Monitoring of asparaginase activity levels in patients with acute lymphoblastic leukemia

Akutna limfoblastna levkemija (ALL) je najpogostejša rakava bolezen pri otrocih, za katero v Sloveniji v povprečju zboli od 10 do 15 otrok na leto. K izboljšanemu preživetju pediatričnih bolnikov z ALL je pripomoglo tudi zdravljenje z asparaginazo, ki katalizira hidrolizo asparagina v asparaginsko kislino in amonijak. Levkemični blasti imajo zaradi zmanjšane aktivnosti asparagin sintetaze zmanjšano sposobnost sinteze asparagina v primerjavi z normalnimi limfoblasti in so močno odvisni od zunanjih virov asparagina. Da se doseže zadostno zmanjšanje asparagina v krvnem obtoku, ki privede do celične smrti levkemičnih blastov, mora biti aktivnost asparaginaze v krvi vsaj 100 U/L. Veliko oviro pri zdravljenju ALL predstavlja razvoj protiteles proti asparaginazi, kar vodi v inaktivacijo encima. Zaplet se klinično izrazi kot preobčutljivostna reakcija, pri tihi inaktivaciji encima pa ni izraženih simptomov. Pomembno je, da se zmanjšana aktivnost asparaginaze ugotovi čimprej in se temu primerno prilagodi zdravljenje z zamenjavo vrste asparaginaze. Namen magistrske naloge je bilo ugotoviti ali je encimska spektrofotometrična metoda primerna za spremljanje encimske aktivnosti asparaginaze pri bolnikih z ALL. Optimizirali smo postopek za določanje asparaginazne aktivnosti v serumu, pogoje shranjevanja substrata (L-aspartat ß-hidroksimat) in pogoje merjenja absorbance indooksina. Ocenili smo natančnost (koeficient variacije med serijami 9,6%znotraj serije 6,0%), točnost (relativna napaka med serijami 8,3%znotraj serije 11,6%) in merilno negotovost (celokupna analitska napaka 24,2%) optimizirane metode. Vrednosti so bile v skladu s smernicami za validacijo bioanalitske metode, ki jih je izdala Evropska agencija za zdravila in medicinske pripomočke. Pri 8 bolnikih, ki so se zdravili s PEG asparaginazo smo v 33 vzorcih določili encimsko aktivnost v serumu. Visoko stopnjo aktivnosti v 14 dneh po aplikaciji zdravila smo izmerili pri 7 bolnikih. Tiho inaktivacijo smo odkrili pri enem izmed bolnikov. Optimizirana metoda je primerna za klinično diagnostiko in omogoča identifikacijo bolnikov, pri katerih je potrebna sprememba vrste asparaginaze zaradi tihe inaktivacije. V prihodnosti, ko bodo določene smernice za spremljanje zdravljenja, pa bo metoda omogočala tudi individualno prilagajanje odmerjanja asparaginaze.Acute lymphoblastic leukemia (ALL) is the most common cancer in children. There are on average 10 to 15 newly diagnosed cases in Slovenia per year. Treatment with asparaginase - an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia, has contributed to the improved survival rates of pediatric patients with ALL. Due to decreased activity of asparagine synthetase, leukemic blasts have a reduced ability to synthesize asparagine compared to normal lymphoblasts and are highly dependent on external sources of asparagine. In order to achieve a sufficient reduction of asparagine in the blood circulation that would lead to leukemic cell death, asparaginase activity levels in the blood should be at least 100 U/L. A major disadvantage in ALL treatment is the occurrence of antiasparaginase antibodies leading to inactivation of the enzyme. This complication is clinically expressed as a hypersensitivity reaction, whereas with the silent inactivation of the enzyme there are no symptoms. It is important to detect the reduced asparaginase activity as soon as possible to adjust treatment appropriately by switching asparaginase preparation. The purpose of the study was to determine whether the enzymatic spectrophotometric method is suitable for monitoring asparaginase activity levels in patients with ALL. We optimized the procedure for determining serum asparaginase activity, the storage conditions of the substrate (L-aspartic acid β-hydroxamate) and the absorbance measurement conditions of indooxine. We assessed precision (coefficient of variation between-run 9,6%within-run 6,0%), accuracy (relative error between-run 8,3%within-run 11,6%) and measurement uncertainty (total error 24,2%) of the optimized method. Verification parameters were in accordance with the guidelines on bioanalytical method validation issued by European Medicines Agency. We measured serum enzyme activity in 33 samples from 8 patients treated with PEG asparaginase. High activity levels 14 days post-administration were determined in 7 patients. We identified one case of silent inactivation. The optimized method is suitable for clinical diagnostics use and enables identification of patients that require switching asparaginase preparation due to silent inactivation. In the future, when guidelines for therapeutic monitoring will be determined, this method will also enable individual adjustments of asparaginase dosing