Antileukemic Efficacy of Continuous vs Discontinuous Dexamethasone in Murine Models of Acute Lymphoblastic Leukemia

<div><p>Osteonecrosis is one of the most common, serious, toxicities resulting from the treatment of acute lymphoblastic leukemia. In recent years, pediatric acute lymphoblastic leukemia clinical trials have used discontinuous rather than continuous dosing of dexamethasone in an effort to reduce the incidence of osteonecrosis. However, it is not known whether discontinuous dosing would compromise antileukemic efficacy of glucocorticoids. Therefore, we tested the efficacy of discontinuous dexamethasone against continuous dexamethasone in murine models bearing human acute lymphoblastic leukemia xenografts (n = 8 patient samples) or murine BCR-ABL+ acute lymphoblastic leukemia. Plasma dexamethasone concentrations (7.9 to 212 nM) were similar to those achieved in children with acute lymphoblastic leukemia using conventional dosages. The median leukemia-free survival ranged from 16 to 59 days; dexamethasone prolonged survival from a median of 4 to 129 days in all seven dexamethasone-sensitive acute lymphoblastic leukemias. In the majority of cases (7 of 8 xenografts and the murine BCR-ABL model) we demonstrated equal efficacy of the two dexamethasone dosing regimens; whereas for one acute lymphoblastic leukemia sample, the discontinuous regimen yielded inferior antileukemic efficacy (log-rank p = 0.002). Our results support the clinical practice of using discontinuous rather than continuous dexamethasone dosing in patients with acute lymphoblastic leukemia.</p></div

Antileukemic efficacy of continuous, discontinuous, and no dexamethasone for xenografted patient ALL samples.

<p>Red indicates continuous dexamethasone (Cont dex), green indicates discontinuous dexamethasone (disc dex), and blue indicates no dexamethasone. Squares on the curve indicate leukemic events, and + indicates censoring due to toxicity, accidental death, or the end of the experiment. Red and green boxes indicate treatment period—red indicates continuous dexamethasone (4 mg/L) and green indicates discontinuous dexamethasone (8 mg/L for 3.5 days, 0 for 3.5 days). P-values shown were calculated with the log-rank test, comparing the continuous and discontinuous dexamethasone treatment arms, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135134#pone.0135134.t001" target="_blank">Table 1</a> for comparisons of dexamethasone vs no dexamethasone.</p

Anti-leukemic survival was equivalent between dexamethasone treatment regimens in the murine ALL model.

<p>Mice of the 129X1SvJ strain (A) and C57Bl/6 strain (B) were injected with <i>Arf</i>-/- BCR-ABL+ luciferase+ leukemia and treated with continuous (Cont dex, red), discontinuous dexamethasone (Disc dex, green), or no dexamethasone (blue) regimens. Boxes indicate leukemic events, while + indicates censored events. P-values indicated are log-rank p-values comparing the continuous and discontinuous dexamethasone regimens, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135134#pone.0135134.t001" target="_blank">Table 1</a> for comparisons of dexamethasone vs no dexamethasone.</p

Leukemia cells generally became less sensitive to dexamethasone after <i>in vivo</i> treatment.

<p>Dexamethasone sensitivity of leukemic bone marrow cells at diagnosis (orange), the passage prior to <i>in vivo</i> treatment (purple), or at the time of sacrifice after <i>in vivo</i> treatment (continuous dexamethasone, red; discontinuous dexamethasone, green; no dexamethasone, blue). Symbols indicate individual mice, the horizontal line indicates the mean, and error bars indicate standard error of the mean (SEM). Incubation time for all diagnosis samples in the MTT assay was 4 days. Incubation time for cells from the mice receiving continuous dexamethasone, discontinuous dexamethasone, and no dexamethasone was 4 days for SJTALL021916, SJTALL033, SJBALL215, 3 days for SJTALL030, and 2 days for SJMLL005 and SJMLL009. <i>Ex vivo</i> sensitivity was not tested in SJE2A007 due to poor viability after two days <i>ex vivo</i> (<50%), and SJHYPO123 due to resistance of the sample at the time of diagnosis (LC50 > highest concentration tested, 15 uM, which was recapitulated in the xenograft sample prior to <i>in vivo</i> treatment).</p

Median leukemia-free survival (LFS) and growth delay factor (GDF) for each murine model.

<p>Cont dex: mice treated continuously with 4 mg/L dexamethasone. Disc dex: mice treated discontinuously with 8 mg/L dexamethasone for half of each week. N, number of mice per treatment group. LFS, median leukemia-free survival. LFS, Range, and GDF are shown in days. Dex days indicates the days the mice were treated with dexamethasone, with the injection of leukemia being day 0 (the same total dose was administered in the two regimens for each xenograft). LC50-D indicates the dexamethasone LC50 in the patient bone marrow at the time of ALL diagnosis. LC50-X indicates the dexamethasone LC50 in the xenograft bone marrow from the passage prior to the efficacy experiment or in the case of the mouse cell lines, the passage that was injected into the mice. Details on the calculation of LFS and GDF are found in the methods. NA indicates that the median LFS could not be calculated because of too few leukemic events.</p><p>* indicates that the GDF was not calculated because the median LFS was not calculated.</p><p>Median leukemia-free survival (LFS) and growth delay factor (GDF) for each murine model.</p

Plasma dexamethasone concentrations in mice during treatment were similar to those achieved in patients and corticosterone was suppressed in dexamethasone treated mice.

<p>Plasma dexamethasone (A) and corticosterone concentrations (B) in mice engrafted with SJTALL033 cells. Blue shaded area indicates the concentration of dexamethasone found in patient samples (20–200 nM). Plasma dexamethasone (dex) concentrations were determined by LC-MS twice weekly, just prior to the change of medicated water. N = 10 in the dexamethasone treatment groups and N = 5 in the untreated controls (no dex group). Treatment began on day 7 after plasma was taken, and is indicated by the boxes below the x-axis. Green indicates discontinuous dexamethasone and red indicates continuous dexamethasone. Dexamethasone was in the drinking water of the discontinuous (disc) dexamethasone group from day 7–10.5, 14–17.5, 21–24.5, 28–31.5, and 35–39.5. Bar height indicates mean and error bars indicate standard error of the mean (SEM).</p

Continuous dexamethasone was more effective than discontinuous dexamethasone for SJE2A007 cells but not SJTALL021916 cells.

<p>Red indicates continuous dexamethasone (Cont dex), green indicates discontinuous dexamethasone (disc dex), and blue indicates no dexamethasone. A, ventral luminescence over time. Symbols indicate median luminescence and error bars indicate interquartile range. Red and green boxes near the x-axis indicate treatment period (continuous dexamethasone in red, discontinuous dexamethasone in green). B, hCD45% in the blood over time. Symbols indicate median and error bars indicate interquartile range. C, spleen weight on day 62 (SJE2A007, left) or at the time of sacrifice (SJTALL021916, right). Symbols indicate individual mice, and horizontal line indicates median. P values were calculated with a two-tailed Mann Whitney test.</p