generation of proteasome inhibitors (Pis) with improved pharmacological properties was needed. Recently, several of these new agents have been introduced into clinics including carfilzomib, marizomib and ixazomib. Further, new orally administered second-generation PI oprozomib is being investigated. This review provides an overview of main mechanisms of action of Pis in MM, focusing on the ongoing development and progress of novel anti-proteasome therapeutics. Keywords: multiple myeloma • new-generation proteasome inhibitors • bortezomib Introduction The degradation of cellular proteins is a tightly regulated and complex process that plays a central role in regulating cellular function and maintaining homoeostasis in every eukaryotic cell [1]. The ubiquitin-proteasome pathway (UPP) represents the major pathway for intracellular protein degradation. More than 80% of cellular proteins are degraded through this pathway, including those involved in the regulation of numerous cellular and physiological functions, such as cell cycle, apoptosis, transcription, DNA repair, protein quality control and antigens [2,3]. Proteasome as a new cell structure was described by Harris group in the beginning of 1970s as a hollow cylinder and single-torus proteins [4]. Later, it was elucidated that the function of proteasome is 'Correspondence to: Sabina SEVCIKOVA, Tel.: +420 5 4949 3380 Babak Myeloma Group, Department of Pathological Physiology, Fax: +420 5 4949 8480 Faculty of Medicine, Masaryk University, Brno, Czech Republic. E-mail: sevcik@med.muni.cz doi: 10.1111/jcmm.12279 © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. an ATP-dependent degradation of intracellular proteins, and its specificity is determined by interaction only with such proteins that are labelled by polyubiquitin chain or contain a specific amino acid sequence [1, 5]. For this important discovery, Ciechanover, Hershko and Rose received the Nobel Prize in chemistry in 2004. Human 26S proteasome is formed by 20S proteolytic core region and 19S regulatory particle. 20S proteasome is an abundant, barrel-shaped molecule consisting of four highly homologous rings that enclose a central catalytic chamber with proteolytic active sites. Each of the rings contains seven subunits a and p, which are arranged one above the other in the order of a-p-p-a (Fig. 1). While the outer two a-rings surround a small opening through which only denatured polypeptide substrates may pass, two central p-rings contain multiple proteolytic sites that function together in protein degradation [6, 7]. Each of these two p rings comprises three proteolytic sites - pi (caspase-like, C-L), p2 (trypsin-like, T-L) and p5 (chymotrypsin-like, CT-L) [8, 9]. Exposing cells to few stimuli, such as interferon-y, tumour necrosis factor-a (TNF-a) and bacterial lipopolysaccharides, induces the synthesis of other catalytic subunits that are together incorporated into alternative proteasome form - immunoproteasome, which is preferentially expressed in cells of lymphoid origin and plays a role in major histocompatibility complex class I antigen presentation and other constitutive proteolytic activities [10-12]. In the 20S immunoproteasome (i20S), proteolytically active subunits pi, p2 and p5 are substituted by their equivalents pli (LMP2) p2i (MECL-1) and p5i (LMP7; Fig. 1) [13]. Although proteasome contains multiple catalytic sites, to inhibit its function at the constitutive or immunoproteasome level, it is sufficient to block only the P5/LMP7 subunit (CT-L) [14, Fig. 1 Structure of 26S proteasome and immunoproteasome and its catalytic subunits. 26S proteasome consists of regulatory particle and proteolytic core region containing four subunits (arranged as a-p-p-a) In cells of hematopoietic origin, various stimuli, such as interferon (IFN)-y and tumour necrosis factor (TNF)-a induce synthesis of immunoproteasome. Arrangement of proteolytically active subunits: p1 (pi i) - caspase-like subunit, p2 (p2i) - trypsin-like subunit, p5 (p5i) -chymotrypsin-like subunit of proteasome and immunoproteasome is displayed. 15]. Proteins intended for degradation are incorrectly folded proteins and proteins with short half-life and mostly regulatory function that are being cut into oligopeptide chains with an average length of 8-12 amino acids [16,17]. Proteasome inhibitors in multiple myeloma It has become evident that defects within the UPP pathway are associated with a number of diseases, including cancer; thus, inhibitors of this pathway should prevent malignant cells from proliferation [18, 19]. The biggest group of proteasome inhibitors (Pis) is short peptides containing covalently attached pharmacophore - a group of atoms that binds to the catalytic sites of proteasome and thus prevents proper proteasome function [20]. Inhibition of proteasome is particularly useful for the treatment of multiple myeloma (MM), a haematological malignancy caused by malignant transformation of B-lymphocytes into pathological clonal plasma cells (PCs) that accumulate in the bone marrow (BM) and secrete high amounts of monoclonal immunoglobulin (Ig) [21]. As both normal and malignant PCs are highly secretory cells, they require a well-developed endoplasmic reticulum (ER), expansion of secretory apparatus and production of chaperone proteins that ensure proper Ig translation and folding [22]. A stress signalling pathway called the unfolded protein response (UPR) ensures that the PCs can handle the proper folding of proteins and prevent the aggregation of accumulating misfolded proteins. These proteins are then transported out of the ER and degraded by proteasome [23, 24]. It was shown that treatment of MM cells with Pis results in the accumulation of misfolded Ig within the ER, because of inhibition of proteasome function [25]. Such stress activates the UPR pathway, which is mediated by activation or translational repression of several transcription factors, such as XBP-1, ATF6 and PERK/elF2a [25, 26]. Generally, UPR allows the cell to survive reversible environmental conditions, such as chemical insult or nutrient deprivation. However, during prolonged stress caused by Pis, UPR activation leads to cell cycle arrest [27] and induction of apoptosis [28]. Pis initiate UPR leading to apoptosis preferentially in cells with high Ig production; thus, partial inhibition of proteasome in vivo, which is not toxic to patients' normal cells, is sufficient to kill MM PCs [29]. Further, the therapeutic success of Pis in MM relies on their pleio-tropic effects, which decrease both growth and survival of MM cells and the interaction between MM cells and BM microenvironment (MM cells adhesion, formation of new blood vessels and cytokine circuits; Fig. 2). Treatment with Pis has been associated with reports of increased bone formation markers and decrease in markers of bone resorption, which are the consequences of enhanced osteoblastogen-esis and reduced number of osteoclasts [30,31]. The ability of Pis to kill MM PCs and restore proper bone formation led to their use in clinics as one of the therapeutic approaches. Since the approval of first-class PI bortezomib for the treatment of MM, response rates and median survival of MM patients have considerably improved [32, 33]. Further, new-generation Pis, such as 2 © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol XX, No X 2014 Inhibition of NF-kB Fig. 2 Effects of proteasome inhibitors (Pis; bortezomib) on multiple myeloma (MM) cells and bone marrow (BM) micro-environment. Bortezomib affects MM cell survival and signalling pathways eventually leading to apoptosis. Also, bortezomib influences surroundings of MM cells as it inhibits adhesion of MM cells to BM microenvironment, angiogenesis and cyto-kine-mediated interactions. Table 1 Characteristics of proteasome inhibitors evaluated for multiple myeloma treatment Inhibitor of proteasome Active moiety Proteasome target Key celullar effects Binding References Bortezomib Boronate Preferentially CT-I7LMP7, C-IVLMP2 subunit, less T-L/MECL-1 subunit NF-kB, caspase-8, 9, p21, p27, p53, Bid and Bax, caveolin-1, p-H3, EZH2, miR-29b, miR-15a Reversible [87] Carfilzomib Epoxyketone Preferentially CT-I7LMP7 subunit Caspases-3, 7, 8 and 9, JNK, elF2, NOXA Irreversible [74] Marizomib ß-lactone Preferentially CT-I7LMP7 subunit, T-L/MECL-1 subunit, less C-L/LMP2 subunit Caspase-8, NF-kB Irreversible [119] Ixazomib Boronate Preferentially CT-L/LMP7 subunit, less C-L/LMP2 and T-L/MECL-1 subunit Caspase-8, 9 and 3, p53, p21, NOXA, PUMA, E2F, cyclin D1 and CDK6, Bip, CHOP, miR-33b Reversible [82, 87] Oprozomib Epoxyketone CT-L/LMP7 subunit Caspases-8, -9, -3, PARP, JNK, NF-kB Irreversible [89, 120] Delanzomib Boronate CT-L/LMP7 subunit NF-kB Reversible [90] carfilzomib, ixazomib, marizomib and oprozomib, are based on differ- reduced toxicity for MM patients (Table 1). Some of them are cur-ent chemical moieties than bortezomib and have modified pharmaco- rently approved for the treatment of MM, the others are being investi-logic properties, potentially resulting in better clinical outcome and gated in multiple ongoing clinical studies (Table 2). © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 3 Table 2 Clinical development of the drugs and ongoing pivotal trials in MM (according to myeloma.org and clinicaltrials.gov) Bortezomib Carfilzomib Marizomib Ixazomib Oprozomib Delanzomib Stage of development Phase III Phase III Phase 1 Phase III Phase l/l I Phase l/l I Pivotal ongoing trials Clinical trials for use with transplant, induction, consdolidation and maintenance therapy Various phase III clinical trials including a trial comparing carfilzomib versus bortezomib NCT00461045: Clinical trial of NPI-0052 in patients with relapsed or relapsed/ refractory MM NCT01850524: MfN9708 in patients with newly diagnosed MM NCT01564537: MfN9708 in relapsed/ refractory MM NCT01832727: Multicentre, open-label study of oprozomib and dexamethasone in patients with relapsed and/or refractory MM Studies have been terminated Approval EMA: front-line, non-transplant, relapse FDA: all settings FDA: relapse Not approved Not approved Not approved Not approved Bortezomib Bortezomib (Velcade), formerly known as PS-341 (Millennium Pharmaceuticals, Cambridge, MA, USA), is the first-class PI that was approved in 2003 for treatment of refractory MM. In 2005, it was approved for treatment of MM patients who had received at least one prior therapy and in 2008 for the treatment of MM patients in first line [34-36]. further, in 2012, FDA approved subcutaneous administration of bortezomib in all approved indications [37]. Chemically, it is a peptide boronate with molecular formula C19H25BN404 (Fig. 3A). Bortezomib was synthesized for the first time in the mid-90s of the last century by Myogenics/ProScript (today Millennium Pharmaceuticals). An in vitro study on 60 cancer cell lines confirmed its high specificity, efficiency and oxidative stability [38]. Further, it was shown to potently inhibit cell proliferation in different MM cell lines, either drug sensitive or drug resistant [39]. The first clinical trial using bortezomib in the treatment of haematological malignancies was launched in November 1999. In this study, Orlowski et al. showed that low doses of bortezomib, used originally to verify its safety, led to complete remission in a 47-year-old MM patient. Moreover, further eight patients of 11 enrolled in the study showed at least minimal response or stable disease [40]. This result was important as it led to accelerated approval of bortezomib for the treatment of relapsed and refractory MM, after verification in further phases of clinical trials. Mechanism of action Central mechanism of bortezomib function is its covalent binding with high affinity to CT-L (|35) subunit of proteasome or LMP7 subunit of immunoproteasome; however, its binding to C-L (61) and T-L (|32) subunits with lower affinity has been observed as well [41]. The differences in its affinity are because of different interactions of its side chains with each of the subunits [42]. When bound, bortezomib adopts an anti-parallel p sheet conformation, which is stabilized by direct hydrogen bond between the conserved residues (Gly47N, Thr21N, Thr210, and Ala490) of the p-type subunits and main chain atoms of the drug. The actual inhibition is mediated by a pharmacophore group, in this case boronic acid derivative. The boronic acid moiety of the drug ensures increased specificity for the proteasome. The boron atom covalently interacts with the nucleophilic oxygen lone pair of Thr1 Oy, while Gly47N, stabilizing the oxyanion hole, is hydrogen-bridged to one of the acidic boronate hydroxyl groups. The tetra-hedral boronate adduct is further stabilized by a second acidic boronate hydroxyl moiety, which hydrogen-bridges the N-terminal threonine amine atom, functioning as a catalytic proton acceptor. Then, the resulting adduct is characterized by a low degree of dissociation, and therefore remains stable for several hours, even if it is a reversible reaction [42]. Today, the mechanism of action and molecular targets of bortezomib are well characterized. The downstream biological effects of proteasome inhibition