DNA methylation and hypomethylating agents in high-risk myelodysplastic syndromes and acute myeloid leukemia

Epigenetic alterations are common in cancer. One example is aberrant hypermethylation of the promoters of tumor suppressor genes and hence silencing of gene expression. Azacitidine, a DNA hypomethylating drug, has been shown to prolong survival in patients with high-risk myelodysplastic syndromes (MDS) compared to conventional care regimens, and is now recommended as first-line therapy for patients not eligible for allogeneic stem cell transplantation. Azacitidine has DNA hypomethylating properties and has been shown to re-induce expression of aberrantly silenced genes in various cell lines. However, its mechanism of action in primary hematopoietic progenitors in vivo is relatively unknown. This thesis aimed to assess the prognostic value of DNA methylation in high-risk MDS and in de novo acute myeloid leukemia (AML), to evaluate the effect and feasibility of maintenance treatment with azacitidine in patients in complete remission (CR) after induction chemotherapy, and to study mechanisms of action of azacitidine in primary MDS and normal bone marrow (NBM) progenitors. We show, for the first time, a correlation between promoter methylation patterns and outcome of induction chemotherapy in a cohort of 60 patients with high-risk MDS or AML following MDS (MDS-AML). Patients who were hypermethylated in the E-cadherin (CDH) promoter had lower CR rates than those without methylation (P=0.008). CDH methylation was also associated with shorter survival (P=0.003). By contrast, in a material of 107 patients with de novo AML CDH methylation had no impact on survival or on outcome of induction chemotherapy. In fact, promoter hypermethylation of P15ink4b, previously reported as a poor prognostic marker in MDS and MDS-AML, as well as genome wide promoter methylation corresponded to a better survival (P=0.001 and 0.005, respectively). Another novel finding was that de novo AML patients with a low degree of global DNA methylation had a poorer response to induction chemotherapy (P=0.005). These differences in the prognostic value of methylation status in MDS/MDS-AML and de novo AML suggest important differences in disease biology and response to treatment between the two entities. Several mechanisms of action for azacitidine have been suggested, including induction of apoptosis, differentiation of blasts, histone modification, immunomodulation and DNA and RNA demethylation. However, the majority of data results from cell line experiments or from sequential bone marrow sampling during azacitidine treatment. In paper IV of this thesis we exposed primary MDS and normal bone marrow (NBM) progenitors to azacitidine in vitro. Interestingly, azacytidine caused marked up-regulation of gene expression in MDS but not in NBM CD34+ marrow cells. Compared to cell line experiments, induction of apoptosis as well as global and gene-specific hypomethylation was less pronounced in primary cells. Interestingly, azacitidine in doses up to 5 μM had no negative effect on proliferation in suspension cultures, and doses up to 0.5 μM even had a positive effect on colony growth. This is a useful finding since it may support the use of the drug for patients with low-risk MDS, and as maintenance after allogeneic stem cell transplantation

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 48 weeks and a transfusion need of ≥ 4 units over 8 weeks were included. Aza 75mgm -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 ≥ one 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 nonresponders. 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

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

Prolonged survival after second autologous transplantation and lenalidomide maintenance for salvage treatment of myeloma patients at first relapse after prior autograft.

Autologous stem cell transplantation (ASCT) as part of the primary therapy in multiple myeloma (MM) is standard practice. In contrast, the role of a second ASCT (ASCT2) and subsequent lenalidomide maintenance for relapsed disease remains unclear. In this study, we analysed 86 consecutive MM patients with a first relapse after prior ASCT receiving either a second ASCT or conventional chemotherapy. After a median follow-up of 37.7 months since first relapse, 54 (62.8%) patients were still alive and 29 (33.7%) without progression. Sixty-one (71.0%) patients received ASCT2 and had better progression-free survival (PFS) (30.2 versus 13.0 mo; P = .0262) and overall survival (OS) rates (129.6 versus 33.5 mo; P = .0003) compared with 25 (29.0%) patients with conventional treatment. Patients relapsing later than 12 months after ASCT1 benefitted from a second ASCT with better PFS2 (P = .0179) and OS2 (P = .0009). Finally, lenalidomide maintenance after ASCT2 was associated with longer PFS (41.0 vs 21.6 mo; P = .0034) and better OS (not yet reached vs 129.6 mo; P = .0434) compared with patients without maintenance. Our data suggest that a second ASCT and lenalidomide maintenance given at first relapse in MM after prior ASCT are associated with better survival rates