Page 70 - 2019_01-Haematologica-web

J. Stomper et al.
Introduction
Statistical methods
We used non-parametric Spearman correlation coefficients (rs) to assess the association between HbF levels and other variables. To address the hypothesis that HbF after course 2 of decitabine treatment may predict hematologic responses after course 4, we employed linear regression models with HbF levels as the inde- pendent variable, and platelet count, hemoglobin concentration, neutrophil counts, and bone marrow blasts as dependent vari- ables. Results are displayed graphically. The evaluation of possible outliers was based on studentized residuals.18 No potential outliers were excluded from the statistical analyses. The HbF value distri- butions between responders and non-responders were compared using the Wilcoxon two-sample test. Before-after differences in binary variables such as elevated HbF (yes/no) were assessed stat- ically using the McNemar test.
Time-to-event endpoints comprised overall survival, progres- sion-free survival, and AML-free survival. We performed land- mark analyses starting at the time of HbF measurements after two cycles of decitabine treatment in order to avoid the so-called immortal-time bias.19 Thus, they were relevant only for patients who reached this time point. The Kaplan-Meier method was used to estimate distributions of overall, progression-free and AML-free survival according to HbF value (> versus ≤1%), and to compute median estimations. A log-rank test was used to assess the prog- nostic importance of HbF value, and a Cox model to estimate the hazard ratio (HR) and its 95% confidence interval (95% CI).
Statistical analyses (all conducted at the CTU Freiburg, Germany) were performed with SAS 9.2 (SAS Institute Inc., Cary, NC, USA), and IBM SPSS Statistics 22.
Cell lines and in vitro treatment
K562 and HEL cells (DMSZ, Braunschweig, Germany) were cul-
tured in RPMI1640 containing 10% heat-inactivated fetal calf serum. Cells were treated with three 24-h pulses of 100 and 20 nM decitabine (Sigma Aldrich) and harvested 144 h after the first treat- ment, as described previously.20 Hemin (Sigma Aldrich), a positive control for induction of erythroid differentiation,21 was added to the culture medium at a final concentration of 50 mM.22 Cell viabil- ity was tested by 0.4% trypan blue staining. Megakaryocytic dif- ferentiation was induced by exposure to 5 nM phorbol ester (PMA, Sigma Aldrich) for 48 h, as described previously.23 .
Further information on the methods, including transcriptome profiling, immunoblotting, and methyl-CpG immunoprecipitation sequencing, is provided in the Online Supplement.
Results
Kinetics of in vivo induction of HbF in patients with myelodysplastic syndrome/acute myeloid leukemia receiving decitabine
In 40 patients (15 with higher-risk MDS, 25 with AML), HbF was measured serially twice or more during treat- ment (in a single patient, HbF before treatment was not available). The patients’ characteristics and those of their diseases are presented in Table 1.
When analyzing HbF kinetics over time (Figure 1A, Online Supplementary Figure S1), the first on-treatment time point with robust HbF induction was at the end of the sec- ond course of decitabine treatment. Compared to the median pre-treatment HbF of 0.8%, we observed overall induction to 1.1% after course 2. The median fold-change of HbF concentration at the end of the second course of decitabine treatment as compared to pre-treatment levels was 1.67 (P=0.088).
DNA-hypomethylating agents (HMAs) alter gene expression of malignant cells by gene reactivation, e.g. of epigenetically silenced tumor suppressor genes resulting in induction of apoptosis or senescence,1-4 or of cancer/testis antigens and retroviral sequences, thus elic- iting an immune response against cancer cells,5,6 and by reduction of oncogene overexpression.7 In cell line models of acute myeloid leukemia (AML), they also induce gran- ulocytic maturation although this effect has yet to be val- idated in vivo.8
Probably the earliest proof of therapeutic gene reactiva- tion by an HMA was the demonstration of induction of fetal hemoglobin (HbF) in a patient with severe beta-tha- lassemia through demethylation and transcriptional acti- vation of the gamma-globin gene locus,9 the developmen- tal regulation of which is governed by DNA methylation. This ability of HMAs to de-repress epigenetically silenced gamma-globin expression in (polyclonal) erythropoietic precursors has also been observed in patients with solid tumors10,11 and has prompted clinical trials on the use of HMAs in hemoglobinopathies.12,13
Several groups have shown that in patients with myelodysplastic syndrome (MDS) or AML receiving azac- itidine, HbF is induced and may be a marker of treatment effect.14,15 Very recently, we observed elevated HbF levels also in MDS/AML patients treated with decitabine.16 We now dissected the kinetics of the in vivo induction of HbF in these patients with high-risk MDS or AML, in order to evaluate the prognostic value of HbF induction. We observed that HbF levels after two courses of treatment were of prognostic value regarding hematologic responses of the different lineages, and, in MDS, for overall survival from this time point on [hazard ratio (HR)=0.2; 95% con- fidence interval (95% CI): 0.1-0.9; P=0.03]. The cellular source of HbF in responders appeared to be predominant- ly non-malignant erythroid precursors. To model HbF reactivation in vitro, we investigated two transformed leukemia cell lines for the induction of an erythroid differ- entiation program that also encompassed HbF protein synthesis induced by decitabine.
Methods
Patients, treatments and response evaluations
Details on the patients, the treatments given and evaluation of responses are provided in the Online Supplement. Both the thera- peutic studies and the translational investigations were approved by the institutional review board (ethics committee) of the University of Freiburg Medical Center. Patients (all treated at the Freiburg study site) provided their written informed consent to be included in the respective clinical studies, and to related translational investigations according to the Declaration of Helsinki.
Hemoglobin quantification by high-performance liquid chromatography
HbF levels were measured by high-performance liquid chro- matography (HPLC) before treatment and after the end of each treatment course (every 6 weeks) as described previously.17 Patients were grouped by normal (HbF ≤1%) or elevated HbF levels (HbF >1%), in accordance with reference values for the University of Freiburg Medical Center Central Laboratory.
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haematologica | 2019; 104(1)