are multifactorial, with direct effects on both MM cells and MM cell microenvironment, and key signalling pathways influenced by bortezomib are described further in this review. NF-kB pathway The initial rationale to use bortezomib in cancer was its inhibitory effect on inflammation-associated transcription factor, nuclear factor-kB (NF-kB) through stabilization of its inhibitor I-kB [43]. NF-kB not only regulates various immune and inflammatory responses, but it is also involved in several tumour-related processes, such as suppression of apoptosis and induction of angiogenesis, proliferation and migration. NF-kB is present in the cytoplasm as an inactive complex with its inhibitor I-kB and is activated by proteasomal degradation of I-kB [44]. As Pis inhibit function of proteasome, they prevent degradation of I-kB, subsequent translocation of NF-kB to the nucleus and 4 © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol XX, No X 2014 Fig. 3 Chemical structures of proteasome inhibitors. (A) Bortezomib, (B) Carfilzomib. (C) Ixazomib, (D) Marizomib, (E) Oprozo-mib, (F) Delanzomib. binding to the promoters of target genes (such as anti-apoptotic genes, interleukin-6 efc.) [45]. It was elucidated that MM cell adhesion to BM stromal cells (BMSCs) induces NF-KB-dependent up-regu-lation of interleukin-6 (IL-6) expression by BMSCs [46, 47], Therefore, inhibition of NF-kB could prevent IL-6 expression, which triggers terminal differentiation of normal B-cells and stimulates growth of MM cells [48]. Although pre-clinical and clinical studies with bortezomib showed down-regulation of transcriptional targets of NF-kB, further studies demonstrated that bortezomib is able to induce I-kB down-regulation that occurred at a transcriptional or post-transcriptional level in MM cell lines [49]. This study further showed that effect of bortezomib is cell dependent, as it triggered NF-kB activation via the canonical pathway, associated with down-regulation of I-kB in peripheral blood mononuclear cells, but significantly inhibited NF-kB in BMSCs. Further, it was demonstrated that bortezomib promotes non-proteasomal degradation of I-kB, as it activates two upstream NF-kb-activating kinases (RIP2 and IKKfj) and therefore is able to directly or indirectly (via RIP2) activate IKKp, which subsequently phosphorylates I-kB leading to its degradation [49]. A hypothesis that instead of I-kB stabilization, bortezomib induces I-kB degradation was confirmed by a later study in which I-kB degradation by bortezomib occurred early before induction of apoptosis and could be prevented by calpain inhibitors. Therefore, in the presence of calpain inhibitors, the apoptosis-inducing activity of bortezomib was dramatically enhanced [50]. As bortezomib inhibits inducible NF-kB activity in MM cells, but enhances constitutive NF-kB activity via activation of the canonical pathway, bortezomib-induced cytotoxicity cannot be completely attributed to inhibition of canonical NF-kB activity in MM because inhibition of both canonical and non-canonical pathways is necessary to efficiently block total activity [49,51]. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 5 Apoptotic pathway Inhibition of proteasome promotes programmed cell death of MM cells, as bortezomib is a potent activator of three distinct apoptotic pathways: the intrinsic pathway mediated by caspase-9 activation, the extrinsic pathway mediated by caspase-8 and death receptors (DR) activation and thirdly, activation of ER stress response pathway that involves caspase-2 (Fig. 4) [52-55]. In the first case, bortezomib induces Bax (pro-apoptotic member of the Bcl-2 family) accumulation, its translocation from cytosol to mitochondria, conformational change and oligomerization. Such changes lead to inhibition of anti-apoptotic Bcl-2, release of cytochrome c/Smac from mitochondria and activation of caspase-9 [56, 57]. Further, it was elucidated that bortezomib induces caspase-dependent apoptosis by promoting up-regulation of NOXA (pro-apoptotic BH3 member of Bcl-2 family), and down-regulation of apoptosis inhibitors, such as XIAP, Bcl-2 or c-FLIP via NF-kB blockade [58]. Bortezomib-induced cell death is also linked to the accumulation of ASF1B, Myc, ODC1, BNIP3, Gadd45a, p-SMC1A, SREBF1 and p53 [59]. Also, bortezomib induces p53-dependent apoptosis in MM cells, as it activates and stabilizes the tumour suppressor p53 protein via cleavage of the ubiquitin-ligation enzyme MDM2. Another mechanism of bortezomib-mediated apoptosis is via activation of extrinsic apoptotic pathway, as was demonstrated by an increased activity of c-Jun N-terminal kinase (JNK) and increase in death-inducing receptors Fas and DR5 that further enhanced Fas-mediated signalling and caspase-8 activation [58, 60, 61]. It was further elucidated that bortezomib activates caspase-2, which is associated with ER stress-initiated apoptosis. As caspase-2 functions upstream rather than downstream of mitochondria, it stimulates release of cytochrome c, changes within mitochondrial membrane and further caspase-9 activation [55]. Cell cycle and migration In replicating cells in vitro, bortezomib seems to cause cell cycle arrest at the transition of G2/M phase, which further leads to apoptosis in inhibited cells [62]. Bortezomib has also been shown to stabilize the cyclin-dependent kinase (CDK) inhibitors, such as p21 and p27 as it inhibits their degradation by proteasome, leading to disruption of cell cycle progression, that eventually cause apoptosis as well [39, 61]. This effect is partly mediated through inhibition of the NF-kB pathway [38]. Bortezomib in vitro triggered inhibition of VEGFand IL-6 secretion by MM patient-derived endothelial cells (MMECs); therefore, it inhibited function of BM milieu relevant to angiogenesis. The observation was also confirmed using an in vivo model [63]. Notably, bortezomib is a potent inhibitor of cell migration, as it is able to decrease caveo-lin-1 expression and prevent phosphorylation of caveolin-1 in MM cell lines. Caveolin-1 is a protein involved in cell motility or migration in a number of tissues and its activation requires VEGF-triggered thyro-sine phosphorylation. As bortezomib also decreases VEGF secretion in the BM microenvironment, it prevents activation of caveolin-1 [64]. Using GEP70 and GEP80 models, Shaughnessy era/, found proteasome 26S subunit, non-ATPase4 (PSMD4) and two other Fig. 4 Mechanism of antitumour activity of bortezomib in multiple myeloma (MM) cell. Inhibition of proteasome with bortezomib impairs turnover of multiple proteins resulting in their accumulation in the cell and disruption of multiple signalling pathways within the cell. Consequently, bortezomib-activated signalling pathways lead to disruption of cell cycle and apoptosis. 6 © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol XX, No X 2014 proteasome genes to be up-regulated by bortezomib but not by immunomodulatory agents, dexamethasone or melphalan. Function of PSMD4 is binding and selecting ubiquitin-conjugates for destruction. Further analysis revealed that expression levels of PSMD4 (mapped to 1q21 region) are highly sensitive to copy number, as patients with high PSMD4 expression had four copies of 1q21 and patients with low expression had two copies. Authors anticipated that observed up-regulation of PSMD4 could be caused by preferential killing of normal PCs with two copies of 1q21, as more than 90% of PCs of MM patients tend to have more than two copies of 1 q21. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely [65]. Effect of bortezomib on MIV1 side population Bortezomib was also shown to reduce tumourigenicity of MM as it targets the side population (SP) fraction of MM cells. The MM SP cells show high tumourigenic potential and self-renewal capability and are further characterized by up-regulation of polycomb-related genes, such as EZH2 and EPC1. As bortezomib can reduce levels of p-histone H3 and EZH2, it effectively increased apoptosis and induced G2/M arrest in MM SP [66]. Bortezomib and microRNA It was shown in wfrothat bortezomib influences also microRNA (miR-NA) expression, as the treatment of MM cell lines with bortezomib led to a dose-dependent increase in apoptotic cells and up-regulation of miR-29b. In this study, enforced expression of miR-29b also strongly increased bortezomib-induced growth inhibition, and thus potentiated its anti-MM activity. Moreover, phosphatidylinositol-3-kinase (PI3K)/ AKT pathway played a major role in the regulation of miR-29b-Sp1 (transcription factor Specificity protein 1) loop and induction of apoptosis in MM cells [67]. Two other miRNAs, miR-15a and miR-16, are down-regulated in primary MM cells, their expression inversely correlated with the expression of VEGF and their ectopic overexpression in vivo resulted in inhibition of tumour growth and angiogenesis [68]. In vitro treatment of MM cells by bortezomib led to up-regulation of miR-15a in MM cells, although it was inhibited by MM-BMSCs. Interestingly, as MM-BMSCs are able to suppress miR-15a expression, they provide survival support and protect MM cells from bortezomib-induced apoptosis, as they block repression of bortezomib downstream targets (VEGF, cyclin D, Bcl-2; Fig. 5) [69]. Further effects Bortezomib prevents repair of damaged DNA, reduces adhesion of MM cells to BM cells by inhibiting the mitogen-activated protein kinase (MAPK) signalling pathway and inhibits tumour angiogenesis [39, 70]. Angiogenesis in the BM environment plays an important role in MM pathogenesis and disease progression. It was shown in preclinical models using MMECsthat bortezomib inhibited cell prolifera- Fig. 5 Effect of bortezomib on miR-15a and miR-29b. Bortezomib up-regulates expression of miR-15a and miR-29b, which supports bortezomib-mediated effects on cell differentiation, proliferation and survival. However, expression of miR-15a is suppressed by bone marrow stromal cells (BMSCs) that protect multiple myeloma (MM) cells from bortezomib-induced apoptosis, as they block repression of bortezomib downstream targets (VEGF, cyclin D, Bcl-2-B-cell lymphoma. Sp1—transcription factor Specificity protein 1). tion, chemotaxis, adhesion and capillary formation, which further supported its angiogenic inhibitory activity in vivo. Furthermore, it inhibited the expression and secretion of several pro-angiogenic factors, including VEGF [63]. Bortezomib also participates in osteoclasts apoptosis and osteoblasts differentiation. It was elucidated that it promotes matrix mineralization and calcium deposition by osteoprogenitor cells and primary mesenchymal stem cells (MSCs) via Wnt-independent activation of p-catenin/TCF (transcription factor) signalling and nuclear accumulation of p-catenin. Both these factors are required for promoting MSCs differentiation into osteoblasts [71]. Second-generation of proteasome inhibitors The phenomenal success of bortezomib in MM treatment increased the interest of scientific community in Pis. Optimization of the doses of bortezomib and its combination with other anticancer therapeutics reduced its negative side effects and partially suppressed resistance. Further, new generation of Pis was developed and was expected to bring even better results. Carfilzomib, ixazomib and marizomib represent the second-generation Pis and offer many benefits in terms of increased overall effectiveness, reduced negative off-target effects and overcoming resistance to bortezomib because of their different chemical structure, biological properties, mechanism of action, irre-versibility/reversibility of proteasome inhibition and usage [72]. