| Abstract | Akutna mijeloična leukemija (AML) jest heterogena skupina malignih klonskih bolesti
krvotvornog sustava koje nastaju malignom transformacijom mijeloidnih progenitorskih stanica
zbog oštećenja kromosoma ili mutacije genâ. Specifične citogenetičke promjene i nalaz
specifičnih mutacija danas predstavljaju glavne prognostičke čimbenike i temelj suvremene
klasifikacije AML. Međutim, budući da se u značajnog broja bolesnika s AML navedene
promjene ne nalaze, to znanstvenicima pruža prostora za istraživanje drugih još uvijek nepoznatih
gena i biološki relevantnih obilježja leukemijskih stanica u cilju što bolje terapijske i
prognostičke stratifikacije bolesnika. Sukladno tome, u ovom su radu ispitani izražaj i aktivnost
medijatora kemorezistenicje P-glikoproteina (P-gp) te razina fosforilacije unutarcitoplazmatskih
signalnih molekula Akt, ERK1/2 i p38 u blastima bolesnika s novootkrivenom AML metodom
protočne citometrije. Specifični ciljevi rada bili su analizirati navedena obilježja leukemijskih
stanica u odnosu na poznate prognostičke kliničko-laboratorijske parametre bolesnika s AML,
njihovu međusobnu povezanost kao i njihov potencijalni prognostički utjecaj na preživljavanje
bolesnika s AML. Istraživanjem je pokazan izražaj P-gp na blastima 34%, a aktivnost u blastima
25% bolesnika s AML. Potvrđeno je da izražaj i aktivnost P-gp koreliraju sa starijom dobi, većim
brojem leukocita pri dijagnozi, izražajem nezrelih biljega (CD34 i TdT) i citomofološki
nezrelijim oblicima AML te da predstavljaju prognostičke čimbenike za kraće ukupno
preživljavanje. Konstitutivna fosforilacija Akt signalnog puta PI3K/Akt nađena je u blastima
48% bolesnika, a signalnog puta MAPK u 39% (ERK1/2), odnosno 44% (p38) bolesnika, pri
čemu nalaz fosforilacije Akt korelira s nalazom fosforilacije molekule p38, ali ne i molekule
ERK1/2. Konstitutivna fosforilacija Akt, ERK1/2 i p38 u blastima korelira s pokazateljem
tumorske mase – serumskim LDH pri dijagnozi, fosforilacija Akt s ukupnim preživljavanjem i
preživljavanjem bez znakova bolesti, fosforilacija ERK1/2 s mlađom dobi bolesnika, a
fosforilacija p38 s brojem eritrocita pri dijagnozi. Štoviše, nađena je i negativna korelaciju
između razine fosforilacije p38 i aktivnosti P-gp u blastima AML što bi indirektno moglo
govoriti u prilog potencijalne uloge p38 u regulaciji P-gp. U multivarijatnoj analizi s pomoću
Coxovog regresijskog modela pokazano je da je aktivnost P-gp nezavisni negativni prediktor za
ukupno preživljavanje bolesnika, fosforilacija ERK1/2 nezavisni pozitivni prediktor za ukupno
preživljavanje i preživljavanje bez pojave bolesti, a fosforilacija Akt nezavisni negativni
prediktor za ukupno preživljavanje bolesnika. To govori u prilog da aktivnost P-gp i razina
konstitutivne fosforilacije signalnih putova Akt i ERK1/2 u kombinaciji s poznatim kliničkolaboratorijskim
čimbenicima bi mogli predstavljati dodatne parametre za prognostičku
stratifikaciju bolesnika s AML. |
| Abstract (english) | Acute myeloid leukemia (AML) is a heterogeneous group of malignant clonal disorders of the
hematopoietic system originating from malignant transformation of the myeloid precursor cell
due to chromosome alterations or gene mutations. Cytogenetic rearrangements and specific
mutations are the most important prognostic factors and form the basis for latest AML
classification system. Nevertheless, since genetic alterations are not present in a significant
number of AML patients, it provides space for scientists to investigate other biological
characteristics of leukemic cells with the ultimate goal of improvement of patients' risk
stratification and treatment regimens. Accordingly, this study investigated the expression and
activity level of chemotherapy resistance mediator P-glycoprotein (P-gp), as well as the
phosphorylation level of intracytoplasmic signalling molecules Akt, ERK1/2 and p38 in leukemic
blasts of patients with de novo AML by flow cytometry. Specific objectives of the study were to
analyze those leukemic cells’ features in relation to the known prognostic clinical and laboratory
parameters of patients with AML, their interconnection as well as their potential prognostic
impact on the survival of patients with AML. This research showed P-gp expression in 34% and
its activity in 25% of AML patients. It was confirmed that the expression and activity of P-gp
correlated with older age, higher number of leukocytes, immature cell markers expression (CD34
and TdT) and cytomorphologically immature AMLs, and that they represent unfavourable
prognostic factors for overall survival. Constitutive phosphorylation of Akt molecule of the PI3K
signalling pathway was found in 48%, while MAPK signalling pathway was activated in 39%
(ERK1/2) and 44% (p38) of all tested AML samples, respectively. Significant correlation was
found between Akt and p38 phosphorylation levels, but not with the ERK1/2 molecule. Serum
lactat-dehydrogenase (LDH) level, an indicator of tumor burden, correlated with the
phosphorylation levels of all three signalling molecules. In addition, Akt phosphorylation
correlated with overall survival and relapse-free survival, ERK1/2 with younger patient age,
while p38 correlated with the number of red blood cells at diagnosis of AML. Moreover, negative
correlation was found between levels of p38 phosphorylation and P-gp activity in AML,
indicating a possible role of p38 in P-gp regulation. Multivariate Cox regression analysis showed
that P-gp activity and Akt phosphorylation are independent negative predictors of overall
survival, while phosphorylation of ERK1/2 is an independent positive predictor of overall
survival and disease free survival of AML patients. This P-gp activity and level of constitutive
phosphorylation of the signalling pathways molecules in combination with other previously
established clinical and laboratory features could represent additional parameters for the
prognostic stratification of patients with AML. |
