Clinical and molecular effects of azacitidine in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) constitute a heterogeneous group of malignant bone marrow disorders characterized by peripheral cytopenia and increased risk of leukemic progression. In higher-risk MDS, Azacitidine has been shown to prolong survival and modulate the epigenome, although the precise mechanisms by which the drug exerts its effect are unknown. Paper I reports the result from a Nordic study enrolling 30 transfusion-dependent Epo-refractory lower-risk MDS patients. Patients were treated with 6 cycles of Azacitidine and terminated the study if they reached transfusion independence, while non-responding patients received another 3 cycles combined with Epo. Five (21%) and one patient responded after Azacitidine and the combined treatment, respectively, and only 2 patients (10%) responded for more than 6 months. Toxicity was substantial, mainly consisting of infections. We conclude that Azacitidine can be effective in this cohort of patients but that the low response rate and relatively high toxicity precludes its recommendation as standard treatment. Targeted sequencing revealed a high frequency of recurrent MDS mutations without clear relation to response. In paper II we cultured CD34+ progenitors from higher-risk MDS and normal bone marrow (NBM) with or without Azacitidine and studied the effects on DNA methylation and histone acetylation. We showed that the MDS genome at the global level is hypermethylated compared to NBM and that Azacitidine induced profound demethylation. Histone acetylation was decreased by treatment, which theoretically would counteract the transcriptional activation resulting from reduced DNA methylation. To further explore these effects, we repeated the same culture experiment in paper IV to study the effects of Azacitidine on both DNA methylation and gene expression. We confirmed the marked demethylating effect of Azacitidine, and by using RNA seq we could show that Azacitidine significantly increases gene expression but without association with demethylated regions. Interestingly, the repressive histone mark H3K9me3 increased in three demethylated genes without increased expression, providing a potential explanation for the lack of association between demethylation and increased expression. In paper III we searched for factors associated with response to Azacitidine by studying clinical parameters (n=134); mutations (n=90); and DNA methylation (n=42) in patients treated with Azacitidine. Among the clinical variables, only disease duration before treatment predicted for poor response and survival. The group of mutations involved in histone modulation (ASXL1, EZH2, MLL) was associated with prolonged survival, contrasting previous reports on mixed MDS cohorts. Furthermore, DNA methylation profiles differed significantly between responding and non-responding patients. Analysis of 200 differentially methylated regions showed enrichment in pathways involved in differentiation and development. Methylation level of the most significant DMR, the HOXA5/A6-locus, was associated with survival. To summarize, these studies show that epigenetic modifications play a significant role in the pathogenesis and response to treatment in MDS and that further understanding of chromatin modifications will be important in order to develop therapeutic strategies in MDS

Comprehensive mapping of the effects of azacitidine on DNA methylation, repressive/permissive histone marks and gene expression in primary cells from patients with MDS and MDS-related disease

Azacitidine (Aza) is first-line treatment for patients with high-risk myelodysplastic syndromes (MDS), although its precise mechanism of action is unknown. We performed the first study to globally evaluate the epigenetic effects of Aza on MDS bone marrow progenitor cells assessing gene expression (RNA seq), DNA methylation (Illumina 450k) and the histone modifications H3K18ac and H3K9me3 (ChIP seq). Aza induced a general increase in gene expression with 924 significantly upregulated genes but this increase showed no correlation with changes in DNA methylation or H3K18ac, and only a weak association with changes in H3K9me3. Interestingly, we observed activation of transcripts containing 15 endogenous retroviruses (ERVs) confirming previous cell line studies. DNA methylation decreased moderately in 99% of all genes, with a median beta-value reduction of 0.018; the most pronounced effects seen in heterochromatin. Aza-induced hypomethylation correlated significantly with change in H3K9me3. The pattern of H3K18ac and H3K9me3 displayed large differences between patients and healthy controls without any consistent pattern induced by Aza. We conclude that the marked induction of gene expression only partly could be explained by epigenetic changes, and propose that activation of ERVs may contribute to the clinical effects of Aza in MDS.Peer reviewe

"Randomized phase II study of azacitidine +/- lenalidomide in higher-risk myelodysplastic syndromes and acute myeloid leukemia with a karyotype including Del(5q)"