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 7 Carfilzomih Carfilzomib, an irreversible inhibitor of proteasome (also known as PR-171, Kyprolis; Onyx Pharmaceuticals, San Francisco, CA, USA), is a tetrapeptide epoxyketone with molecular formula C40H57N5O7 (Fig. 3B). Peptide epoxyketones are the most advanced and specific Pis known to date, as were shown in pre-clinical studies, where carfilzomib targeted haematological-specific immunoproteasome. Its promising characteristics and potential to overcome drug resistance led in 2012 to its approval by FDA for MM treatment (www.fda.gov). Mechanism of action Carfilzomib binds to CT-L (p5) catalytic subunit of the proteasome or LMP7 subunit of immunoproteasome with higher selectivity than bortezomib [73]. In this case, inhibition is an irreversible process that decreases proteasomal activity to less than 20%; therefore, restoration of proteasome activity in cells is only possible by new synthesis of individual subunits and their further compilation into new proteasome [74]. Carfilzomib forms a unique six-atom ring structure with p5 sub-unit leading to intramolecular cyclization and morpholino adduction. This intermolecular cyclization is a two-step mechanism. In the first step, oxygen from hydroxyl group of Thr1 nucleophilically attacks carbon of epoxyketone, which subsequently leads to formation of hemiacetal. The second step is a nucleophilic attack of the a-amino nitrogen of Thr1 to C2 carbon-epoxide ring, resulting in the formation of the morpholine adduct [75, 76]. Compared to bortezomib, carfilzomib has only little off-target activity outside the proteasome, can induce apoptosis of bortezomib-na'i've and even bortezomib-pre-treated MM cells without increased toxicity and is also more effective in xenograft models, which is consistent with its higher affinity for the proteasome [74, 77]. In MM cells exposed to carfilzomib, induction of both external and internal apoptotic cascade was observed, with significant elevations of caspases-3, -7, -8 and -9. Programmed cell death has been associated with activation of JNK, mitochondrial membrane depolarization and cytochrome c release. Moreover, an initial decrease in phosphor-ylated elF2 was observed, in connection with the ER stress that is induced by accumulation of non-functional proteins and increased levels of NOXA (pro-apoptotic member of Bcl-2 family) [15, 74]. Recently, it has been elucidated that carfilzomib promotes MSCs differentiation into osteoblasts with a mechanism similar to that used by bortezomib. It was also shown that carfilzomib does not affect p-catenin gene expression, implying that it induces activation of p-catenin/TCF activity by blocking p-catenin degradation [78]. Ixazomib Ixazomib (MLN9708, Takeda/Millenium Pharmaceuticals), an analogue of boric acid, is the first orally administered, reversible PI, which has demonstrated greater potential activity against MM cells than bortezomib in in vivo pre-clinical studies [79]. This second-generation PI with chemical formula C20H23BCI2N2O9 is immediately hydrolyed in aqueous solution or plasma to MLN2238, a biologically active form (Fig. 3C) [80]. Therefore, it is capable of a wider distribution in blood in a stable form and has greater pharmacodynamic effects in tissues [81]. Mechanism of action Ixazomib (its active form MLN2238), just like bortezomib, inhibits particularly the CT-L (p5) subunit of the 20S proteasome. Moreover, in higher concentrations, it is able to inhibit C-L (pi) and T-L subunit (P2) and induce accumulation of ubiquitinated proteins [79, 82]. It has a shorter 20S proteasome dissociation half-life than bortezomib and an improved pharmacokinetic and pharmacodynamic profile. Both ixazomib and bortezomib showed time-dependent reversible proteasome inhibition; however, proteasome dissociation half-life for ixazomib was determined to be about 6-fold faster than that of bortezomib (half-life of 18 and 110 min. respectively) [82]. Ixazomib is responsible for caspase-dependent induction of apoptosis and inhibition of cell cycle in MM cells. Administration of the drug leads to activation of caspase-8, -9 and -3, increased levels of p53, p21, pro-apoptotic proteins NOXA, PUMA, transcription factor E2F and vice versa reduced levels of cyclin D1 and CDK6. Treatment with ixazomib also induced expression of Bip and CHOP - heat shock protein and transcription factor connected with ER, which expression is induced by cellular stress and is involved in mediating apoptosis. Further, ixazomib effectively inhibits the canonical and non-canonical NF-kB pathways in MM supporting cells, thus influencing cytokines important for growth and survival of MM cells secreted by BMSCs. In this way, cyto-protective effects of BM microenvironment on MM cells are disrupted. It was also shown that ixazomib inhibits tumour-associated angiogenic activity, as the number of VEGFR2- and PECAM-positive cells (cells containing two distinct markers of angio-genesis) was reduced [79]. Study on mouse models revealed that unlike bortezomib, ixazomib possibly relieves bone osteolysis, the most common symptom of MM [80]. MicroRNA profiling of MM cells treated with ixazomib showed increased expression of miR-33b. Increased expression of this miRNA is associated with reduced migration and viability of MM cells as well as with increased apoptosis and sensitivity of MM to ixazomib. Moreover, overexpression of miR-33b led to negative regulation of oncogene PIM-1. Therefore, Tian et al. proposed that miR-33b acts as a tumour suppressor which is involved in the apoptosis of MM cells induced by ixazomib treatment, leading to inhibition of tumour growth and increased survival of human MM xenograft models [83]. Marizomib Marizomib, also known as NPI-0052 or Salinosporamid A (Nereus Pharmaceuticals), is a secondary metabolite of obligate marine bacterium, actinomycetes Salinispora tropica; it is the first natural PI, which has been included in MM clinical research [84]. Chemically, marizomib is bicycle p-lactone-y-lactam with molecular formula C15H20CINO4 (Fig. 3D). Unlike all others Pis, it does not contain a 8 © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol XX, No X 2014 peptide chain in its structure; therefore, it is structurally distinct from bortezomib and carfilzomib. In pre-clinical studies with MM cell lines, marizomib was shown to be highly effective [85]. The combination of bortezomib with marizomib would allow using individual drugs in such concentrations that are non-toxic for patients and improve combined anti-myeloma effect of drugs [86]. Mechanism of action Unlike bortezomib and carfilzomib, which are selective for the CT-L activity of the proteasome, marizomib inhibits all three enzymatic activities of the proteasome as it binds irreversibly with high affinity to the CT-L ((55) and T-L ((52) catalytic sites as well as with lower affinity to the C-L ((51) subunit [87]. It contains a (5-lactone ring that is uniquely substituted with a chloroethyl group playing a role in its irreversible properties. This group binds to the S2 binding pocket of the active site, and as chlorine behaves as a leaving group, it is eliminated to render a stable cyclic ether end product following acylation of the catalytic enzyme active site ThMCF by the (5-lactone of the inhibitor [42]. Comparably to bortezomib, marizomib inhibits the canonical NF-kB pathway and related secretion pathways, such as IL-6, TNF-a and IL-1(5. [87]. On the other hand, unlike bortezomib, which activates both caspase-8 and -9, the apoptotic effect of marizomib is mainly mediated by caspase-8 activation and, to a lesser extent, by caspase-9. As the mechanism of caspases activation by marizomib is different than in bortezomib-mediated activation and relies primarily on caspase-8 activation, it allows to overcome the resistance of MM cells to apoptosis also with Bcl-2 mutations, leading to overexpres-sion of Bcl-2. It was shown that overexpression of Bcl-2 in MM cells confers drug resistance and partially protects MM cells against bortezomib; however, caspase-9 activation by marizomib is minimally affected by Bcl-2 overexpression [87]. Apoptotic signal leads to the release of cytochrome c and Smac proteins from mitochondria to the cytoplasm, generation of oxygen radicals and activation of caspases. Moreover, marizomib is able to induce apoptosis in MM cells even in the presence of MM growth factors IL-6 and insulin growth factor-1 (IGF-1) and is involved in blocking IL-6 secretion in BMSCs without affecting their viability. Notably, marizomib significantly blocks MM cells migration induced by VEGF and thus confirms its anti-angio-genic effect [87]. Novel proteasome inhibitors Oprozomib Oprozomib (ONX0912; Onyx Pharmaceuticals), a new orally bioavail-able and selective peptide epoxyketone PI, represents a deriváte of carfilzomib, which irreversibly inhibits proteasome resulting in longer duration of inhibition compared with bortezomib (Fig. 3E). Just like bortezomib and carfilzomib, oprozomib is highly selective for CT-L ((55) subunit of proteasome; however, in contrast with bortezomib, it specifically inhibits only N-terminal threonine active proteasome subunits [88]. The orally bioavailable oprozomib inhibits proteasome with the same efficacy as intravenously delivered carfilzomib, although in higher concentrations of the drug. It is able to activate JNK and inhibit NF-kB pathways [88, 89]. Further in vitro study using MM cell lines showed that oprozomib inhibits growth, migration and induces apoptosis of MM cell lines and its activity is associated with activation of caspase-8, -9 and -3, and poly(ADP) ribose polymerase (PARP). Oprozomib, suchlike carfilzomib, directly inhibits osteoclasts differentiation and function in vitro. Conversely, it directly stimulates transforming growth factor-(5 (TGF-(5) and MAPK signalling pathways leading to increased activity of UPR, which results in enhanced osteoblasts differentiation and matrix mineralization. Therefore, in MM, oprozomib in a similar way as carfilzomib, shifts the BM microenvi-ronment from catabolic to anabolic state. Efficacy of oprozomib was also tested in vivo using mouse models. Comparably to carfilzomib, oprozomib inhibited MM growth, prolonged survival of mouse models, decreased tumour burden and inhibited bone resorption [31, 88]. Moreover, oprozomib in combination with low-dose bortezomib showed a synergistic anti-MM activity. However, mechanisms mediating combined anti-MM activity of both Pis remain to be defined [88]. Delanzomib Delanzomib (CEP-18770; Teva Pharmaceuticals, North Wales, PA, USA) is an orally active, reversible, boronic acid-based PI. Suchlike bortezomib, it exhibits high potency primarily against CT-L ((55) and then C-L ((51) activity (Fig. 3F). An in vitro study showed that it effectively decreases NF-kB activity and expression of several NF-kB downstream effectors; furthermore, it has strong anti-angiogenic activity and potently represses receptor activator of NF-kB ligand (RANKL)-induced osteoclastogene-sis [90]. Further in vitro study on MM cell lines compared delanzomib with bortezomib in terms of specificity and activity profiles. While the two Pis show comparable proteasome inhibitory effects on cell lines, ex vivo study on pre-clinical mouse model of human MM showed that delanzomib induced an improved response in MM tumours and is active also against bortezomib-resistant cells [91]. Delanzomib was shown in pre-clinical and clinical studies to be effective in combination with melphalan or bortezomib with favourable cytotoxicity profile [92, 93]. Although it showed promising effect and favourable toxicity in the initial studies, its further research has been suspended because of unmanageable toxicity (Teva, personal communication). Mechanism of resistance and cross-resistance of Pis Despite high efficacy of bortezomib, there are still MM patients that are primarily resistant or develop secondary resistance to bortezomib during treatment [94]. So far, several molecular mechanisms of resistance development have been identified. One of them is Ala49Thr © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 9 mutation in the |35 subunit (PSMB5) of the proteasome, which is situated in a binding site for bortezomib and leads to excessive synthesis of PSMB5. Higher levels of PSMB5 at the RNA or protein level were shown to be connected with resistance to bortezomib [95, 96]. High-throughput RNA screen of MM cell lines further revealed a panel of genes - their suppression enhanced bortezomib sensitivity. These genes included proteasome subunits a- and p-type (PSMA5, PSMB2, PSMB3 and PSMB7) as well as Aurora kinase A, CDK5 and modulators of the aggresome pathway. Moreover, the authors confirmed that CDK5 knockdown sensitized MM cell lines to bortezomib and other Pis [97]. In addition, factors downstream of proteasome enzymatic complex can mediate resistance to bortezomib, as was observed in a study where increased resistance of tumour cells to the treatment correlated with elevated levels of anti-apoptotic proteins from the Bcl-2 family and heat shock proteins Hsp27, Hsp70 and Hsp90 [87, 98], Zhang et al. revealed that in bortezomib-adapted cell lines, the treatment continued to inhibit proteasome enzymatic activity. However, it did not lead to induction of UPR and accumulation of pro-apoptotic proteins p53, Mcl-1 S and NOXA because the cells displayed increased expression of factors protecting them from bortezomib-mediated ER stress [99]. More recently, other signalling pathways have been described to mediate bortezomib resistance. It was evaluated that interactions between Notch receptors on MM cells and Notch ligand DM expressed on MM-BMSCs could contribute to bortezomib resistance. Activation of Notch signalling leads to up-regulation of CYP1A, a cytochrome P-450 enzyme involved in the metabolism of a variety of xenobiotic compounds [100]. Blockade of Notch pathway and inhibition of CYP1A expression was able to increase sensitivity of MM cells to bortezomib in vitro [101]. Further in vitro study on MM cell line with no mutation in |35 subunit revealed evidence that increased IGF-1 signalling through enhanced IGF-1 secretion and IGF-1R activation was also associated with resistance to bortezomib [102]. Newly, the focus is on POMP (proteasome maturation protein), which is involved in addition of catalytically active p subunits to the hemipro-teasome ring initially formed by structural a subunits. In bortezomib-resistant cell lines the levels of POMP mRNA are enhanced compared to their drug-sensitive counterparts. POMP overexpression, that is influenced by NF erythroid-2 (NRF-2), contributes to PI resistance in MM [103]. It was described that