Monitoring of thiopurine S-methyltransferase activity and dosing of 6-mercaptopurine in patients with acute lymphoblastic leukemia

Tiopurini so pomembna skupina zdravilnih učinkovin, ki jih med drugim uporabljamo za zdravljenje akutne limfoblastne levkemije (ALL). V njihovem metabolizmu sodeluje veliko encimov, ki vodijo do nastanka aktivnih metabolitov, 6-tiogvanozinov. Deaktivacijska pot, pri kateri je ključen encim tiopurin S-metiltransferaza (TPMT), vodi do nastanka metiliranih produktov. Pri okoli 11 % posameznikov je aktivnost tega encima oslabljena, pri 0,3 % pa celo nezaznavna. Znižana aktivnost TPMT je posledica genetskih polimorfizmov posameznih nukleotidov, med katerimi so najpogostejši polimorfizmi alelov TPMT*3. Poznavanje aktivnosti TPMT je pri bolnikih, ki prejemajo zdravljenje s tiopurini, pomembno za določitev pravega odmerka, ki bo dosegel želen terapevtski izid, hkrati pa ne bo povzročil hudih neželenih učinkov. V sklopu magistrske naloge smo ovrednotili metodo za merjenje aktivnosti TPMT, pri čemer smo kot substrat uporabili 6-tiogvanin (6-TG). Zanimalo nas je, ali obstaja korelacija med rezultati nove metode (kjer nastaja 6-metiltiogvanin (6-MTG)) in že vzpostavljene metode, kjer kot substrat uporabljamo 6-merkaptopurin (6-MP). Preverili smo, kako se aktivnost TPMT razlikuje med skupinami posameznikov z različnimi genotipi TPMT. Pri bolnikih z ALL, ki so prejemali zdravljenje s tiopurini, smo ugotavljali, ali obstaja povezava med aktivnostjo TPMT in kumulativnimi odmerki tiopurinov, odmerki 6 MP, odmerki metotreksata (MTX), koncentracijo levkocitov in aktivnostmi jetrnih transaminaz. Z uporabo tekočinske kromatografije visoke ločljivosti z reverzno fazo smo izmerili aktivnost TPMT v 109 hemolizatih zdravih prostovoljcev iz Estonske genomske banke z uporabo 6-TG kot substrata. Encimsko aktivnost smo preko nastanka 6-MTG izmerili še v hemolizatih bolnikov z ALL. Aktivnost TPMT, izmerjena preko nastanka 6 MTG, korelira z aktivnostjo TPMT, izmerjeno preko nastanka 6 metilmerkaptopurina (6-MMP). Po razvrstitvi zdravih prostovoljcev iz Estonske genomske banke med heterozigote TPMT*3C oz. TPMT*3A in nevariantne homozigote smo ugotovili, da se aktivnost TPMT razlikuje med osebami z različnimi genotipi TPMT in je v skupini z variantnim alelom značilno nižja kot v skupini brez variantnega alela. Pri bolnikih z ALL nismo pokazali statistično značilne povezave med aktivnostjo TPMT ter kumulativnimi odmerki tiopurinov, odmerki 6-MP, odmerki MTX ali aktivnostjo jetrnih transaminaz. Med aktivnostjo TPMT in koncentracijo levkocitov je šibka, a statistično značilna povezava, za katero smatramo, da ni klinično pomembna. Naši rezultati potrjujejo, da je metoda za merjenje aktivnosti TPMT, kjer kot substrat uporabljamo 6-TG, hitra, občutljiva in primerna za rutinsko spremljanje aktivnosti TPMT. Pred uvedbo v rutinsko klinično prakso bo potrebno metodo še nekoliko optimizirati s ciljem izboljšati celokupno ponovljivost. Potrebna je še dodatna validacija metode.Thiopurines are an important group of active substances used, among others, in the treatment of acute lymphoblastic leukemia (ALL). Many enzymes are involved in their metabolism, leading to the formation of active metabolites, 6-thioguanosins. The deactivation pathway, in which the enzyme thiopurine S-methyltransferase (TPMT) is a key player, leads to the formation of methylated products. The activity of this enzyme is impaired in about 11% of individuals and even undetectable in 0,3%. Reduced TPMT activity is due to genetic single nucleotide polymorphisms, the most common of which are polymorphisms in TPMT*3 alleles. Knowledge of TPMT activity in patients on thiopurine therapy is important to determine the right dose, which will achieve the desired therapeutic outcome while avoiding serious adverse effects. As part of our Master\u27s thesis, we evaluated a method for measuring TPMT activity using 6-thioguanine (6-TG) as a substrate. We were interested in whether there is a correlation between the results of the new method (based on the formation of 6 methylthioguanine (6-MTG)) and the already established method using 6 mercaptopurine (6-MP) as a substrate. We examined how TPMT activity differs between groups of individuals with different TPMT genotypes. In ALL patients on thiopurine therapy, we investigated, whether there is a correlation between TPMT activity and cumulative thiopurine doses, 6-MP doses, methotrexate doses, leukocyte counts and liver transaminase activities. With the usage of reversed-phase high-performance liquid chromatography, we measured TPMT activity in 109 haemolysates from healthy volunteers from the Estonian Genome Bank using 6-TG as a substrate. The enzyme activity was also measured in haemolysates from ALL patients via the formation of 6-MTG. The TPMT activity measured via the formation of 6-MTG correlated with the TPMT activity measured via the formation of 6-methylmercaptopurine (6-MMP). After classifying healthy volunteers from the Estonian Genome Bank into TPMT*3C/TPMT*3A heterozygotes and non-variant homozygotes, we discovered that TPMT activity differs between subjects with different TPMT genotypes and is significantly lower in the group with the variant allele than in the group without the variant allele. In ALL patients, we found no statistically significant association between TPMT activity and cumulative doses of thiopurines, 6-MP, MTX or liver transaminase activity. There is a weak but statistically significant association between TPMT activity and leukocyte counts, which we consider not clinically relevant. Our results confirm that the method for measuring TPMT activity using 6-TG as substrate is rapid, sensitive, and suitable for routine monitoring of TPMT activity. The method will need some further optimization to improve overall reproducibility before it can be introduced into routine clinical practice, and further validation of the method is still needed