Non peer reviewe

Postazacitidine clone size predicts long-term outcome of patients with myelodysplastic syndromes and related myeloid neoplasms

Azacitidine is a mainstay of therapy for MDS-related diseases. The purpose of our study is to elucidate the effect of gene mutations on hematological response and overall survival (OS), particularly focusing on their post-treatment clone size. We enrolled a total of 449 patients with MDS or related myeloid neoplasms. They were analyzed for gene mutations in pre- (n=449) and post- (n=289) treatment bone marrow samples using targeted-capture sequencing to assess the impact of gene mutations and their post-treatment clone size on treatment outcomes. In Cox proportional hazard modeling, multi-hit TP53 mutation (HR, 2.03; 95% CI, 1.42-2.91; P<.001), EZH2 mutation (HR, 1.71; 95% CI, 1.14-2.54; P=.009), and DDX41 mutations (HR, 0.33; 95% CI, 0.17-0.62; P<.001), together with age, high-risk karyotypes, low platelet, and high blast counts, independently predicted OS. Post-treatment clone size accounting for all drivers significantly correlated with International Working Group (IWG)-response (P<.001, trend test), except for that of DDX41-mutated clones, which did not predict IWG-response. Combined, IWG-response and post-treatment clone size further improved the prediction of the original model and even that of a recently proposed molecular prediction model, IPSS-M (c-index, 0.653 vs 0.688; P<.001, likelihood ratio test). In conclusion, evaluation of post-treatment clone size, together with pre-treatment mutational profile as well as IWG-response have a role in better prognostication of azacitidine-treated myelodysplasia patients

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer1,2. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease3,4, rapid transformation to acute myeloid leukemia (AML)5, resistance to conventional therapies6–8 and dismal outcomes9. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations10. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R)11. Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response

Limited clinical efficacy of azacitidine in transfusion-dependent, growth factor-resistant, low- and Int-1-risk MDS: Results from the nordic NMDSG08A phase II trial

This prospective phase II study evaluated the efficacy of azacitidine (Aza)+erythropoietin (Epo) in transfusion-dependent patients with lower-risk myelodysplastic syndrome (MDS). Patients ineligible for or refractory to full-dose Epo+granulocyte colony stimulation factors for >8 weeks and a transfusion need of greater than or equal to4 units over 8 weeks were included. Aza 75 mg m−2 d−1, 5/28 days, was given for six cycles; non-responding patients received another three cycles combined with Epo 60 000 units per week. Primary end point was transfusion independence (TI). All patients underwent targeted mutational screen for 42 candidate genes. Thirty enrolled patients received greater than or equal toone cycle of Aza. Ten patients discontinued the study early, 7 due to adverse events including 2 deaths. Thirty-eight serious adverse events were reported, the most common being infection. Five patients achieved TI after six cycles and one after Aza+Epo, giving a total response rate of 20%. Mutational screening revealed a high frequency of recurrent mutations. Although no single mutation predicted for response, SF3A1 (n=3) and DNMT3A (n=4) were only observed in non-responders. We conclude that Aza can induce TI in severely anemic MDS patients, but efficacy is limited, toxicity substantial and most responses of short duration. This treatment cannot be generally recommended in lower-risk MDS. Mutational screening revealed a high frequency of mutations

Single-Cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy

Altres ajuts: Cellex Foundation, CEL007; Asociación Española Contra el Cáncer, AC18/000002Alterations in epigenetic marks, such as DNA methylation, represent a hallmark of cancer that has been successfully exploited for therapy in myeloid malignancies. Hypomethylating agents (HMAs), such as azacitidine (AZA), have become standard-of-care therapy to treat myelodysplastic syndromes (MDS), myeloid neoplasms that can evolve into acute myeloid leukemia (AML). However, our capacity to identify who will respond to HMAs, and the duration of response, remains limited. To shed light on this question, we have leveraged the unprecedented analytical power of single-cell technologies to simultaneously map the genome and immunoproteome of MDS samples throughout clinical evolution. We were able to chart the architecture and evolution of molecular clones in precious paired bone marrow MDS samples at diagnosis and post-treatment to show that a combined imbalance of specific cell lineages with diverse mutational profiles is associated with the clinical response of MDS patients to hypomethylating therapy