Mutacijska analiza v genu ABL1

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1 Mutacijska analiza v genu ABL1 Asist. Mag. Tadej Pajič Spec.med.biokem. Specializirani hematološki laboratorij kliničnega oddelka za hematologijo Interna klinika Univerzitetni klinični center Ljubljana

2 BCR-ABL1 t(9;22)(q34;q11): fuzijski gen BCR-ABL1 Kronična mieloična levkemija (KML) > 95 % Akutna limfoblastna levkemija celic B (B-ALL) 3 % (otroci) 25 % (odrasli) Akutna mieloična levkemija (AML) < 1 % Zdravljenje: zdravilo kot so imatinib, nilotinib, dasanitib in druga zdravila ter drugi načini zdravljenja (presaditev krvotvornih matičnih celic) Groffen et al. Cell, 1984; 36(1):93-9; Konopka et al.,cell, 1984; 37(3): ; Shtivelman et al. Nature, 1985; 315(6020):550-4

3 Delovanje imatiniba veže se v vezavno mesto za ATP, prepreči fosforilacijo AK tirozin

4 Mejniki uspešnosti zdravljenja in rezistenca na zdravljenje z imatinibom pri KML Mejnike uspešnosti zdravljenja: National Comprehensive Cancer Network (NCCN) in EuropeanLeukemiaNet. Določen ustrezen, nezadovoljiv odgovor, neuspeh zdravljenja Čas Ustrezen odgovor Po 3 mesecih: popolni hematološki odgovor (krvna slika) Po 6 mesecih: vsaj delni citogenetski odgovor (Ph+ 35 %) Po 12 mesecih: popolni citogenetski odgovor (Ph+ 0 %) Po 18 mesecih: glavni molekularni odgovor (RT-qPCR; BCR-ABL1/ABL1 (referenčni gen) 0,1 % mednarodne skale). Primarna rezistenca: O primarni rezistenci na zdravljenje z imatinibom govorimo,če nikoli ne dosežemo želenih odgovorov. Sekundarna rezistenca: Nekateri bolniki sprva dosežejo ustrezne odgovore na zdravljenje z imatinibom, nato doseženo stanje usahne.

5 Vzroki za pojav odpornosti na zdravljenje z imatinibom Klonska evolucija (dodatne kromosomske spremembe) in pojav točkovnih mutacij v genu BCR-ABL1 Mutacije najpogosteje odkrijemo pri bolnikih v pospešenem poteku bolezni ali blastni krizi. vplivi na farmakokinetiko IM aktivacija alternativnih signalnih poti, ki vodijo do rasti celic neodvisno od BCR-ABL1. Bixby D. and Talpaz M. Mechanisms of resistance to tyrosine kinase inhibitors in chronic myeloid leukemia and recent therapeutic strategies to overcome resistance. ASH Education Book. 2009:

6 Pojav točkovnih mutacij Mutacije se pojavijo v področju tirozin kinazne domene, ki pripada genu ABL1 gena BCR- ABL1. Zmanjšajo afiniteto vezave ali preprečijo vezavo imatiniba in njemu sorodnih zdravil v aktivno mesto fuzijskega proteina. KML Določene v približno 50 % bolnikov, ki se jim je razvila rezistenca pri zdravljenju z imatinibom. Bolj pogosta pri bolnikih z bolj napredovalo obliko bolezni. ALL Ph + Določene v 80 do 90 % ob ponovitvi bolezni bolnikov zdravljenih z imatinibom. Z občutljivimi načini zaznave določili nizek nivo mutacij v levkemičnih celich pred terapijo v 40 %.

7 Pogostnost pojavljanja mutacij v genu ABL1 KML in ALL z Ph + pri zdravljenju z imatinibom KML: 219 bolnikov ALL Ph + : 26 bolnikov Blood, 2006, Vol. 108, No. 1, pp

8 Analiza mutacij Kdaj? KML Priporočilo ekspertov (evropska mreža za levkemije): v primeru neuspeha zdravljenja, neustreznega odgovora ali potrjenem porastu nivoja prepisa BCR-ABL1 (za 5 do 10x), sprememba načina zdravljenja (drugi TKI ali drugi načini). ALL Ph + Porast nivoja prepisa BCR-ABL1, sprememba načina zdravljenja (TKI druge generacije) (ni tako izdelanih priporočil). Izsledki mutacijske analize pomagajo pri odločitvi o nadaljnjem zdravljenju. Pomembno: identifikacija mutacije ne pomeni vedno vzrok za rezistenco. Niso vse mutacije klinično enako pomembne. Izbira drugega TKI lahko temelji: na moči delovanja inhibitorja tirozinske kinaze določene v pogojih in vitro IN na osnovi kliničnih preiskav.

