Vpliv kemoterapije na izražanje celičnih označevalcev na levkemičnih celicah pri otrocih z akutno limfoblastno levkemijo B

Prelog, Tomaž (author), Kloboves Prevodnik, Veronika (mentor), Jesenko, Tanja (comentor)

Namen dela Z raziskavo smo ugotavljali vpliv citostatikov na izražanje celičnih označevalcev na površini malignih celic, izoliranih iz kostnega mozga otrok z akutno limfoblastno levkemijo B (B-ALL). V prvem delu raziskave smo in vitro analizirali spremembe izražanja celičnih označevalcev, ki imajo glede na podatke iz literature prognostični pomen ali pa so terapevtsko pomembni, saj vplivajo na učinkovitost imunoterapije. Poleg tega smo analizirali tudi označevalce, katerih prognostični ali terapevtski pomen pri obravnavi otrok z B-ALL še ni poznan. V drugem delu raziskave smo ugotavljali spremembe v izražanju celičnih označevalcev in vivo med indukcijskim zdravljenjem B-ALL ter te spremembe povezali z odzivom na kemoterapijo. Materiali in metode Iz kostnega mozga 35 otrok z B-ALL smo izolirali maligne celice in jih izpostavili enemu izmed citostatikov, ki jih uporabljamo pri indukcijskem zdravljenju B-ALL. Vsak citostatik smo uporabili v treh logaritmično naraščajočih koncentracijah. Po tridnevni kultivaciji smo s kvantitativno pretočno citometrijo določili izražanje celičnih označevalcev CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c in CD137 na površini malignih celic. Šest vzorcev v končno analizo ni bilo vključenih zaradi premajhnega števila izoliranih malignih celic ali zaradi prevelike izgube števila malignih celic med postopkom izolacije in kultivacije. Pri drugem delu raziskave smo ugotavljali izražanje antigenov na malignih celicah kostnega mozga, odvzetega pri bolnikih v času postavitve diagnoze ter na 15. in 33. dan zdravljenja. V tem delu raziskave smo izražanje posameznih celičnih označevalcev opredelili s srednjo intenziteto fluorescence (ang. mean fluorescence intensity: MFI), izmerjeno s pretočno citometrijo. Spremembo v izražanju celičnih označevalcev smo povezali tudi z izmerjeno minimalno rezidualno boleznijo (MRD), določeno na 15. dan zdravljenja, s čimer smo povezali spremembo imunofenotipa z odgovorom bolezni na kemoterapijo. Rezultati Z našim in vitro delom raziskave smo pokazali, da se ob izpostavljenosti daunorubicinu, pronizonu in vinkristinu spremeni imunofenotip malignih celic B-ALL. Daunorubicin je vplival na izražanje vseh opazovanih označevalcev razen na CD66c, saj se je ob tem zmanjšalo izražanje CD10, CD19, CD27, CD34, CD45 in CD58 ter povečalo izražanje CD137. Zmanjšanje izražanja CD20 smo ugotovili le v odvisnosti od koncentracije daunorubicina. Ob izpostavljenosti pronizonu se je zmanjšalo izražanje CD10, CD19, CD27, CD34 in CD58. Ob izpostavljenosti vinkristinu se je zmanjšalo izražanje CD19 in CD58 ter povečalo izražanje CD45. Asparaginaza in metotreksat na imunofenotip nista imela vpliva. Analizirali smo tudi spremembe imunofenotipa, do katerih je prišlo med indukcijskim zdravljenjem B-ALL in vivo . Pokazali smo, da se je do 33. dne zdravljenja povečalo izražanje CD20 in CD45 ter zmanjšalo izražanje CD10, CD34 in CD58. Prognostični pomen spremembe imunofenotipa smo poskušali opredeliti v povezavi s celokupnim preživetjem in stopnjo pojavnosti ponovitve bolezni. Ker pa sta med vključenimi bolniki umrla le dva, pri nobenem od preostalih pa ni prišlo do ponovitve bolezni, prognostičnega pomena spremembe imunofenotipa nismo uspeli opredeliti. Vendar nam je statistični model, ki smo ga uporabili v raziskavi, omogočil, da smo analizirali povezavo med spremembo izražanja celičnih označevalcev in določitvijo MRD. Tako smo ugotovili, da je sprememba v izražanju CD38 povezana s stopnjo minimalne rezidualne bolezni (MRD) na 15. dan zdravljenja in s tem z odgovorom levkemije na indukcijsko kemoterapijo. Zaključki Raziskava nam je omogočila primerjavo sprememb v izražanju posameznih celičnih označevalcev, do katerih pride in vitro ter in vivo. Pokazali smo, da daunorubicin, pronizon in vinkristin zmanjšajo izražanje CD19 in CD58 in vitro, kar bi lahko vplivalo na učinkovitost zdravljenja z blinatumomabom ali s celicami T z izraženim himernim receptorjem (CAR-T), ter da se izražanje CD20 spremeni le v odvisnosti od odmerka daunorubicina, kar je pomembno v primeru uporabe rituksimaba. Poleg tega smo ugotovili, da daunorubicin in vinkristin vplivata na izražanje posameznih celičnih označevalcev neodvisno od koncentracije zdravila, kar nakazuje, da bi lahko že nizki odmerki zdravila vplivali na imunofenotip malignih celic. Potrdili smo tudi, da je sprememba v izražanju CD38 povezana z odzivom levkemičnih celic na indukcijsko kemoterapijo in vivo, kar nam bi lahko omogočilo dodatno razporejanje bolnikov v skupine tveganja in izbiro temu prilagojenega zdravljenja.