bortezomib-resistant cells display a marked cross-resistance to |35-targeted cytotoxic peptides, but not to other classes of therapeutic drugs, therefore cross-resistance of bortezomib-resistant cells is restricted to (peptide) drugs that primarily target the proteasome |35-subunit [96]. Therefore, drugs with similar mechanism of action as bortezomib, such as ixazomib and delanzomib, will unlikely overcome bortezomib resistance. However, new drugs that are based on epoxyketone pharmacophore, such as carfilzomib or oprozomib, differ in terms of their chemical structure and mechanism of action. Moreover, carfilzomib is a more selective inhibitor of the CT-L activity of proteasome and immunoproteasome and shows prolonged irreversible inhibition of proteasome - thus it could overcome resistance to bortezomib [77,104]. In fact, carfilzomib was shown in pre-clinical experiments to overcome bortezomib resistance [74]. Nevertheless, new study using mouse MM model and gene expression profiling revealed that bortezomib-resistant cells show cross- resistance to ixazomib and carfilzomib as well. Results of this study also suggested that resistance to one drug class reprograms resistant clones for increased sensitivity to a distinct class of drugs, such as inhibitors of histone deacetylases [105]. Taken together, the results from pre-clinical studies are contradictory so far; although irreversible Pis demonstrate an ability to overcome some forms of bortezomib-mediated resistance, further studies using e.g. combination of irreversible Pis with other chemotherapeutic agents may identify strategies to enhance efficacy or decrease toxic effects. Further, there is a need for additional analyses from currently ongoing studies, which include bortezomib-refractory patients who are treated with either analogues of boric acid Pis, such as ixazomib, or epoxyketones, such as carfilzomib. Induction of neuropathy Peripheral neuropathy (PN) is a significant and most common dose-limiting toxicity of Pis. The pathophysiology and molecular basis of bortezomib-induced PN is not completely understood and current knowledge is limited. Damage of mitochondria and ER seems to play a key role in bortezomib-induced PN genesis, as bortezomib can activate the mitochondrial-based apoptotic pathway [106]. Generally, dipeptide boronates inhibit active proteasome subunits, but also serine proteases. Although inhibition of proteasome by bortezomib has originally been shown to be several orders of magnitude stronger than inhibition of serine proteases [107], it has been revealed that bortezomib inhibits also an ATP-dependent serine protease in mitochondria - HtrA2/Omi. As HtrA2 protects neurons from apoptosis, it is now believed that its inhibition is the cause of PN in MM [108]. In contrast with bortezomib, peptide epoxyketones, such as carfilzomib and oprozomib, specifically inhibit only N-terminal threonine active proteasome subunits. This difference may be responsible for the favourable toxicity profiles and relatively low rates of PN associated with epoxyketone Pis. Further proposed mechanism of bortezomib-induced PN genesis is dysregulation of neutrophins, as bortezomib inhibits activation of NF-kB and thus blocks the transcription of nerve growth factor-mediated neuron survival [109]. A GEP study compared patients with grade 2-4 late-onset bortezomib-induced PN, patients developing early-onset grade 2-4 bortezomib-induced PN and patients who did not develop bortezomib-induced PN. Results suggested that patients with early-onset grade 2-4 bortezomib-induced PN have dysregulated genes involved in control of transcription, apoptosis and AMPK-mediated signalling. Out of them, AMPK-mediated signalling is of particular interest, because this enzyme stimulates the signalling pathways that replenish cellular ATP supplies in response to low glucose, hypoxia, ischaemia or heat shock, which might be triggered in MM cells in response to bortezomib. On the other hand, patients with late-onset grade 2-4 bortezomib-induced PN showed 27 differentially expressed genes when compared to the first group, with the enrichment of expression of genes involved in transcription regulation as well as in the development and function of the nervous system, including S0D2 and MY05A [110]. Moreover, the authors suggested an interaction 10 © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol XX, No X 2014 Table 3 Key features of different proteasome inhibitors Inhibitor of proteasome IC 50 for CT-L activity IC 50 for C-L activity IC 50 for T-L activity Half-life (minutes) Application Bortezomib 7.9 ± 0.5 nM 53 ± 10 nM 590 ± 67 nM 110 Intravenous Carfilzomib <5 nM 2400 nM 3600 nM <30 Intravenous Marizomib 3.5 ± 0.3 nM 430 ± 34 nM 28 ± 2 nM 10-15 Intravenous Ixazomib 3.4 nM 31 nM 3500 nM 18 Oral Oprozomib 36 nM/82 nM ND ND 30-90 Oral Delanzomib 3.8 nM ND ND ND Oral between MM-related factors and the patient's genetic background in the development of bortezomib-induced PN, as several of single nucleotide polymorphisms (SNPs) were associated with early-onset bortezomib-induced PN (SNPs located in caspase 9, RDM1, AL0X12, /GF/fiand LSM1 genes). In addition, SNPs associated with late-onset bortezomib-induced PN were preferentially located in DNA repair genes (SNPs in ERCC3, ERCC4, ATM, BRCA1, EX01 and MRE11A genes) [110]. Apart of mechanisms mentioned above, another possible mechanism for different rates of PN induced by Pis is their diverse proteasome dissociation half-life, pharmacokinetics and pharmacondynamics (Table 3). It was shown that in the group of boronic acid Pis, ixazomib has a six-fold faster proteasome dissociation half-life than bortezomib, greater overall tumour pharmacodynamic effect than bortezomib and prolonged overall survival in a mouse model. All of these features might stand for lower toxicity [82]. Further, as was tested on a fruit fly model, the group of epoxyketone Pis exerts significantly milder impact on neuromusculatory system than bortezomib [111]. Proteasome inhibitors in other diseases Besides MM, bortezomib has been described to be effective in several other lymphoid malignancies; it demonstrated clinical potential in the treatment of mantle cell lymphoma (MCL) and non-Hodgkin lymphoma and is effective also in treatment of newly diagnosed Waldenstrom macroglobulinaemia (WM) [112-114]. In 2007, FDA granted approval to single-agent bortezomib for the treatment of relapsed/ refractory MCL patients [115] and currently it is tested in multiple clinical trials as a component of cytotoxic chemotherapeutic regimens [116]. Further experience with second-generation Pis, such as carfilzomib is limited. Carfilzomib in combination with other drugs was tested in B-cell lymphomas (DLBCL, MCL) to overcome bortezomib resistance with promising results [117]. Also, it is under investigation in WM patients. Although the data are still immature, the biggest advantage of this drug seems to be the lack of neurotoxicity [118]. Conclusion The inhibition of proteasome has become an impressively successful strategy in MM treatment for the past 10 years, because of the particular sensitivity of MM cells to the mechanism of action of such agents. Today, therapies based on bortezomib belong to the standards of care for both relapsed/refractory and previously untreated MM patients, dramatically improving outcome of these patients. Although the exact mechanisms of action of Pis are not yet fully defined, four major mediators of direct anti-MM activity have been identified: transcription factor NF-kB, pro- and anti-apop-totic factors, p53 protein and UPR leading to ER stress response. However, the effect of Pis should be understood as a complex process involving many signalling pathways as neither the inhibition of NF-kB nor inactivating mutations of p53 are able to evoke apopto-sis of MM cells induced by Pis. Detailed studies of mechanism of Pis action have a great potential for the discovery of possible molecular targets of new-generation drugs. Continuing development of new-generation Pis will likely offer further opportunities and better regimens in terms of treatment efficacy, acceptable tolerability, administration and quality of life of MM patients. Nowadays, Pis are the most effective group of anti-MM drugs, and they will surely be a cornerstone in all combination regimens used in MM treatment even in the future. Acknowledgements This work was supported by grant of the Ministry of Education, Youth and Sports MSM0021622434, IGA grants of the Ministry of Health: MT12130, NT14575, grant MUNI/11/lnGA17/2012 and by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 278570. The authors also wish to thank John B. Smith for proofreading the manuscript. Conflicts of interest The authors confirm that there are no conflicts of interest. © 2014 The Authors. 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Palazzo dei Congressi, Lugano, Switzerland, June 19-22, 2013, abstract 150,2013. 119. Feling RH, Buchanan GO, Mincer TJ, et al. a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus sali-nospora. Angew Chem Int Ed Engl. 2003: 42: 355-7. 120. Zhou HJ, Aujay MA, Bennett MK, et al. Design and synthesis of an orally bioavail-able and selective peptide epoxyketone proteasome inhibitor (PR-047). J Med Chem. 2009; 52: 3028-38. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 15 The miR-29 family in hematological malignancies Fišerová B, Bešše L, Sedlaříková L, Hájek R, Ševčíková S. Biomedical Papers, Univerzita Palackého v Olomouci, 2014. ISSN 1213-8118. doi:10.5507/bp.2014.037. PMID: 25004911 IF v roce 2014: 1,661 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. The miR-29 family in hematological malignancies Barbora Fišerova3, Lenka Kubiczkovaab, LenkaSedlarikova3 b, Roman Hajekab,SabinaSevcikovaab Background. MicroRNAs are short non-coding regulators of gene expression. The human miR-29 family consists of three members: miR-29a, miR-29b and miR-29c. Members of this family were found to be aberrantly expressed in various types of tumors, including hematological malignancies. This family was described to have both oncogenic and tumor suppressor features influencing various pathological processes, such as tumor growth and apoptosis. This review summarizes current knowledge about the miR-29 family in selected hematological malignancies. Conclusion. Recent research of miR-29 family in hematological malignancies has proven its oncogenic as well as tumor suppressive potential. Nevertheless, the level of current evidence is not sufficient, and data remain inconclusive. Key words: microRNA, miR-29 family, hematological malignancy, circulating miRNA Received: December 13, 2013; Accepted with revision: June 12, 2014; Available online: July 4, 2014 http://dx.doi.Org/10.5507/bp.2014.037 "Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 1'Department of Clinical Hematology, University Hospital Brno Corresponding author: Sabina Sevcikova, sevcik@med.muni.cz INTRODUCTION MicroRNAs (miRNAs) are short, non-coding and highly conserved RNAs, approximately 22 bp in size1. The genes for miRNAs represent 1-2% of all known eukaryotic genes2. They regulate gene expression, both at transcriptional and translational levels. A single miRNA molecule can control expression of various genes and vice versa, one gene can be regulated by various miRNAs3. MiRNAs act in a wide range of physiological biological processes, such as cell proliferation, differentiation, apoptosis and hema-topoiesis46. As all of these processes, as well as miRNAs levels, are dysregulated in solid tumors and hematological malignancies, it was confirmed that there is an association between miRNAs and cancer7. Despite the fact that miRNAs were discovered in 1993 (ref.8), it was only in 2002 that miR-15a and miR-16-1 were identified as potential cancer genes in the pathogenesis of chronic lymphocytic leukemia (CLL) highlighting the direct link between miRNAs deregulation and hematological malignancy9. Genes for miRNA are frequently located at fragile sites and genomic regions involved in cancers, such as minimal regions of loss of heterozygosity, minimal regions of amplification (minimal amplicons), or common breakpoint regions, explaining the contribution of miRNAs to cancerogenesis10. Such localization may lead to upregulation of miRNAs levels or their downregulation during pathological processes. Further, depending on the mRNA target which miRNAs bind and regulate, they can act either as oncogenes (also called oncomirs) or as tumor suppressors11. Due to overexpression of miRNA targeting tumor suppressor gene, anti-oncogenic mechanisms can be inhibited, whereas defects of miRNA repressing oncogene can lead to gain of oncogenic features. Both these roles have been demonstrated in tumors1214. In general, miRNAs can affect specific cell development (e.g. B cell) or alter expression of components in miRNA biogenesis in hematological malignancies. Canonical model of the miRNAs biosynthetic pathway involves several steps as shown in Fig. 1. In the nucleus, RNA polymerase II transcribes miRNA genes into long primary precursors - pri-miRNAs. These are recognized and cleaved by microprocessor complex including ribo-nuclease Drosha and dsRNA-binding protein Pasha (or DGCR8) (ref.1516). Secondary precursors are short, about 70 nucleotides stem-loop structures, known as pre-miR-NAs that are further actively transported to cytoplasm by exportins, Ran-GTP dependent transporters. In the cytoplasm, pre-miRNAs are processed near the terminal loop by RNase III type endonuclease Dicer, which is in complex with dsRNA-binding protein TRBP (TAR RNA binding protein), and this generates mature miRNA/ miRNA* duplexes1718. Subsequently, one strand of mature miRNA (so-called guide strand), which is less stable at the duplex 5'end, is incorporated into the Argonaut protein, a central part of multiprotein complex miRISC (miRNA-induced silencing complex). The other strand, called the passenger strand (miRNA*), is released from the duplex and degraded. The question of gene fate now lies in the RISC complex with incorporated mature miRNA because this is the site where gene mRNA and miRNA pair. If the complementarity between the seed sequence of miRNA (2-8 nucleotides at 5'end) and the 3'UTR of the target mRNA is perfect, mRNA is cleaved and degraded. In the other case, low degree of complementarity leads to inhibition of mRNA translation (Fig. 1) (ref.19). 