Dexamethasone in hyperleukocytic acute myeloid leukemia

Abstract

Patients with acute myeloid leukemia and a high white blood cell count are at increased risk of early death and relapse. Because mediators of inflammation contribute to leukostasis and chemoresistance, dexamethasone added to chemotherapy could improve outcomes. This retrospective study evaluated the impact of adding or not adding dexamethasone to chemotherapy in a cohort of 160 patients with at least 50×10⁹ white blood cells. In silico studies, primary samples, leukemic cell lines, and xenograft mouse models were used to explore the antileukemic activity of dexamethasone. There was no difference with respect to induction death rate, response, and infections between the 60 patients in the dexamethasone group and the 100 patients in the no dexamethasone group. Multivariate analysis showed that dexamethasone was significantly associated with improved relapse incidence (adjusted sub-HR: 0.30; 95% CI: 0.14-0.62; P=0.001), disease-free survival (adjusted HR: 0.50; 95% CI: 0.29-0.84; P=0.010), event-free survival (adjusted HR: 0.35; 95% CI: 0.21-0.58; P<0.001), and overall survival (adjusted HR: 0.41; 95% CI: 0.22-0.79; P=0.007). In a co-culture system, dexamethasone reduced the frequency of leukemic long-term culture initiating cells by 38% and enhanced the cytotoxicity of doxorubicin and cytarabine. In a patient-derived xenograft model treated with cytarabine, chemoresistant cells were enriched in genes of the inflammatory response modulated by dexamethasone. Dexamethasone also demonstrated antileukemic activity in NPM1-mutated samples. Dexamethasone may improve the outcome of acute myeloid leukemia patients receiving intensive chemotherapy. This effect could be due to the modulation of inflammatory chemoresistance pathways and to a specific activity in acute myeloid leukemia with NPM1 mutation.

Figure 1.

Study flowchart. y: years; WBC: white blood cell; OS: overall survival; CR: complete response.

Figure 2.

Estimates of survival end points and incidence of relapse. (A) Kaplan–Meier curves for overall survival in patients treated with dexamethasone (black line) or not (solid line in red). (B) Show event-free survival, (C) disease-free survival and (D) cumulative incidence of relapse. It is worth nothing that the follow up concerning relapse seems to be equal in the dexamethasone and no dexamethasone groups. This is due to the competing risk analyses. Indeed, in the competing risk analyses, a subject having a non-relapse death was not censored at the date of death and was still virtually considered to be at risk of having a relapse (and was still in follow up).

Figure 3.

Impact of dexamethasone on chemoresistance. (A) Leukemia long-term culture initiating cell (L-LTC-IC) frequency in acute myeloid leukemia (AML) samples upon dexamethasone treatment (#38 to 49, Online Supplementary Table S8). (B) Expression of CD34 and CD38 upon dexamethasone treatment in co-culture with AML samples and MS-5 stromal cells. (C) Expression of lineage and CD14/11b markers upon dexamethasone treatment in co-culture with AML samples and MS-5 stromal cells. (D) Seven AML cell lines incubated in a co-culture system with MS-5 stromal cells were treated for 1 week with vehicle, dexamethasone, cytarabine, or dexamethasone plus cytarabine. (E) Gene ontology enrichment analysis of down-regulated and up-regulated genes from RNA expression profiles of viable AML cells following cytarabine versus vehicle-treated AML-patient-derived xenograft (PDX) mice, by 1.5-fold or more. (F and G) Gene-to-small molecule associations that are significantly enriched within residual post-cytarabine AML cells (Figure 3F and Online Supplementary Table S5) or in relapse (compared to pairwise diagnosis, Figure 3G and Online Supplementary Table S6) using a data-mining algorithm (Genomatix) from GSE97631 or GSE66525 publicly accessible transcriptomic databases, respectively. These two graphs shown a gene signature ranking assessed by the number of observed versus expected genes significantly modulated in transcriptomes after treatment with diverse small molecules and significantly enriched in AML transcriptomes of residual post-cytarabine AML cells or of relapse. (H) Treatment of PDX models from 2 AML samples collected at diagnosis (black dots: normal karyotype, NPM1 mutation, wild-type for FLT3-ITD, DNMT3A-exon23, CEBPA, IDH1, and IDH2, red dots: normal karyotype, NPM1 mutation, DNMT3A-exon23 mutation, FLT3 wild type) with vehicle, dexamethasone (10 mg/kg/day, 5 days), cytarabine (30 mg/kg/day, 5 days), or dexamethasone plus cytarabine. At day 8, the reduction of the total AML cell burden was assessed by the absolute quantification of the hCD45⁺hCD33⁺mCD45.1⁻ cell population in bone marrow and spleen. Mann Whitney test: ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; ns: not significant.

Figure 4.

Activity of dexamethasone against acute myeloid leukemia (AML). (A) Anti-leukemic effect of dexamethasone on AML cell lines cultured in minimum essential medium alpha whose cytogenetic and molecular characteristics are shown in Online Supplementary Table S4 (two-way ANOVA test, P<0.001). OCI-AML3 and KG1a were significantly more sensitive compared to the other AML cell lines (Tukey multiple comparison test, P<0.001). (B) Kaplan–Meier curves for survival in a xenotransplantation mouse model of AML using the OCI-AML3 cell line (NPM1 and DNMT3A-R882C mutated) and NSG mice treated with vehicle, dexamethasone (10 mg/kg/day, 5 days), cytarabine (30 mg/kg/day, 5 days), or dexamethasone plus cytarabine. P-values of the log-rank test are vehicle versus cytarabine (P=0.0105), vehicle versus dexamethasone (P=0.0014), vehicle versus dexamethasone plus cytarabine (P<0.0001), cytarabine versus dexamethasone (P=0.1474), cytarabine versus dexamethasone plus cytarabine (P=0.0001), and dexamethasone versus dexamethasone plus cytarabine (P=0.0943). (C) Apoptosis induction by dexamethasone in primary AML samples (#1 to 37, Online Supplementary Table S8) according to NPM1 mutational status (16 samples with NPM1 mutations and 18 samples that were wild-type for NPM1). AML samples were incubated with or without 100 and 300 nM of dexamethasone for 24 hours and then processed for apoptosis studies using annexin-V/propidium iodide staining. Results are presented as mean percentages of annexin V-positive cells in treated versus untreated cells (P=0.049 and P=0.04 for 100 and 300 nM when comparing NPM1 mutated versus NPM1 wild-type samples, respectively). (D) Gene ontology enrichment analysis using the data set of Verhaak et al. in AML with NPM1 mutations. (E) Gene-gene associations between mutant NPM1 up-regulated target genes and small molecule gene signatures and a data-mining algorithm (Genomatix). Gene expression analyses revealed that the NPM1 mutation gene signature was highly enriched in genes responsive to treatment using small molecules, such as all trans-retinoic acid (ATRA), retinoic acid (RA), dexamethasone, and to a lesser extent dactinomycin (Online Supplementary Table S7).