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.
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