1 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. RNA polymerase nucleus I cytoplasm mRNA degradation translational inhibition Fig. 1. MiRNA biogenesis. miRNA genes are transcribed in the nucleus, into long primary precursors - pri-miRNAs. Then, they are cleaved by Drosha and Pasha. Secondary precursors are short, stem-loop structures, known as pre-miRNAs that are further actively transported to cytoplasm by exportins. In the cytoplasm, pre-miRNAs are processed near the terminal loop by RNase 111 type endonuclease Dicer, and this generates mature miRNA/miRNA* duplexes. THE MIR-29 FAMILY In the human miR-29 family, the precursors are transcribed into two clusters, miR-29a/miR-29b-l from chromosome region 7q32, and miR-29b-2/miR-29c from chromosome region lq32. As the only difference between miR-29b-l and miR-29b-2 is their localization in different parts of genome, they both form identical mature miR-29b. The first discovered member of the family was miR-29a in HeLa cells in 2001 (ref.20), followed by miR-29b and miR-29c (ref.2122). The members of the miR-29 family have identical seed sequence, similar expression patterns as well as function. The only differences among miR-29 members were reported in their expression levels in various cancerous tissues as some studies claim that miR-29a or miR-29c do not follow the same expression pattern as miR-29b (ref.23). It was described that in lung cancer, only miR-29b-2 was differentially expressed24. Further, miR-29b was found to be differentially expressed in cholangiocytes and in brain malignancies2526. Another study demonstrated that expression of miR-29a and miR-29c in cervical cancer was decreased27. These results suggest that miR-29 is not tissue-specific. MIR-29 UNDER PHYSIOLOGICAL CONDITIONS The miR-29 family regulates several signaling pathways that are involved in various physiological and pathological processes. Physiologically, it takes part in regulation of cell cycle and proliferation27 30, senescence3132, differentiation33,34, apoptosis25'28'30'35,36, metastasis37,38, DNA methyla-tion3941 and immune regulation42,43, as well as regulation of extracellular matrix (ECM). MiR-29 in cell cycle, proliferation and differentiation Progression through the eukaryotic cell cycle is driven by cyclin-dependent kinases (CDKs), which are regulated by interaction with oscillatory expressed proteins called cyclins44. In cell cycle progression, from Gl to S phase, cyclin D1 binds CDK6 and CDK4 which then phosphory-late and inactivate Rb protein. These CDKs are essential for response to mitogenic stimuli, therefore the loss of CDK6 affects production of terminally differentiated cells (Fig. 2) (ref.45). It was demonstrated that 3'UTR of CDK6 contains 2 conserved sequence motifs with perfect homology to miR-29 seed sequence; therefore, CDK6 was suggested as a direct target of miR-29 (ref.29). In terms of its role in cell differentiation, miR-29b has multiple functions in osteoblastogenesis - to control collagen expression during ECM maturation is one of them. However, this process does not happen in immature cells. Instead, miR-29b helps to maintain the differentiated phe-notype in osteoblasts through regulating collagen. On the other hand, miR-29b downregulates negative regulators of signaling pathways to promote osteoblastogenesis. Both these miR-29b roles regulate osteoblast differentiation46. In another study, miR-29a and miR-29c were shown to be induced by the Wnt pathway that is critical in osteoblast differentiation. During the late phases of osteoblast differentiation, the expression of these miRNAs is upregulated and increased. Beside this, miR-29a and miR-29c down-regulate osteoblast differentiation by targeting osteonectin, an essential protein for bone remodeling33. Apart from osteoblast differentiation, miR-29 was reported to play a role in muscle cells development. The miR-29 family enhances myogenic differentiation through its involvement in the NF-kB-YYI regulatory loop. In myo-genesis, downregulated transcription factors NF-kB and Yin Yang 1 (YY1) decrease miR-29 levels and this in turn induces differentiation by targeting YY1 (ref.34). Another experiment showed that miR-29 together with miR-142 also regulates monocytic and granulocytic cyclin D E3 phosphorylation status + phosphorylation status B Fig. 2. MiR-29 function in cell cycle. (A) Cell cycle without miR-29 influence, (B) MiR-29 inhibits Cdk6 which cannot bind with cyclin D and phosphorylate Rb; therefore, cell is not differentiated. 2 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. (myeloid) differentiation. Targeting CDK6 by miR-29, as well as targeting cyclin T2 (CCNT2), a component of positive transcription elongation factor b (P-TEFb), by either miR-29 or miR-142 increases myeloid differentiation47. Aging processes and senescence MiR-29 might be a pro-aging miRNA as it accumulates during aging and its upregulation is associated with DNA damage response. MiR-29 is part of the signaling pathway involving Ppmld/Wipl phosphatase, a key DNA damage response regulator, and the p53 tumor suppressor (Fig. 3) (ref.31). Further effects of miR-29 during cellular senescence were described in association with B-Myb which is an oncogene and a transcription factor for various genes involved in proliferation48. Besides these functions, B-Myb is able to induce senescence by inhibition of its transcription49. One of the options for repressing abundant B-Myb mRNA is through binding of Rb-E2F complexes to B-Myb promoter50. The other option of B-Myb repression at the posttranscriptional level involves miRNAs. MiR-29 together with miR-30 directly targets B-Myb 3'UTR and reduce its expression in cells undergoing senescence32. These facts are also consistent with miR-29 suppressor function in cancer. ECM regulation ECM regulation includes formation of extracellular matrix key proteins, e.g. various collagen (COL) isoforms, elastins, metalloproteinases, etc (ref.23,46). In osteoblasts, miR-29 regulates essential proteins of bone ECM. It mediates translational inhibition and decreases C0L1A1, C0L5A3, C0L4A2 synthesis; furthermore, it maintains differentiated phenotype in mature cells46. The broad spectrum of collagens and other related genes, e.g. matrix me-tallopeptidase 2, which are miR-29 targets, was confirmed and even extended in a study done on rats. There were 20 genes for collagen predicted as miR-29 targets which makes this miRNA unique because no other miRNA targeted more than 11 collagen genes23. Regulation of these proteins by miR-29 is implicated in the development of fibrosis in many organs5153 and Fig. 3. Inhibition of p53 pathway by miR-29. Upregulation of miR-29 with increasing age and DNA damage inhibits Wipl phosphatase. After that, Wipl phosphatase cannot repress DNA damage responsefactors and p53. Wipl and mdm2 are not transcribed by p53 which is not phosphorylated. Cell cycle is arrested. systemic sclerosis54. Not only in mice developing liver fibrosis, but also in patients with hepatic fibrosis, the miR-29 family was significantly downregulated and inhibited collagen expression in hepatic stellate cells52. A significant decrease was also found in the lungs of idiopathic pulmonary fibrosis patients53. MIR-29 IN HEMATOLOGICAL MALIGNANCIES Despite the range of physiological processes the miR-29 family is involved in, most studies concentrate on its pathological function and tumor suppressive or oncomir (oncogenic miRNA) effects in various cancers. In terms of solid tumors and hematological malignancies, both these roles have been proven; they are believed to depend on cellular context or tissue specificity. Although expression of miR-29 was found to be altered in cancer, its role in pathogenesis of hematological malignancies is still poorly understood55. There is, however, a predominance of publications supporting the tumor suppressor role of the miR-29 family. By targeting oncogenes, the miR-29 family helps prevent carcinogenesis; therefore, in cancer, its levels are downregulated (Table 1) (ref.12'56'57). Furthermore, it was observed that miR-29 is associated with some cytogenetic aberrations. MiR-29, among other miRNAs, was found to be down-regulated in acute myeloid leukemia (AML) patients with llq23 balanced translocation compared to AML patients without this translocation56. Further, Garzon et al. observed that miR-29 a and miR-29b are downregulated in primary AML samples with monosomy of chromosome 7. However, forced expression of these miRNAs had first anti-prolif-erative effects and later anti-apoptotic effects in AML cell lines and primary AML blasts, thus inhibiting cell growth and induced apoptosis by targeting Mcl-1 (Myeloid cell leukemia-1) (ref.28). In AML patients with monosomy of chromosome 7 or deletion of 7q, a link between miR-29a and oncogene Ski was described as the nuclear oncogene Ski is upregulated and miR-29a located on 7q32 is downregulated in these AML patients. Further, it was shown that miR-29a targets Ski, as their expression is inversely correlated, which suggest the tumor suppressive role of miR-29a (ref.58). Although previous study reported also tumor suppressor miR-29 family to be upregulated in AML patients with mutations in the nucleophosmin (NPM) gene when compared to wild type NPM (ref.59); however, this was not confirmed and miR-29 downregulation was described in AML patients independently of the NPM status60. A genome-wide profiling study on CLL (chronic lymphocytic leukemia) revealed that miR-29 precursors are upregulated61. Afterwards, another study demonstrated downregulated miR-29a in aggressive CLL compared to indolent CLL (ref.35). Interestingly, miR-29a was found to be the second and miR-29c the fifth most represented miRNA among the most expressed miRNAs in CLL (ref.62). Despite some knowledge about miR-29 in other hema- 3 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. tological malignancies, little is known about this miRNA in mantle cell lymphoma (MCL) and further research in this field is needed. One report showed notably decreased miR-29 levels in MCL patients, which were associated with higher levels of its target CDK6 and with shorter overall survival of MCL patients. Therefore, the use of miR-29 as a prognostic marker and pathogenetic factor in MCL was suggested29. Besides studying direct miR-29 family effects and its participation in regulation, there is an effort to apply miR-29 as novel biomarkers. The analysis of miR-29a together with miR-142-3p indicated that these miRNAs could be used as AML molecular diagnostic markers. Because of their key role in regulation of normal myeloid differentiation, miR-29a and miR-142-3p abnormal expression was shown to be involved in AML development, as it directly affected target genes important in AML (ref.60). Recently, the first evidence of miR-29 a as an unfavorable prognostic marker in AML was indicated, as downregulation of miR-29a was shown to be associated with advanced clinical features and poor prognosis in pediatric AML patients63. Apoptosis in hematological malignancies Majority of studies show that miR-29 family effects in cancer are anti-apoptotic. However, in studied hematological malignancies, the miR-29 levels were lower than in physiological conditions. Therefore the tumor suppressive impact of miR-29 on cancer cells is poor (Table 1). MiR-29 family was described to target genes involved in regulation of apoptosis, such as Bcl-2 (B-cell leukemia/ lymphoma) family members and a key anti-apoptotic protein Mcl-1 that are often dysregulated in malignant cells (Table 2). Constitutive Mcl-1 expression can cause malignant transformation as was demonstrated in transgenic mice64. MiR-29b negatively regulates Mcl-1 protein expression; low miR-29b levels upregulate Mcl-1 expression and thus induce anti-apoptotic signals and may play a role in tumor development. On the other hand, experiments with enforced miR-29b expression showed sensitivity