9 Mutacije v genu ABL1 in moč delovanja TKI Haematologica 2008;93:161 IC 50, relativna koncentracija substance, ki inhibira 50 % encimske aktivnosti

10 Mutacije v genu ABL1 in klinične preiskave Predlagana tabela za izbiro TKI druge generacije (nilotinib, dasatinib) glede na izsledke mutacijske analize in osnovi trenutnega razumevanja iz kliničnih raziskav: Mutacijski status Dasatinib Nilotinib Ni mutacije DA DA T315I NE NE F317L/I/V NE DA Y253H, E255K/V, F359 V/C DA NE T315A NE DA V288L NE DA G250E DA DA? Q252H? DA Druge mutacije DA DA Hughes T.P. and Branford S. Monitoring disease response to tyrosine kinase inhibitor therapy in CML, ASH Education Book 2009; p:

11 Analiza mutacij druga generacija TKI Kdaj? Ni izdelanih priporočil kot pri zdravljenju z imatinibom Splošno: porast nivoja prepisa BCR-ABL1

12 Načini ugotavljanja mutacij v genu ABL1 Tehnologija Občutljivost (%) Sekvencioniranje Kloniranje in sekvencioniranje Denaturirajoča visoko ločljiva te kočinska kromatografija (D - HPLC) Pirosekvencioniranje 5 ++ Specifičnost Blood, 2006, Vol. 108, No. 1, pp Dvojno gradientna denaturirajoča elektroforeza 5 ++ Flourescentni PCR Alel no -specifi čni PCR (ASO -PCR) Klinično so verjetno pomembne le mutacije, ki se pojavljajo v % levkemičnih celic.

13 Alelno specifični PCR (ASO-PCR) Za eno mutacijo izvedemo dve PCR: En za divji tip ( se vedno pomnoži) Drugi za mutacijo ( se pomnoži, če je mutacija prisotna) Panel mutacij: M244V, L248V, G250E,Y253F,Y253H, E255V, E255K M343T, Q252Ha, Q252Hb, T315I, F317L, M351T, E355G, F359V, H396R Manjkajo: V299L, F317I/V, T315A, F359C Izvedemo elektroforezo Omejitev metode: zaznamo lahko le prisotnost določenih mutacij Kang, H.J. et al. Haematologica 2006; 91 (5); p:

14 ASO-PCR-rezultati, mutacija T315I S W M W M W M W M MS Negativna kontrola K - C000 C0001 NTC V1 V2 Negativna kontrola Negativen vzorec mutacija ni prisotna Pozitiven vzorec mutacija je prisotna Sliko pripravila dr. Martina Fink, SHL KO za hematologijo

15 Sekvenčna analiza-postopek 1. Del: - 1.PCR: uporabimo cdna bolnika in naredimo 1. PCR, kjer pomnožimo BCR-ABL1 cdna odsek, - V 2.PCR damo del 1.PCR pridelka (vgnezdeni PCR) in pomnožimo regijo gena ABL1, kjer iščemo mutacije. Pridelke PCR očistimo. 2. Del: - naredimo sekvenčno reakcijo in očiščene pridelke te reakcije ločimo z kapilarno elektroforezo, - dobimo elektroferogram z nukleotidnim zaporedjem, ki ga primerjamo z nukleotidnim zaporedjem referenčnega zaporedja v bazi podatkov na svetovnem spletu. - zazanamo spremembe v aminokislinskem zaporedju od mesta 217 do 421 Omejitev metode: za zaznavo mutacij verjetno potrebujemo raven izražanja BCR ABL1 vsaj 5 % na IS; mutacija prisotna v 20 % levkemičnih celic. Soverini, S et. al. Clinical Chemistry 2004; 50; p:

16 Primer elektroferograma Ni mutacije Sliko pripravila dr. Martina Fink, SHL KO za hematologijo

17 Primer : analiza nukleotidnega zaporedja Ni mutacije Sliko pripravila dr. Martina Fink, SHL KO za hematologijo

18 ASO-PCR: pregled dosedanjih rezultatov, obdobje julij 2009 do marec 2010 KML Panel: Neg.: 10 bolnikov Poz.: 1 bolnik M343T 1 bolnik M351T nezanesljiv rezultat ALL Ph + Panel: Neg.: 2 bolnika Poz.: 1 bolnik: F317L, Y253H pozitiven (potrjeno s sekvencioniranjem); T315I nezaneslijiv, Y253H T315I poz. Samo T315I: Neg.: 2 bolnika Poz.: ni Samo T315I: Neg.: ni Poz.: 1 bolnik

19 Zaključek Analiza pridobljenih mutacij v genu ABL1: 1. KDAJ? IMATINIB KML - v primeru neuspeha zdravljenja, neustreznega odgovora ali potrjenem porastu nivoja prepisa BCR-ABL1 (za 5 do 10x), - sprememba načina zdravljenja (drugi TKI ali drugi načini). ALL Ph + - porast nivoja prepisa BCR-ABL1, sprememba načina zdravljenja NILOTINIB, DASATINIB KML in ALL Ph + - porast nivoja prepisa BCR-ABL1 2. Način: -sekvencioniranje, drugi načini, kot npr. ASO-PCR.

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