• Language: Slovenian
• Keywords: spreminjanje imunofenotipa, kemoterapija, imunoterapija, B-ALL otroške dobe
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2024
• PID: 20.500.12556/RUL-159269
• Publication date in RUL: 05.07.2024

Secondary language

• Language: English
• Title: The influence of chemotherapy on antigen espression on leukemic cells in pediatric acute

Abstract

Objective We have investigated the influence of cytostatic drugs used in the induction therapy of acute lymphoblastic leukaemia (ALL) on the immunophenotype of malignant cells isolated from the bone marrow of children diagnosed with B-cell ALL (B-ALL). In vitro experiment allowed us to analyse changes in the expression of antigens, which are either important for the immunotherapy, or of prognostic importance based on literature data. We also analysed markers whose prognostic significance in the treatment of children with B-ALL is not yet known. In the second part of the study we analysed changes in the expression of cell markers in vivo during induction treatment of B-ALL and correlated these changes with treatment response. Materials and Methods Malignant cells were isolated from the bone marrow of 35 children with B-ALL. Cells were cultivated and exposed to one of the cytostatic drugs used in B-ALL induction treatment. Each cytostatic was used in three logarithmically increasing concentrations. After three days of incubation, we performed quantitative flow cytometry and determined the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c, and CD137. Six samples were not included in the final analysis due to either a small number of isolated malignant cells or significant cell loss during the isolation and cultivation process. In the second part of the study, we assessed the expression of antigens on malignant cells taken at the time of diagnosis and on days 15 and 33 of induction treatment in vivo. We performed flow cytometry and defined the expression of individual cell markers (CD10, CD19, CD20, CD34, CD38, CD45, CD58 in CD99) by mean fluorescence intensity (MFI). We identified changes in the expression of cell markers and correlated those with measured minimal residual disease (MRD) on day 15 of treatment, which enabled us to define the correlation between change in immunophenotype and disease's response to treatment. Results Our study demonstrated that daunorubicin, prednisone, and vincristine cause alteration in the antigen expression on the malignant cells. Daunorubicin caused down-modulation of CD10, CD19, CD27, CD34, CD45 and CD58 and up-modulation of CD137. It did not cause any changes in the expression of CD66c. We could also identify a decrease in CD20 expression only after analysing the effect of the concentration of daunorubicin. Exposure to prednisone down-modulated the expression of CD10, CD19, CD27, CD34, and CD58. Exposure to vincristine triggered down-modulation of CD19 and CD58 and up-modulation of CD45. Asparaginase and methotrexate had no impact on the immunophenotype. We also analysed in vivo changes during B-ALL induction treatment. We showed that up-modulation of CD20 and CD45 and down-modulation of CD10, CD34, and CD58 appeared by day 33 of therapy. We also tried to define the prognostic significance of the immunophenotypic shifts by correlating changes in antigen expression and overall survival of patients and relapse rate. However, since only two of the included patients died and no relapse was diagnosed in the remaining cases, the prognostic significance of the immunophenotypic changes could not be determined. Nevertheless, the statistical model used in the study allowed us to analyse the correlation between changes in the expression of antigens and the MRD. We found that the change in CD38 expression was related to MRD on day 15 of treatment. Conclusions The study enabled us to compare changes in the expression of individual cell markers occurring in vitro and in vivo. We demonstrated that daunorubicin, prednisone, and vincristine caused down-modulation of CD19 and CD58, potentially affecting the effectiveness of treatment with blinatumomab or CAR-T cells. Additionally, the concentration of daunorubicin was important for down-modulation of CD20 expression, which is crucial when therapy with rituximab is planned. Furthermore, we confirmed that daunorubicin and vincristine affect the expression of individual cell markers independently of drug concentration, suggesting that even minimal doses could trigger changes in the immunophenotype of malignant cells. We also confirmed that changes in CD38 expression are associated with the MRD on day 15, which could enable additional stratification of patients into risk groups and the selection of personalized therapy accordingly.

• Keywords: immunophenotypic changes, chemotherapy, immunotherapy, paediatric B-ALL