to cell death which might be valuable in cancer therapy25. In multiple myeloma (MM), a plasma cell malignancy, the miR-29b tumor suppressor effects are implicated as well. It was shown that miR-29b is downregulated in MM; however, its overexpression can downregulate Mcl-1 expression and is associated with caspase-3 activation. By targeting critical oncogenic pathways, miR-29b inhibits growth and induces apoptosis of MM cells12. Another miR-29 family target is Tell (T-cell leukemia/ lymphoma 1) gene), a significant oncogene involved in CLL pathogenesis. Tell operates as a coactivator of the Akt oncoprotein that is important in the anti-apoptotic pathway in B- and T-cells65,66. Pekarsky et al. demonstrated that miR-29 family members are partly natural Tell inhibitors and that downregulated miR-29 levels in aggressive CLL might be a causal event in disease pathogenesis35. Another study suggested that the downregulation of miR-29 upregulates Tell in aggressive CLL, and thus develops aggressive phenotype14. Amodio et al. recently identified new miR-29b target Spl, a transcription factor that participates in cell cycle regulation and apoptosis67. In MM, Spl is involved in cell survival and promotes MM cell growth68. Spl is downregulated by miR-29 but it was demonstrated that the forced expression of miR-29b in cell lines inhibited cell growth and triggered apoptosis in vitro and in vivo in a murine model. Besides this, miR-29b-Spl regulatory loop was described. Not only miR-29b influences Spl but also Spl negatively regulates miR-29b. Upregulated Spl transcriptionally inhibits miR-29b and silenced Spl increases miR-29b levels. All of this may prevent the tumor formation in a model of MM (ref.67). A study done by Garzon et al. describes the effects of miR-29 on both apoptosis and proliferation in AML cells. The forced expression of miR-29a and miR-29b led to cell growth inhibition and induction of apoptosis. After the transfection of the miRNAs, the first observed effect was inhibition of apoptosis. It was confirmed that Mcl-1 and other anti-apoptotic genes are miR-29 targets and that this miRNA also upregulates proapoptotic genes. The anti-proliferative effect was observed later after the transfection, which means that the miR-29-dependent proliferation is not a result of apoptosis. For studying miR-29 Table 1. MiR-29 involvement in cancer. Target MiR-29 function MiR-29 regulation Cancer development Oncogene Tumor suppressor upregulated no downregulated induced Tumor suppressor Oncomir upregulated induced downregulated no Table 2. Oncogenes as miR-29 targets in hematological malignancies. Target Function miR-29 member References Mcl-1 Cell survival, proliferation, apoptosis miR-29b 12,25,64 Tcl-1 Apoptosis not specified 35 Spl Cell growth, survival miR-29b 67 SKI Nuclear corepressor complexes miR-29a 58 CDK6 Cell cycle not specified 29 4 Biomed Pap Med FacUniv Palacký Olomouc Czech Repub. 2014; 158:XX. impact on proliferation, CDK6, a miR-29 target, was chosen. Transfection of miR-29b into AML cells indirectly led to decreased Rb phosphorylation through CDK6, which resulted in decreased proliferation28. Tumor initiation and growth Although tumor suppressor effect of miR-29a was elucidated, some studies show that the miR-29 family has also tumor promoting effects, but this oncomir function still remains poorly understood. The first example of a miRNA initiating AML in vivo was reported by Han et al. They showed that miR-29a was highly expressed in human AML and its overexpression led to higher incidence of AML. MiR-29 a can induce AML by converting myeloid progenitors into self-renewing leukemia stem cells, thus showing oncogenic potential13. The same was demonstrated in CLL where miR-29 a was overexpressed in indolent CLL in comparison to normal B cells. However, a hypothesis of solely miR-29 initiating leukemia was not confirmed14. As for MM, one of its main characteristics is bone disease which is a result of imbalance between osteoblasts and osteoclasts bone formation caused by MM cells. It was found that miR-29b expression decreases during osteoclast differentiation in vitro and suppresses its targets c-Fos and metalloproteinase 2. miR-29b-based treatment of MM-related disease was suggested when the results showed enforced miR-29b expression disrupting osteoclast differentiation and overcoming osteoclast activation69. CIRCULATING MIR-29 In 2008, the discovery of miRNA present in body fluids was reported. MiRNAs were found in almost all body fluids, e.g. serum, plasma, saliva, urine, etc (ref.70" 73). Interestingly, under unfavorable conditions, such as boiling, storage at room temperature, low or high pH or repeated cycles of freeze-thawing, plasma miRNAs were found to be unconventionally stable. Possibly, there are two mechanisms by which circulating miRNAs are protected from degradation. The first possibility is to form ri-bonucloeprotein complexes of miRNA and RNA-binding protein, e.g. Ago2 (ref.74), NPM-1 (ref.75) or high-density lipoproteins (HDLs) (ref.76). The other option is packaging in small vesicles. Depending on the size and form of release, these small vesicles can be exosomes, which are released from endosome membrane, or microvesicles, that are shed directly from plasma membrane, or even apop-totic bodies77 80. Current evidence shows that the majority of circulating miRNAs are bound to proteins rather than found in vesicles. This aside, it seems that cells can actively select which miRNAs will be released from cells and which will stay within the cell81. However, little is known about circulating miRNA origins and factors in their regulation and other underlying mechanisms need to be determined. Our own data showed the presence of circulating serum miR-29a in MM patients. Serum levels of miR-29a were able to distinguish MM patients from healthy donors. Although further analysis is required, it is possible that circulating miRNAs represent a novel and easily accessible putative marker82. Such a marker would be important and highly clinically relevant in diseases such as MM, where frequent testing of bone marrow is not ethically permissible. The question of comparing established biomarkers and circulating miRNAs was investigated in patients with CLL (ref.83). A set of 3 miRNAs, including miR-29a, was able to distinguish healthy controls from CLL patients. Furthermore, another set of miRNAs, including miR-29 a, was compared with IgVH and zeta-associated protein (ZAP) status, an established clinical risk stratifier in CLL (ref.84). This miRNA set could separate ZAP-70+ and ZAP-70" samples but did not correlate with IgVH mutation status83. CONCLUSION First dismissed as a type of junk RNA, miRNAs were demonstrated to be pivotal in gene regulation. In recent years, miRNAs have also been discovered to be important players in cancer pathogenesis and understanding of their significance has broadened. MiRNAs function both as tumor suppressors and oncomirs. MiR-29 is involved in various physiological processes, such as proliferation, differentiation, apoptosis and senescence. It has also been shown that the miR-29 family is deregulated in hematological malignancies as well as in solid tumors. The analyses are influenced by heterogeneity of the diseases, detection methods used, various genetic background of patients/control groups, and different disease stage. In some cases, small data sets may impair data validation. However, its specific role in hematological malignancies remains unclear. ACKNOWLEDGEMENT The work was supported by the grant of The Ministry of Education, Youth and Sports MSM0021622434, by grant of the Ministry of Health NT12130 and NT14575, and grant MUNI/ll/InGA17/2012. The authors would like to thank John B. Smith for proofreading the manuscript. Autorship contributions: BF: literature search and manuscript writing; LK, LS, RH: manuscript writing; SS: final approval. Conflict of interest statement: None declared. 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ZenzT, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer 2010;10(1):37-50. 8 Combination of serum microRNA-320a and microRNA-320b as a marker for Waldenstrom macroglobulinemia Kubiczkova-Besse L, Sedlarikova L, Kryukov F, Nekvindova J, Radova L, Almasi M, Pelcova J, Minarik J, Pika T, Pikalova Z, Scudla V, Krejci M, Adam Z, Pour L, Hajek R, Sevcikova S. Am J Hematol. 2014 Nov 26. doi: 10.1002/ajh.23910. PMID: 25428891 IF v roce 2014: 3,477 CORRESPONDENCE Combination of serum microRNA-320a and microRNA-320b as a marker for Waldenstrom macroglobulinemia To the Editor: IgM monoclonal gammopathies are a group of diseases characterized by increased level of IgM immunoglobulin produced by one clone of B cells. These diseases range from benign (monoclonal gammopathy of undetermined significance, MGUS) to malignant, such as Waldenstrom macroglobulinemia (WM) or to a lesser extent multiple myeloma (MM) [1,2]. The criteria that differentiate WM from IgM-MGUS are based on the extent of bone marrow (BM) involvement, amount of serum concentration of the M-protein, presence or absence of symptomatic disease or more recently, MYD88 (L265P) or CXCR4 mutations [3-6]. Despite that, new criteria for the differential diagnosis between these conditions are still needed, circulating microRNAs (miRNAs) being one of them. Circulating miRNAs are present in different body fluids; they reflect physiological or pathological conditions and can be used for patient classification [7,8]. Thus, we aimed to investigate the ability of serum miRNAs to distinguish WM from IgM-MGUS as well as IgM-MM patients and healthy donors (HD). For this purpose, circulating miRNAs were isolated from serum samples and screening of 667 miRNAs using TaqMan Low Density arrays was performed on five WM patients, five IgM-MGUS, five IgM-MM patients, and five HD samples to identify differently expressed circulating miRNAs in WM (Supporting Information Fig. SI). Out of deregulated miRNAs, miR-320b, miR-320a, miR-151-5P, and let-7a were further validated by quantitative real-time PCR (qPCR) on a larger cohort of 21 WM, 15 IgM-MGUS, 10 IgM-MM, and 18 HD serum samples (Supporting Information Table SI), as they were present at the top of the list of deregulated miRNAs between IgM-MGUS, HD, and WM and showed highest fold change and most favorable expression (Ct < 30). In addition, some of the miRNA levels were correlated with clinically important parameters and MYD88 (L265P) mutation status. MiR-320a and miR-320b showed different expression between WM and all other groups of samples (P < 0.05). Let-7a and miR-151-5P were significantly decreased in WM samples as compared with HD (all P < 0.05) and IgM-MGUS samples (all P < 0.05) but not with IgM-MM (P = 0.285 and P = 0.286, respectively). As only miR-320a and miR-320b remained statistically significant, only these two miRNAs were chosen for further analyses (Supporting Information Table S2). Receiver Operating Characteristic curves (ROC) were used to evaluate diagnostic effectiveness of miR-320b and miR-320a and to estimate the appropriate cutoff (Supporting Information Table S3). MiR-320b was more potent than miR-320a to distinguish WM from HD with sensitivity of 85.7% and specificity of 94.4% using cutoff value of 1,072 copies per 1 ng of miRNA/RNA. However, combination of miR-320b with miR-320a improved sensitivity up to 90.5% with specificity of 94.4% using cutoff value — 0.6253 obtained from nominal logistic regression model (Fig. 1A). More importantly, miR-320b discriminated WM from IgM-MGUS with specificity of 73.3% and sensitivity of 85.7% using cutoff value of 1,072 copies per 1 ng of total miRNA/RNA, and combination of miR-320b with miR-320a reached specificity of 73.3% and increased sensitivity up to 90.5% with cutoff defined as -0.2373 (Fig. IB). Furthermore, miR-320b distinguished also WM from IgM-MM with sensitivity of 71.4% and specificity of 80.0% with cutoff 904 copies per 1 ng of total miRNA/RNA, and together with miR-320a, the two-miRNA based combination yielded sensitivity of 81.0% and specificity of 100.0% with cutoff value defined as -1.4322 (Fig. 1C). Additionally, associations between miR-320a and miR-320b expression levels and important disease parameters of IgM-MGUS, WM, and IgM-MM were investigated. In IgM-MGUS group, a positive correlation was found between levels of miR-320b and albumin (P < 0.05; rs = 0.521) and moderate positive correlation between levels of miR-320b and calcium (P < 0.06; rs = 0.509). In WM group, levels of miR-320a negatively correlated with (^-microglobulin (P < 0.05; rs = —0.468) and with percentage of lymphoplasmacytic cells infiltration in the BM (P < 0.05; rs = -0.573). Next, in IgM-MM group, miR-320b and miR-320a negatively correlated with levels of M-Ig (P < 0.05; rs = —0.782 and rs = —0.636, respectively), and there was a positive correlation between levels of miR-320a and lactate dehydrogenase (P < 0.05; rs = 0.818). All associations are present in Supporting Information Table S4. We also evaluated mutation status of MYD88 (L265P) in all available PB samples (Supporting Information Fig. S2). Levels of miR-320a were significantly lower in MYD88 (L265P) positive