Low Levels of Global (LINE) and CDH13 Methylation at Diagnosis and Rapid Clearance of Marrow Blasts Correlate with A Better Haematological Response to Azacitidine in Patients with Newly Diagnosed and Refractory/Relapsed AML Not Eligible for or Resistant to Chemotherapy: A Multi-Centre Phase I/II-Study of the East German Haematology and Oncology Study Group (OSHO).

Leukaemogenesis is commonly accompanied by global DNA hypomethylation together with hypermethylation and inactivation of specific regulatory genes. Therapy with methylation inhibitors can reactivate regulatory gene expression and restore normal control programmes. 5-azacitidine (Aza) is a demethylating agent approved for effective treatment of MDS. Here, we report the initial status and early changes in global DNA methylation (GDM) via analysis of LINE repetitive sequences and in the methylation of the cadherin 13 (CDH13) locus in a multi-centre study evaluating the efficacy of Aza in elderly AML patients (pts) with newly diagnosed and refractory/relapsed AML not eligible for intensive chemotherapy. Changes in methylation were correlated with marrow blast clearance, cytogenetics, response and survival.

From April-October, 2008, 40 pts with AML [19m/21f, median age 72 years] were included. Median WBC was 3.6 (range 0.7–187)×10⁹/L and median marrow blasts were 42%. High-risk cytogenetics were found in 12/40 (30%). All pts received Aza 75 mg/m²/day sc for 5 days every 4 weeks, amounting to a total of 203 treatment-courses. Haematological response according to the International Working Group Criteria for AML was assessed at 8 week intervals. For methylation studies, bone marrow mononuclear cells were collected from 33 pts prior to treatment (d0) and on day15 (d15). CpG methylation levels for LINE and CDH13 were determined by quantitative methylation-specific PCR of bisulfite treated DNA.

After a median follow-up of 13 months, overall response was 68% and occurred after a median of 2 cycles. Median response duration was 6 (range 2- not reached) months. CR, PR, and haematologic improvement (HI) occurred in 12 (30%) and stable disease (SD) in 15 (38%) pts. Response in newly diagnosed AML was 77% compared to 15% in refractory/relapsed disease (p=0.07). Interestingly, there was a strong correlation between marrow blasts on d15 and subsequent response (p=0.002), with patients achieving CR/PR/HI having a median of 13%, and those achieving SD 52% blasts. Marrow blasts <45% at d15 was highly predictive of later CR/PR/HI. Median survival time for pts with SD was 4 months while that for pts achieving CR/PR/HI has not been reached (p<0.000). High-risk cytogenetics had no negative impact on response but tended to be associated with an inferior survival (p=0.05). Median initial GDM (24.6%) decreased to 20% by d15 (p<0.000), with 82% of pts showing a decrease. The median initial methylation level of CDH13 was 29% and was unchanged by d15, with 48% of pts showing a decrease and 42% an increase. Interestingly, GDM and CDH13 methylation levels on d0 did not correlate with initial blast count. Similarly, methylation levels on d15 did not correlate with blast count by d15. On the other hand, both GDM and CDH13 methylation tended to be lower on d0 (p=0.06) and d15 (p=0.03) in pts who went on to achieve a later CR/PR/HI compared to those achieving SD only. Similarly, initial GDM and methylated CDH13 levels were lower in those pts in whom blasts dropped below 45% by d15, compared to those in whom blasts remained above 45% (p=0.05). Interestingly, the changes in initial LINE and CDH13 methylation by d15 did not correlate with blasts on d15 or later response. High-risk cytogenetics was not associated with higher initial or d15 GDM or CDH13 methylation levels compared to intermediate-risk cytogenetics. Neither the levels of methylation nor the early changes in methylation correlated with survival.

Aza resulted in an overall response in 77% of elderly pts with newly diagnosed AML with a survival rate of 39% at one year. Haematological responses occurred rapidly with bone marrow blasts at d15 being strongly indicative of subsequent clinical response. While global (LINE) methylation levels were reduced by the first cycle of Aza, it is the initial, pre-treatment degree of methylation rather than the treatment-associated changes which were found to be indicative of subsequent responses, suggesting that pre treatment methylation analysis may help identify patients who will benefit most from Aza therapy. These findings also suggest that clinical response might be the result of mechanisms other than hypomethylation such as an RNA effect with rapid down regulation of NFκB, IL6, IL6R etc as previously reported from in vitro studies in multiple myeloma.

Hoppe:Celgene GmbH: Employment. Niederwieser:Celgene GmbH: Speakers Bureau. Al-Ali:Celgene GmbH: Honoraria, Research Funding; Chugai Pharma: Research Funding.