The effect of short-course high-dose methylprednisolone on peripheral blood lymphocyte subsets in children with acute leukemia during remission induction treatment☆

1. Introduction 

Although corticosteroids (CS) have been used successfully in conventional doses for the treatment of acute lymphoblastic leukemia (ALL) for a long time, more favorable results have been obtained with high-dose methylprednisolone (HDMP) alone in the treatment of children with ALL even in children who failed to respond to conventional doses of steroids, or in resistant and in relapsed cases [1], [2], [3]. HDMP combined with other antileukemic agents improved the prognosis of these children [4], [5]. Furthermore, remarkable antileukemic effect of HDMP treatment to which induce differentiation and apoptosis of leukemic cells has been demonstrated in children with acute myeloblastic leukemia (AML) in vivo and in vitro [6], [7], [8]. More over, HDMP has been shown to increase the leukocyte count [9] and the number of CD34+ hematopoietic progenitor cells in children with ALL and AML [10], [11], [12]. Various clinical and experimental studies have indicated the importance of antileukemic T cell activity in patients with leukemia. Because of the above studies we assume that the potential antileukemic effects of HDMP could also be mediated by T cell activation. In this study we evaluated the effect of short-course HDMP treatment on peripheral blood (PB) T lymphocyte subsets during the remission induction therapy in children with AML and ALL.

2. Material and methods 

Twenty-three children (14 boys, nine girls) with newly diagnosed acute leukemia (16 with ALL, seven with AML) were enrolled in this study. The patients ranged in age from 3 to 14 years (mean, 5.6 years). None of them had any signs of infection. Diagnosis was made by morphology according to the French–American–British (FAB) classification, by cytochemistry, and by cell surface marker analysis. With informed consent, methylprednisolone sodiumsuccinate (Prednol-L® 30mg/kg per day) as a single agent was administered orally once a day to all children for the first 4 days of induction therapy. Since then treatment was continued with HDMP containing chemotherapy regimens according to our institutional ALL and AML protocols.

Complete blood counts were performed by using an automatic analyzer (STKS Coulter) and manual differential counts were performed on wright-stained PB smears. Surface marker analysis was performed by using a flow cytometry (FACScan, Becton Dickinson, San Jose, CA, USA) with a panel of monoclonal antibodies [13]. The numbers of CD3+, CD4+ (inducer/helper T cells), CD8+ (cytotoxic/suppresor T cells), CD16+56+ (natural killer cells), and CD45RA+, CD45RO+ T lymphocytes were determined in anticoagulated peripheral venous blood samples before and after 4 days of HDMP treatment. If BM leukemic cells has lacked T cell markers at diagnosis, all PB T cell marker positive cells after HDMP treatment were accepted as from nonleukemic origin. The absolute counts for the circulating lymphocyte subsets per mm3 were calculated by multiplying the percentage of lymphocyte subsets by the absolute number of mononuclear cells. The final results were expressed as mean±S.D.

Statistical analysis were performed by Wilcoxon test.

3. Results 

Changes in the absolute number of PB T lymphocyte subsets, after 4 days of HDMP treatment in children with AML and ALL are shown in Fig. 1. After 4 days of HDMP treatment the number of PB blast cells decreased significantly (P<0.05) in children with either AML (12868±8930/μl versus 2389±993/μl) or ALL (23700±9495/μl versus 2572±230/μl). In contrast, a statistically significant increase was observed in the absolute number of PB CD3+, CD4+, and CD8+ T cells (P<0.05) in patients with AML and ALL. In addition the number of CD45RA+, CD45RO+ and NK cells also increased in children with AML. Among those, the increase in the number of CD45RA+ cells (2800±177/μl versus 8787±410/μl) was statistically significant (P<0.05). However, in children with ALL, the numbers of CD45RA+ cells and NK cells increased very slightly, whereas CD45RO+ cells decreased insignificantly (P>0.05) (Fig. 1).

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Fig. 1. Changes in absolute number of peripheral blood T lymphocyte subsets in children with AML (n=7) and ALL (n=16), after 4 days of HDMP treatment.

4. Discussion 

In the present study, we have shown that administration of a short course of HDMP treatment at the beginning of induction therapy increased the PB CD3, CD4 and CD8 T lymphocytes in children with AML and ALL. On the other hand, a significant decrease in the number of PB blast cells was observed after 4 days of HDMP treatment which was consistent with our previous observations in children with ALL and AML treated with HDMP [4], [6]. Since HDMP treatment increased the number of CD8+ cytotoxic T cells, it can be expected to provide an antileukemic effect, in a short period of time. Increased number of CD4+ (helper/inducer) T lymphocytes after short-course HDMP treatment, may contribute to this effect by further stimulating cytotoxic T lymphocyte functions. An increase of the CD4+ and CD8+ cells after short-course HDMP treatment has also been shown in children with idiopathic thrombocytopenic purpura [14] and in neutropenic children with acute leukemia during maintenance treatment [15]. Furthermore, a high dose of glucocorticoids has also been shown in vitro to rescue peripheral T cells from antigen-induced cell death [16].

The increases in PB CD3+, CD4+ and CD8+ lymphocytes may be attributed to HDMP stimulation of the PB and BM CD34+ progenitor cells [11], [12], [17], and is possibly due to an increase in production of endogenous G-CSF and GM-CSF [18], [19]. It has been reported that HDMP increases the level of IL-2 levels in children with chronic myelomonocytic leukemia [20], possibly by activation of T cells. The administration of standard doses of corticosteroid results in marked but transient lymphopenia [21]. We have previously shown that the increase in CD34+ progenitor cells and polymorphonuclear cells are significantly higher in patients treated with HDMP compared to those received standard dose steroid [10]. This might explain why, high doses of corticosteroid increases lymphocyte numbers in children with acute leukemia.

As seen in Fig. 1, we also observed an increase in the number of CD45RA+, CD45RO+ cells and NK cells after short-course HDMP treatment in children with AML and ALL with the exception of CD45RO+ cells in children with ALL. The expression of CD45RA and CD45RO isoforms has been reported to be associated with T cell maturation [22], [23]. However, the significance of changes in CD45 isoforms following short-course HDMP treatment observed in the present study should be clarified in further studies in larger number of patients. Previous studies demonstrated that untreated children with acute leukemia have low numbers and defective functions of NK cells [24]. In the present study, as observed in children with chronic idiopathic thrombocytopenic purpura [14], NK cells increased slightly after 4 days of HDMP treatment which may also contribute to antileukemic effect of HDMP treatment.

In conclusion, short-course HDMP treatment is effective in the dramatic eradication of the leukemic blasts in children with ALL and AML as well. This beneficial antileukemic effects of HDMP in induction therapy may be partly due to an increase in T cell subsets.