patients (P = 0.032), and there was an identical trend for miR-320b, although not significant (P = 0.079). Both miR-320a and miR-320b were observed in higher concentrations in cellular fractions in comparison with cell-free fractions, and interestingly, more copies of these microRNAs were observed in the CD19-fraction. Additionally, both miR-320a and miR-320b were present in exosomes as well as in exosome-depleted samples; however, their levels tend to be increased in exosomal fractions (Supporting Information Fig. S3). The investigation of molecular features of IgM-monoclonal gammopathies is essential to identify specific risk markers for disease development. Routinely, differentiation of IgM-MGUS from WM is possible only by trepanobiopsy, as neither BM aspiration nor flow-cytometry provide sufficient data for diagnosis. Therefore, in this study, we focused on the role of circulating serum miRNAs as biomarkers of WM. Combination of miR-320a and miR-320b served as the best indicator for WM as it was able to distinguish WM from HD, but more importantly WM from premalignant IgM-MGUS and malignant IgM-MM. Our data suggest that such miRNAs combination might be a novel effective tool for WM discrimination which, however, needs further validation and study. Although the amount of patients with mutant MYD88 (L265P) was small, it can be hypothesized that mutation in MYD88 might be connected to lower miRNA levels; however, underlying biology again needs to be elucidated. It still remains an open question, if studied miRNAs are actively or passively secreted from tumor cells; nevertheless, it is plausible to assume that they are actively transported in vesicles, as they are present primarily in exosomes. Association of low levels of these miRNAs with WM diagnosis suggests their involvement in the disease; however, the origin of such miRNAs in circulation still remains unclear. Nonetheless, considering the minimally invasive character of serum sampling, reproducibility, and easy detection of circulating miRNA, they may provide a convenient and inexpensive method to establish the diagnosis of WM or to predict the evolution of IgM-MGUS. A) B) C) WM vs HD WM vs IgM-MGUS WM vs IgM-MM 0 20 40 60 80 100 0 20 40 60 60 100 0 20 40 60 SO 100 100-Specificily 100-Specifieity 100-Specificity Figure 1. Receiver operating characteristics (ROC) curves. ROC curves for combination of miR-320a and miR-320b distinguishing (A) WM from HD with area under the curve (AUC) = 0.921, (B) WM from IgM-MGUS with AUC = 0.743, (C) WM from IgM-MM with AUC = 0.924. ©2014 Wiley Periodicals, Inc. doi:10.1002/ajh.23910 American Journal of Hematology, Vol. 00, No. 00, Date 2014 1 Kubiczkova-Besse et al CORRESPONDENCE ■ Acknowledgments The authors would like to thank the patients involved in this study and their caregivers. The authors would also like to thank Barbora Sablikova, Božena Hanákova, and Marcela Stouracova for technical support. John B. Smith proofread the manuscript. Lenka Kubiczkova Besse1'2, Lenka Sedlarikova1'2, Fedor Kryukov1'3, Jana Nekvindova4 Lenka Radova5, Martina Almasi1'2, Jana Pelcova6, Jiri Minarik7, Tomas Pika7 Zuzana Pikalova7, Vlastimil Scudla7, Marta Krejčí6, Zdenek Adam6 Luděk Pour6, Roman Hajek1'2'3 and Sabina Ševčíková1'2* 1 Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; 2Department of Clinical Hematology, University Hospital Brno, Brno, Czech Republic; ^Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic; institute for Clinical Biochemistry and Diagnostics, University Hospital Hradec Kralove, Czech Republic; 5Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 6Department of Internal Medicine - Hematooncology, University Hospital Brno, Brno, Czech Republic; 7Department of Hematooncology, University Hospital Olomouc, Olomouc, Czech Republic Conflict of interest: Nothing to report. Contract grant sponsor: The Ministry of Health (IGA Grants); Contract grant numbers: NT12130, NT13190, NT14575; Contract grant sponsor: The Ministry of Health, Czech Republic - Conceptual Development of Research Organization; Contract grant number: FNBr, 65269705 ^Correspondence to: Sabina Ševčíková; Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic. E-mail: sevcik@med.muni.cz Additional Supporting Information may be found in the online version of this article. Received for publication: 20 November 2014; Accepted: 24 November 2014 Published online: 26 November 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.23910 ■ References 1. Kyle RA. Sequence of testing for monoclonal gammopafhies. Arch Pathol Lab Med 1999;123: 114-118. 2. Kyle RA, Garton JP. The spectrum of IgM monoclonal gammopafhy in 430 cases. Mayo Clin Proc 1987;62:719-731. 3. Ansell SM, Kyle RA, Reeder CB, et al. Diagnosis and management of Waldenstrom macro-globulinemia: Mayo stratification of macroglobulinemia and risk-adapted therapy (mSMART) guidelines. Mayo Clin Proc 2010;85:824-833. 4. Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia 2009;23:3-9. 5. Treon SP, Xu L, Yang G, et al. MYD88 L265P somatic mutation in Waldenstrom's macroglobulinemia. N Engl J Med 2012;367:826-833. 6. Roccaro AM, Sacco A, Jimenez C, et al. C1013G/CXCR4 acts as a driver mutation of tumor progression and modulator of drug resistance in lymphoplasmacytic lymphoma. Blood 2014; 123:4120-4131. 7. Gilad S, Meiri E, Yogev Y, et al. Serum microRNAs are promising novel biomarkers. PLoS One 2008;3:e3148. 8. Kubiczkova L, Kryukov F, Slaby O, et al. Circulating serum microRNAs as novel diagnostic and prognostic biomarkers for multiple myeloma and monoclonal gammopafhy of undetermined significance. Haematologica 2014;99:511-518. 2 American Journal of Hematology, Vol. 00, No. 00, Date 2014 doi:10.1002/ajh.23910 MikroRNA u mnohočetného myelomu Kubiczková L, Ševčíková S, Hájek R. In: Slabý et al MikroRNA v onkológii. Praha (ČR). Galén, 2012. s. 271-280. 271 18. MikroRNA u mnohočetného myelomu Lenka Kubiczková, Sabina Ševčíková, Roman Hájek Mnohočetný myelom (MM) je maligní lymfoproliferativní onemocnění charakterizované infiltrací kostní dřeně patologickými plazmocyty, osteolytickými lézemi skeletu a přítomností monoklonálního imunoglobulinu v séru a/nebo moči. Incidence MM je 4/100 000 obyvatel v ČR. Incidence vzrůstá s věkem, s mediánem 65 let při diagnóze [ 1 ]. MM je považován za obtížně léčitelné, nicméně již vyléčitelné onemocnění s pětiletým mediánem přežití nižším než 40% [2]. Jedna skupina MM pacientů (10-15%) je považována za vysoce rizikovou (high-risk), jelikož u nich dochází k rapidní progresi onemocnění, pacienti dosahují kratší remise s následným vývojem refraktorní nemoci [3,4]. Pro MM je typická genomická nestabilita. Cytogenetická analýza MM buněk ukazuje na časté mutace a chromozomální aberace. Aneuplodie je velice častá, mezi nejčastější změny patří reciproké chromozomální translokace IgH lokusu, monosomie chromozomu 13, ztráta krátkého raménka chromozomu 17 a zisk dlouhého raménka chromozomu 1. Ve srovnání s ostatními hematologickými malignitami, které jsou charakterizovány malým počtem genetických aberací, jsou tyto změny u MM časté, některé jsou navíc používány jako prognostické markery [5,6]. Právě heterogenita tohoto onemocnění je pravděpodobně spojena s molekulární charakteristikou maligního klonu [3]. V poslední době přichází do popředí zájmu také problematika miRNA v patoge-nezi a progresi MM. Z metodického hlediska jsou dva základní přístupy ke studiu miRNA u MM, a to screeningový (spíše translační) a funkční (základní výzkum). V této kapitole se nejdříve budeme věnovat výsledkům globálního profilování exprese miRNA a dále konkrétním miRNA a jejich souvislosti s patogenezí MM. 18.1. MikroRNA v patogenezí mnohočetného myelomu První abstrakta zabývající se úlohou miRNA v patogenezí MM byla prezentována v roce 2005 na setkání Americké hematologické společnosti. Jako první byly popsány expresní profily miRNA u myelomových linií a vzorků pacientů a bylo zjištěno, že jak buněčné linie, tak maligní, CD138+ plazmatické buňky (PC - plasma cells) pacientů mají odlišnou expresi některých miRNA (miR--125b, miR-133a, miR-1 nebo miR-124a) ve srovnání s PC zdravých dárců [7]. 02Speciélní-komplet-1.indd 271 31.5.2012 10:44:41 272 Speciální část / MikroRNA v hematoonkologii Další práce, ve které byla použita kvantitativní PCR (qRT-PCR), popisuje zvýšenou expresi let-7a, miR-16, miR-17-5p a miR-19b, a naopak sníženou expresi miR-372, miR-143 a miR-155 u MM pacientů a buněčných linií ve srovnání se zdravými kontrolami [8]. Exprese miR-15 amiR-21 sevtéto studii významně nelišila mezi zdravými dárci a nemocnými, což je v rozporu s pozdější studií, která identifikovala miR-21 jako onkogen s antiapoptotickou funkcí [9]. Pomocí chromatinové imunoprecipitace bylo zjištěno, že se STAT3 podílí na regulaci exprese miR-21 v IL-6 závislých PC po přídavku IL-6. Zdá se, že u těchto buněk je transkripce miR-21 kontrolována pomocí IL-6 a zprostředkovaná aktivací STAT3, což napomáhá přežívání maligních buněk. Navíc ektopická exprese miR-21 za nepřítomnosti IL-6 vedla ke snížení apoptózy buněk, což potvrzuje účast miR-21 v procesu apoptózy, která je zprostředkovaná pomocí STAT3 [9]. V pilotní studii zabývající se úlohou miRNA v maligní transformaci PC byla pomocí miRNA mikročipů a následné qRT-PCR srovnávána exprese miRNA jak u zdravých dárců, tak u osob s monoklonální gamapatií nejasného významu (MGUS - monoclonal gammopathy of undetermined significance), pacientů s MM a u buněčných linií. Byly identifikovány specifické profily miRNA popisující jak PC v MM, MGUS a MM liniích, tak transformaci z MGUS do MM. U MGUS bylo nalezeno 48 miRNA, u MM pacientů již 96 odlišně exprimovaných miRNA ve srovnání se zdravými dárci. U obou skupin, MM i MGUS, byla pozorována zvýšená exprese miR-21, klastru miR-106-25 a miR-181a/b, nicméně pouze u MM byla stanovena zvýšená exprese miR-32 a klastru miR-17-92. Zdá se tedy, že se tyto miRNA podílejí na progresi onemocnění a napomáhají transformaci z MGUS do MM (obr. 18.1.) [10]. V návaznosti na získané poznatky byla provedena (u PC zdravých dárců a MM pacientů) srovnávací analýza expresního profilu miRNA a expresního profilu kó- B-lymfocyt germinálního centra tmiR-21 tmiR-106-25 tmiR-181a/b tmiR-1 tmiR-133a Myc t MDM2I Dicer1 tmiR-181a/b tmiR-17-92 tmiR-32 tmiR-193b-365 lmiR-192-194-215 !miR-15a/16 Obr. 18.1. Schematické znázornění transformace plazmatické buňky. Reprezentativní mikroRNAa geny významně deregulované u jedinců s MGUS a MM ve srovnání se zdravými jedinci. 02Speciélní-komplet-1.indd 272 31.5.2012 10:44:41 MikroRNA u mnohočetného myelomu 273 Tab. 18.1. Deregulované mikroRNA v plazmatických buňkách pacientů s mnohočet-ným myelomem (MM). Převzato z [38]. MikroRNA Lokus Exprese u MM PCs Cílové geny Literatura miR-181a/b 1q32.1 zvýšená PCAF.HOXAII, TCL [10,11,17] miR-1 20q13.33 zvýšená zvýšená u MM pacientů st(14;16)/t(14;20) zvýšená u MM pacientů s t(14;16) [10,18,27] miR-15b 3q25.33 zvýšená snížená u MM relapsů/ refraktorních MM [10,11,16,17] miR-221 Xp11.3 zvýšená zvýšená u pacientů s t(4;14) snížená u pacientů s delecí RB [10,11,17,18,27] miR-222 Xp11.3 zvýšená u MGUS zvýšená zvýšená u pacientů s (4;14) P27, PTEN [10,11,17,27] miR-106b-25 7q22.1 zvýšená P21.BIM, E2F1, PCAF [10,11,16] miR-17-92 13q31.3 zvýšená u miR-17, miR-19a, miR-19b, miR-20a, miR-92a zvýšená u miR-17, miR-18a, miR-19a, miR-20a, miR-92a zvýšená u miR-92a snížená u miR-19a, miR-19b, miR-20a u pacientů s delecí RB P21.BIM, E2F1, PTEN [10,11,16,18] dujících genů (GEP - gene expression profiling), která prokázala souvislost mezi globální zvýšenou expresí miRNA a špatnou prognózou high-risk MM pacientů [11]. Další studie by mohly podpořit tuto souvislost, jelikož bylo pozorováno, že vyšší viabilita MM buněk souvisí s vyřazením z funkce Argonaut (EIF2C2/AG02) komplexu, který je hlavním regulátorem maturace a funkce miRNA a jehož exprese je zvýšená u high-risk MM [12,13]. EIF2C2/AG02 se navíc podílí na diferenciaci B-lymfocytů [14] a je znám jako marker nádorové progrese u MM [15]. Vtéto studii byla také navržena hypotéza, že miRNA mohou působit synergisticky, a tím významně přispívat k progresi MM. Jiná miRNA mikročipová srovnávací studie odhalila zvýšenou expresi klastru miR-193b-365 u PC MM pacientů [16]. Dále byly porovnány expresní miRNA profily PC MM pacientů s profily normálních PC a byla zjištěna významně zvýšená exprese miR-222, miR-221, miR-382, miR-181a a miR-181b a snížená exprese miR-15a a miR-16 [17]. Gutierrez et al. ve své práci porovnali miRNA expresní profil PC 60 MM pacientů s PC zdravých dárců a pozorovali sníženou expresi 11 miRNA (miR-375, miR-650, miR-214, miR-135b, miR-196a, miR-155, miR-203, 02Speciélní-komplet-1.indd 273 31.5.2012 10:44:41 274 Speciální část / MikroRNA v hematoonkologii miR-95, miR-486, miR-10 a miR-196b), z nichž pouze miR-155 byla již dříve popsána v souvislosi s lymfoidními buňkami [18]. Nedávno publikovaná práce popisuje 40 miRNA se sníženou expresí v PC MM pacientů ve srovnání se zdravými dárci, z nichž 6 miRNA (miR-214, miR-135b, miR-196a, miR-155, miR-203 a miR-486) se shoduje s miRNA publikovanými skupinou Gutierreze et al. Navíc výsledky klastrovací analýzy 54 MM pacientů poukázaly na tři miRNA, a to miR-296, miR-194 a let-7f, jejichž zvýšená exprese souvisí s lepším přežíváním pacientů [19]. Stanovené expresní profily PC MM pacientů nejsou jednotné, nicméně některé miRNA byly potvrzeny ve více studiích, jak je znázorněno v tab. 18.1. 