Early and Sustained Response to Azacytidine in High-Risk MDS Patients with Monosomy 7 Correlates with Increased Apoptosis and Not CDKN2B Demethylation.

Disease progression in MDS is associated with CDKN2B (encodes p15^INK4b) promoter methylation and an inhibition of apoptosis. We therefore studied predominantly high-risk MDS patients treated with azacitidine in order to determine whether clinical responses correlated with changes in CDKN2B promoter methylation and bone marrow apoptosis. In all, 24 patients (19 male) with a median age of 66.7 years were treated with azacitidine (75mg/m²/day x 7 days, every 28 days). Patients were FAB RA (n=2), RAEB (n=7), RAEB-T (n=13) and AML (n=2) with 18/24 having an IPSS risk of Int-2 or High. Cytogenetic abnormalities were present in 17 patients (4 patients with monosomy 7; 1 with der(7) as the sole cytogenetic abnormality and 1 as part of a complex karyotype; 4 with trisomy 8; 1 with 11q abnormalities; 1 with 5q-, 1 with 20q, 1 with iso 17p; and the remaining -y or misc). A median of 5 courses of azacitidine was administered (range: 1–13). Complete remission was achieved in 6 patients: 2 with trisomy 8, 3 with monosomy 7, and 1 with der(7). Haematological Improvement (HI) in Platelets occurred in 6 patients, HI-E in 2 patients, and HI-N in 3 patients. Five patients had a reduction in blast percentage. Importantly, even in complete cytogenetic remission bone marrow dysplasia persisted. All patients with monosomy 7/der(7) are in complete remission (median follow up of 10 months) whereas those with trisomy 8 relapsed their response at 2 and 5 months. CDKN2B promoter methylation in patients pre-treatment and at hematological remission was studied by bisulfite genomic sequencing (region: −263 to +243). There was no difference in CDKN2B methylation in CD34⁺ or CD33⁺ cells of responders and non-responders (both had low level, heterogeneous methylation patterns). However, CDKN2B was unmethylated in lymphocytes of responders and methylated in non-responders. Demethylation was not evident following treatment. Baseline bone marrow mononuclear cell apoptosis of 16 patients (12 RAEB, 2 RAEB T, 2 AML) analysed by PI/Annexin V staining (6% mean, 4.06% median); was not significantly different from normal controls (2%: mean, 0.77%; median, n=3) (2 sample T-test 0.103). From 12 treated patients, 8 patients responding to azacitidine (6 CR; 2 blast reduction with HI-E major and HI-N major, regardless of cytogenetic subtype), the mean apoptosis at the time of remission (11.61% mean; 9% median) was significantly higher than the mean apoptosis of 1.82% (median, 1.095%) in 4 non- responders (2 sample t-test p = 0.006). Azacitidine treatment induced sustained responses in all patients with monosomy 7, in whom the CDKN2B promoter is unmethylated. We propose that increased bone marrow apoptosis disrupts the leukaemic clone and leads to disease regression to an earlier stage.