18.2. Rezistence na léčbu a mikroRNA v mnohočetném myelomu Přítomnost miRNA je také spojována s rezistencí vůči některým lékům. Bortezo-mib (Velcade, dříve PS-341, Millennium Pharmaceuticals, Inc.) patří do skupiny inhibitorů proteazomu. Jedná se o dipeptid kyseliny borité, vykazující protiná-dorové účinky [20]. Bortezomib byl schválen k léčbě MM v relapsu i pro léčbu nově diagnostikovaných pacientů [21]. V roce 2009 byly popsány expresní dráhy miRNA, které souvisejí s léčebnou odpovědí k bortezomibu. Srovnání expresních profilů linií rezistentních a citlivých k bortezomibu odhalilo 22 deregulovaných miRNA, z toho zvýšenou expresi měly miR-155, miR-342-3p, miR-181a, miR-181b, miR-128 a miR-20b, naopak snížená exprese byla pozorována u let-7b, let-7i, let-7d, let-7c, miR-222, miR-221, miR-23a, miR-27a a miR-29a. Mezi predikované cíle těchto miRNA patří geny zapojené do buněčného cyklu, buněčného růstu, apoptózy a ubikvitinace. Následně, pro stanovení klinického významu uvedených miRNA, byly korelovány expresní profily miRNA PC pacientů rezistentních a citlivých k bortezomibu s jejich odpovědí na léčbu. Bylo zjištěno, že pacienti citliví k terapii bortezomibem měli stejný profil deregulovaných miRNA jako linie citlivé k bortezomibu a stejně tak profil pacientů rezistentních k bortezomibu inklinoval k profilu stanovenému na liniích [22]. V další studii, zabývající se změnou expresních profilů miRNA během získané lékové rezistence, byly srovnány modelové expresní profily miRNA mezi MM buněčnými liniemi (RPMI-8226 a U266) se získanou rezistencí k doxorubicinu a melfalanu a jejich parentálními liniemi. Výsledky expresní analýzy byly valido-vány pomocí qRT-PCR a významné změny byly pozorovány u miR-21 a miR- 181a a miR-181b. Exprese miR-21 byla zvýšená u obou klonů linií rezistentních k melfalanu. Překvapivě bylo zjištěno, že exprese miR-18la a miR-181b byla snížená u U266 doxorubicin rezistentní linie, ale zvýšená u RPMI-8226 doxorubicin rezistentní linie. Zdá se, že změny vedoucí k lékové rezistenci jsou náhodné a efekt miRNA je závislý na kontextu [23]. 02Speciélní-komplet-1.indd 274 31.5.2012 10:44:41 MikroRNA u mnohočetného myelomu 275 18.3. Mechanizmus deregulace mikroRNA v mnohočetném myelomu Nové studie, navazující na předchozí objevy, částečně vysvětlují mechanizmus deregulace miRNA u MM. Srovnávací mikročipová analýza miRNA a analýza počtu kopií (CNV - copy number variations) DNA nebo GEP MM linií objasnily deregulaci 16 miRNA, jejichž geny leží v oblastech chromozomů, které jsou často předmětem různých alelových změn u MM. Mezi nejčastější změny patřily zisky chromozomů. Bylo zjištěno, že miR-548-1 se vyskytovala s nejvyšší četností (94 %) v oblastech zisku chromozomu, zatímco miR-130b, miR-185, miR-648 a miR-649 (všechny leží v oblasti 22qll.21) jsou zastoupeny v oblastech ztráty chromozomu. Mezi další často deregulované miRNA patřily miR-22 ležící v oblasti 17pl3.3, miR-106b a miR-25 v oblasti 7q22.1, miR-15a v oblasti 13ql4.3, miR-21 v oblasti 17q23.1 a miR-92b, která se nachází v oblasti lq22 [24]. Klastr miR-15a/16-1 byl dále podrobněji studován a bylo zjištěno, že u pacientů s delecí chromozomu 13 zcela chybí miR-15a a miR-16, nicméně u pacientů bez delece chromozomu 13 byla exprese miR-15a a miR-16 také významně snížená [17]. Další studie, srovnávající CNV s čipy mapujícími jednonukleotidové polymor-fizmy (SNP - single nucleotide polymorphism), ukázala, že exprese miR-15a a miR-16 není závislá na statutu chromozomu 13, ale obecně je u MM pacientů exprese zmíněných miRNA oproti normálním PC zvýšená [25]. Byla také nalezena korelace mezi šesti intragenovými miRNA a geny, uvnitř kterých se miRNA nacházejí. Tyto geny jsou deregulovány u MM linií a pacientů a některé jsou důležité v patogenezi MM, jako MEST a miR-335 nebo EVL a miR--342-3p [26]. V jiné práci byla nalezena souvislost mezi 32 intragenovými miRNA a geny, uvnitř kterých leží; některé z těchto genů jsou opět významně deregulovány u MM pacientů. Studie potvrdila již výše zmíněné korelace, navíc byla zjištěna souvislost mezi genem COPZ2 a miR-152 [24]. Získané výsledky naznačují, že změna počtu kopií genu souvisí se zvýšenou expresí jeho intragenových miRNA, což by částečně vysvětlovalo mechanizmus změněné exprese miRNA u MM. Jelikož je myelom velmi heterogenní onemocnění, pro které jsou charakteristické komplexní cytogenetické aberace, je velmi pravděpodobné, že tyto aberace ovlivňují také expresi miRNA. V nedávné studii bylo 60 MM pacientů rozděleno na základě translokačních partnerů IgH genu a statutu RB genu do různých cyto-genetických podskupin a tyto podskupiny pacientů byly srovnány s jejich expresí 365 miRNA. Výsledky klastrovací analýzy poukázaly na zvýšenou expresi miR-1 a miR-133a, které souvisejí s translokací t(14;16) [18]. Změněná exprese jiných miRNA byla dále popsána v souvislosti s translokacemi t(4;14), t(ll;14) nebo t(14;16) [18,27]. Nově bylo popsáno pět miRNA, které byly zvýšeny u pacientů s t(ll;14), a to miR-122a, miR-33, miR-489, miR-519 a miR-555 [19]. Další možností deregulace miRNA je změna v jejich zpracování nebo maturaci. Již dříve zmíněná studie EIF2C2/AG02 komplexu uvádí, že úbytek AG02 souvisí 02Speciélní-komplet-1.indd 275 31.5.2012 10:44:41 276 Speciální část / MikroRNA v hematoonkologii se zástavou růstu a apoptózou u MM buněk [11]. V souladu s tím bylo prokázáno, že změněná hladina enzymu Dicer, ale ne enzymu Drosha, může souviset s progresí MM. Autoři pozorovali podobnou hladinu enzymu Dicer u PC zdravých dárců a pacientů s MGUS, která je však oproti doutnajícímu myelomu a MM pacientům významně zvýšená. Navíc bylo pozorováno, že skupina pacientů s vyšší hladinou enzymu Dicer měla delší dobu do progrese [28]. Zmíněné výsledky jsou však v rozporu s nedávno provedenou studií, ve které nižší exprese genu DICER1 u skupiny MM pacientů souvisí s delší dobou do progrese nemoci [19]. Zdá se tedy, že regulační mechanizmy ovlivňující jak miRNA maturaci, tak jejich funkci se mohou podílet na změněné expresi miRNA, další studie určitě pomohou objasnit zmíněné nesrovnalosti. 18.4. MikroRNA ovlivňující kritické geny u mnohodetného myelomu Mnoho vědeckých skupin se zabývalo otázkami, jak důležité jsou z funkčního hlediska změny v expresi miRNA a jak tyto změny souvisí s patogenezí MM. Pro zodpovězení těchto otázek jsou využívány různé přístupy od predikce cílových genů pomocí in silico modelů až po pokusy s transgenními zvířaty. Je známo, že kódující geny, které se podílejí na procesu kancerogeneze u MM, jsou cílem pro deregulované miRNA. Bylo prokázáno, že klastr miR-17-92, nacházející se v oblasti 13q31-32, ovlivňuje expresi genu PTEN, genu pro transkripční faktor E2F1 a B1M [29,30]. U transgenních myší se zvýšenou expresí tohoto klas-tru v lymfocytech byly pozorovány lymfoproliferativní a autoimunitní onemocnění a časná úmrtí. Dále bylo zjištěno, že purifikované myší CD4+ lymfocyty se zvýšenou expresí miR- Í7-92 obsahovaly snížené množství proteinů PTEN a B1M, což naznačuje, že miR-i7-92 klastr ovlivňuje tyto nádorové supresory [29]. Brzy nato byla publikována další studie, ve které bylo prokázáno, že zmíněný klastr je nezbytný pro vývoj B-lymfocytů. Nepřítomnost miR- Í7-92 vedla ke zvýšené hladině pro-apoptotického proteinu BIM, a tím k zástavě vývoje z pro-B do pre-B stadia [30]. Zdá se tedy, že zvýšená exprese miR- Í7-92 negativně reguluje zmíněné nádorové supresory a přispívá k transformaci PC a progresi MM. Predikce in silico také ukázala, že cílem miR-2i a klastru miR- Í06-25 jsou mezi jinými nádorové supresory PTEN, BIM a p21, a proto je pravděpodobné, že se tyto miRNA mohou podílet na vývoji plně rozvinutého myelomu [10]. Jiná miRNA, miR-19a/b, ovlivňuje dráhu STAT-3/IL-6, která je důležitá v patogenezí MM. Bylo prokázáno, že miR-19a/b přímo ovlivňuje SOCS1 (negativní regulátor IL-6), a tím přispívá k jeho časté deregulaci u MM buněk [10]. Také miR-21, zmíněná výše, působí jako onkogen a podílí se na regulaci této dráhy [9]. Jak již bylo zmíněno dříve, miR-15a a miR-16-l leží v oblasti chromozomu 13ql4.3, která je deletována u více než 50 % pacientů s MM. Tato delece je pova- 02Speciélni-komplet-1.indd 276 31.5.2012 10:44:41 MikroRNA u mnohočetného myelomu 277 žována za primární mutaci, která se podílí na patogenezi MM [31]. MiR-15a/16 jsou považovány za nádorové supresory podílející se na proliferaci MM buněk in vitro i in vivo tím, že inhibují AKT serin/treonin proteinovou kinázu (AKT3), ribozomální protein S6, MAP kinázy a NFkB aktivátor MAP3KIP3 [17]. Dále bylo prokázáno, že miR-15a/16 nejen regulují expresi genů buněčného cyklu, jako jsou cykliny Dl a D2, dále CDC25A, ale rovněž ovlivňují expresi genů spojených s apoptózou: BCL2 nebo MCL1 [32]. Navíc ektopická exprese miR- 15a/16 negativně reguluje angiogenezi pomocí VEGF [17]. Nedávno byla popsána úloha miR-15a/16 v mikroprostředí kostní dřeně. Bylo zjištěno, že exprese miR-15a/16 je v MM buňkách po ovlivnění cytotoxickými látkami vyšší. Nicméně po interakci těchto buněk se stromálními buňkami kostní dřeně odvozenými od MM (MM-BMSC) pacienta, byla pozorována snížená exprese miR-15a/16 u myelomových buněk. Důvodem byla zvýšená produkce IL-6 stromálními buňkami, který inhiboval expresi zmíněných miRNA Zdá se tedy, že mikroprostředí je důležité pro přežití MM buněk a chrání je před působením léků pomocí sekrece IL-6, kterýinhibuje expresi miR-15a/16 [33]. Nově publikované práce se dále zaměřují na vztah miRNA k nádorovému su-presoru p53. Výsledky screeningové metody umožňující identifikovat miRNA, které negativně regulují signalizaci p53 pomocí přímé interakce s genem TP53, naznačily, že miR-25 a miR-30d mohou ovlivňovat p53. Navíc byla exprese miR-25 a miR-30d zvýšená v PC MM pacientů a u miR-25 zvýšená exprese korelovala se sníženou expresí mRNA TP53 [34]. Také miR-181a byla popsána jako negativní regulátor exprese genu TP53, což potvrzuje spojitost mezi p53 a aberantní miRNA expresí [35]. Je známo, že miR-34a je transkripčním cílem p53 zprostředkovávajícím apoptózu [36]. U MM pacientů byla pozorována hypermetylovaná miR-34a v oblasti lp36. Jelikož se u krevních nádorových onemocnění nevyskytuje mutace TP53 tak často jako u solidních nádorů, mohla by hypermetylace miRNA částečně vysvětlit dysregulaci p53 signalizace [37]. V další studii byla nalezena snížená exprese miR-192, miR-194 a miR-215 u části nových diagnóz MM pacientů. Další pokusy in vitro prokázaly, že při použití molekulárních inhibitorů MDM2 mohou být tyto miRNA transkripčně aktivovány pomocí p53 a posléze mohou modulovat expresi MDM2. Je tedy patrné, že miR-192, miR-194 a miR-215 ovlivňují MDM2/ TP53 regulační osu a kontrolují rovnováhu mezi MDM2 a p53. Navíc miR-215 a miR-192 ovlivňují signální dráhu IGF, a tím zabraňují zvýšené migraci PC do kostní dřeně [35]. ■ Závěr Během posledních let bylo provedeno mnoho studií srovnávajících globální profil CD138+PC MM pacientů a zdravých dárců pomocí různých high-throughput screeningových metod, od oligonukleotidových čipů až po qRT-PCR profilování. Každá z metod má své silné a slabé stránky poskytující rozdílné výsledky, ke kterým navíc přispívá velká heterogenita onemocnění. Obecně bylo doposud ve většině prací u myelomu identifikováno více miRNA se zvýšenou expresí u PC než se 02Speciélní-komplet-1.indd 277 31.5.2012 10:44:42 278 Speciální část / MikroRNA v hematoonkologii sníženou expresí. Výjimkou je práce Guttiereze et al, která popisuje více miRNA se sníženou expresí (tab. 18.1.). Dále můžeme říci, že ani identifikace jednotlivých miRNA není jednotná, což může být způsobeno několika faktory. Za prvé je v každé studii rozdílný soubor pacientů a kontrol a rozdílná velikost souboru. Jak již bylo zmíněno, je MM velmi heterogenní onemocnění a každý pacient má jinou kombinaci genetických mutací a cytogenetických aberací, což se může projevit na rozdílné subklasifikaci do skupin ve srovnání se zdravými dárci. Za druhé, pacienti mohou mít v různých stadiích onemocnění odlišné profily miRNA. Například miR-15 byla popsána jako zvýšená u nových diagnóz, ale snížená u relapsů [10,11]. V neposlední řadě se na odlišnostech podílejí rozdíly ve zpracování vzorku, purifikaci PC, extrakci miRNA a dále rozdílné mikročipové platformy a různé verze čipů. Dnes již víme, že změněná exprese miRNA u MM může mít příčiny genetické, cytogenetické nebo epigenetické. Byly také popsány specifické miRNA charakterizující progresi MM, lepší prognózu nebo rezistenci vůči lékům. Mechanizmus deregulace není zatím přesně znám, víme již, že v pozadí stojí jak změna v cílovém genu pro miRNA, tak změny v počtu kopií lokusů, ve kterých se nachází miRNA, defekty v biogenezi miRNA a epigenetické změny. Snahou dalších studií by mělo být objasnění komplexity regulace miRNA a identifikace terapeutických cílů. ■ Poděkování Tato práce byla podpořena výzkumnými projekty MŠMT ČR MSM0021622434, GA ČR GAP304/10/1395 a IGA MZ ČR NT11154 a NT12130. ■ Literatura 1. Hajek R, Krejčí M, Pour L et al. Multiple myeloma. 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